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PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
The 411th Meeting of the Society was held in the Wolfson In8titute, Po8tgraduate Medicail School of
London, on Saturday, 16 December 1961, when the foUowing papers were premented:
COMMUNICATIONS
The Identification and Assay of Noradrenaline in Adipose Tissue
By R. L. SMIT,* R. PAoJ.Er and B. B. BRODIE.
(Laboratory of Chemical Pharmacology, National
Heart In8vtitute, National Inetitutem of Healh,
Bethesda, 14, Maryland, U.S.A.)
Various studies suggest that catecholamines have
a role in fatty acid mobilization from adipose tissue.
The addition of adrenaline or noradrenaline to
adipose tissue incubated in vitro increases the rate
of release of free fatty acids (White & Engel, 1958)
and this effect is abolished by pretreating the
tissue with an adrenergic blocking agent (Schotz &
Page, 1959). Further, the intravenous infusion of
noradrenaline or adrenaline in humans and dogs
increases the level of free fatty acids in plasma
(Havel & Goldfien, 1959). We have analysed
adipose tissue from several mammalian species for
the presence of catecholamines. Extracts of adipose tissue prepared by the procedure similar to
that described by Shore & Olin (1961) were found
to contain a substance which after oxidation with
iodine showed activation (410 mu max.) and
fluorescence (510 mH max.) spectra identical with
those produced by noradrenaline and adrenaline.
The catecholamine has been shown to be noradrenaline rather than adrenaline by comparison
of the fluorescence intensities obtained after
oxidation at pH 3 and pH 5. Dopamine was not
found in significant amounts. A fluometric assay
for noradrenaline in adipose tissue has been
developed, and using this method the amine content of various fat samples was found to be as
follows: rat epididymal fat pad 0.12jug./g., dog
omental fat 0 12j&g./g. and rabbit retroscapular
fat O.19,ug./g.
Havel, R. J. & Goldfien, A. (1959). J. Lipid Res. 1,
102.
Schotz, C. & Page, I. H. (1959). Fed. Proc. 18, 139.
Shore, P. A. & Olin, J. S. (1961). J. Pharmacol. 122,
295.
White, J. E. & Engel, F. L. (1958). Proc. Soc. exp. Biol.,
N.Y., 99, 375.
* Present address:
Department of Biochemistry, St
Mary's Hospital Medical School, London, W. 2.
b
Some Properties of Alkaline Phosphatase
Iso-enzymes
By D. W. Moss and E. J. KmGo. (Posgraduate
Medical School, London, W. 12)
When extracts of human liver, kidney and smallintestinal mucosa, prepared by Morton's (1950)
butanol method and concentrated by dialysis
against polyvinylpyrrolidone powder, are submitted to electrophoresis on starch gel in triscitrate buffer, pH 8-65 (Poulik, 1957) several
alkaline phosphatase fractions are obtained from
each. In each extract one fraction predominates;
in liver this fraction moves slightly more slowly
than the transferrin (a-globulin) fraction, while in
intestine and kidney it migrates in the haptoglobin region. Additional minor bands are seen in
the ,B-lipoprotein region in all three tissues, with
faint bands in the slow x2-globulin zone in liver and
ahead of the main enzyme band, close to the
transferrin protein fraction, in kidney.
Moss, Campbell, Anagnostou-Kakaras & King
(1961) recovered the major alkaline phosphatase
fractions from these tissues and from bone, after
starch-gel electrophoresis, and determined their
Michaelis constants with ,-naphthyl phosphate as
substrate by a spectrofluorimetric method: they
showed that small but reproducible differences
existed between the enzymes from different
tissues. The minor enzyme bands from liver,
kidney and intestine have now been studied by
similar techniques. The differences between tissues
which were shown for the major fractions are
reflected in the minor ones; thus all three phosphatase bands from liver have the Km value
characteristic of that tissue. Similarly, the three
fractions from kidney and the two from intestine
have Km values consistent with their tissues of
origin, although the difference in Km between the
phosphatases of kidney and intestine is less marked
than that between liver and other tissue phosphatases. The respective values are (pM): liver, 67;
intestine, 90; kidney, 103 ( ± 4, S.D.).
The enzymefractions from a given tissue also show
similar properties of pH optima, activation by Mg"+
ions and inactivation by incubation at 55° at pH 7.
There are slight variations between phosphatases
from different tissues in some of these properties.
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
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The differences between iso-enzymes of alkaline
phosphatase from a single tissue appear, therefore,
to be less marked than those between lactic dehydrogenase iso-enzymes, since in the case of the
latter enzyme differences in thermal stability and
in Km values as well as in electrophoretic mobility
have been demonstrated between iso-enzymes from
a given organ (Plagemann, Gregory & Wr6blewski,
1960).
Bowden, C. H., MacLagan, N. F. & Wilkinson, J. H. (1955).
Biochem. J. 59, 93.
Mandl, R. H. & Block, R. J. (1959). Arch. Biochem.
Biophy8. 81, 25.
Werner, S. C. & Block, R. J. (1959). Nature, Lond., 183,
406.
Morton, R. K. (1950). Nature, Lond., 166, 1092.
Moss, D. W., Campbell, D. M., Anagnostou-Kakaras, E. &
King, E. J. (1961). Biochem. J. 81, 441.
Plagemann, P. G. W., Gregory, K. F. & Wroblewski, F.
(1960). J. biol. Chem. 235, 2288.
Poulik, M. D. (1957). Nature, Lond., 180, 1477.
Further Studies on the Nature of the Iodotyrosines Demonstrable in Normal Human
Sera
By ALIcE DIMITRIADou,* V. MANIPOL and R.
FRASER. (Department of Medicine, Po8tgraduate
Medical School, London, W. 12)
Following the findings noted in the preceding
paper (Dimitriadou, Turner, Slater & Fraser, 1962),
other parallel acid-butanol extracts of some of the
normal sera (5 out of 17 normal subjects reported
in the previous paper) and of 7 additional sera
from thyrotoxic patients have also been made and
purified by resin-column procedures, which revealed lesser proportions of the 127I-labelled iodotyrosines from those found with the method
reported in the previous abstract. The validity of
these procedures has been tested by extracting
3121-labelled iodotyrosines from serum.
The possibility of iodotyrosine formation from
iodide and free tyrosine or from degradation of
thyronines during the Werner & Block (1959}
extraction procedure, has also been excluded. To
check whether these 127I-labelled iodotyrosines
could be metabolites of thyroxine rather than precursors of it, serum extracts of myxoedematous
subjects on thyroxine maintenance have been tested.
lodotyrosine-like Material in Serum Detected by Analysis for 1271 but not 1311
By ALICE DIMTRIADOU,* P. C. R. TURNER,
J. D. H. SLATER and R. FRASER. (Department of
Medicine, Postgraduate Medical School, and Radiotherapeutic Research Unit, Medical Research Council,
London, W. 12)
Fasting normal human serum was extracted with
acidified butanol as described by Mandl & Block
(1959) and fractionated by one-dimensional paper
chromatography in butanol-2N-acetic acid (1:1,
v/v) to separate the iodotyrosines and iodide. Where
the subject had received 131I, the paper was first
scanned for distribution of l31I (3 out of 17 subjects). Distribution of 127I was then measured
in a densitometer after spraying the paper
with a ceric ammonium sulphate-arsenious acid
mixture [equal volumes of 5% (w/v) ceric ammonium sulphate in 2N-H2SO4 and 0 2N-arsenious
acid in 1V5N-H2SO4, freshly prepared before use
as modified from Bowden, MacLagan & Wilkinson
Dimitriadou, A., Turner, P. C. R., Slater, J. D. H. &
Fraser, R. (1962). Biochem. J. 82, 20P.
Werner, S. C. & Block, R. J. (1959). Nature, Lod., 183,
406.
(1955)].
The ceric ammonium sulphate-arsenious acid
staining confirmed Werner & Block's (1959) claim
that 30-70% of the 127J was in the iodotyrosine
region (17 subjects).
Distribution of "3"I (3 subjects) was found only in
the iodothyronine and the iodide regions as
expected.
Activation analysis was carried out on paper cut
from the chromatograms with subsequent purification and counting of the 128I produced, against
standards, and this confirmed that the ceric ammonium sulphate-arsenious acid staining was
demonstrating 127J.
*
Supported by a grant from the Weilcome Trust.
Transport of Iodide by Everted Sacs of Rat
Small Intestine.
By J. D. ACLAND. (Department of Pharmacology
and Therapeutic8, Sheffield University, Sheffield 10)
The central region of the rat's small intestine has,
been observed to transport iodide against a concentration gradient from its serosal to its mucosal
side in vivo (Pastan, 1957) and in vitro (Acland &
Illman, 1959).
*
Supported by a grant from the Wellcome Trust.
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
In the present investigation, the everted sac
technique (Wilson & Wiseman, 1954) was used
throughout. Two 200-250 g. sibling male albino
Wistar rats, matched in body weight, were taken
for each comparative observation, one being used
as a control. Krebs bicarbonate buffer (Krebs &
Henseleit, 1932), containing 0.3% glucose and
0-1 mm-K'31I (about 0-05btc/ml.), was used as
incubation medium. The rate of transport of
iodide by each sac was expressed as the increase in
the total iodide content of the mucosal fluid,
measured in ,uequiv., after 1 hr. at 37°.
It was found by radioautography that all the
transported radioactivity appeared in the iodide
spot on paper chromatography in butanol-dioxan2N-NH4OH (4:1:5, by vol.) and on horizontal
paper electrophoresis in 0- lM-phosphate buffer,
pH 8, at a potential gradient of 10 v/cm. for 2 hr.
This confirmed an earlier chromatographic observation of Acland & Illman (1959), using a different
solvent system. In four experiments, transport of
iodide was unaffected by 2 mM-2-mercaptopyrazine, which completely inhibits horseradish peroxidase in vitro at this concentration and is
absorbed when administered to rats by mouth
(Rimington, 1961). Acland & Illman (1959) and
Acland & Johnson (1960) observed that transport
of iodide was unaffected by 6-methyl-2-thiouracil
and by carbimazole (British Schering Ltd), which
inhibit organic binding of iodide in the thyroid
gland. These results indicate that iodide is unchanged chemically within the mucosal cell.
Fasting for 24 hr. (3 experiments) and 48 hr. (3
experiments) caused a marked reduction in the rate
of transport of iodide as compared with control
animals fed on Diet 86 (Oxo Ltd.). Both groups
drank tap water ad libitum. Feeding cow's milk
alone ad libitum for 24 hr. (5 experiments) failed to
maintain body weight and appeared to reduce
slightly the rate of transport of iodide. The nutritional state of the mucosa may consequently be of
importance.
I wish to thank Miss Susan Whiteley for skilled technical
assistance and Professor G. M. Wilson for his interest in
this work. 2-Mercaptopyrazine was synthesized by Dr
G. W. H. Cheeseman and was a gift of Professor C. Rimington, F.R.S.
Acland, J. D. & Illman, 0. (1959). J. Physiol. 147, 260.
Acland, J. D. & Johnson, S. (1960). Biochem. J. 76,
19P.
Krebs, H. A. & Henseleit, K. (1932). Hoppe-Seyl. Z. 210,
33.
Pastan, I. (1957). Endocrinology, 61, 93.
Rimington, C. (1961). In Advances in Thyroid Research,
p. 47. Ed. by Pitt-Rivers, R. Oxford: Pergamon
Press Ltd.
Wilson, T. H. & Wiseman, G. (1954). J. Physiol. 123,
116.
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The Movement of 28Mg2+ Across the
Cell Wall of Guinea-pig Small Intestine in
vitro
By D. B. Ross* and A. D. CARE. (Animal Health
Trust, Farm Livestock Research Centre, Stock,
Essex)
Little is known about factors influencing the
movement of magnesium across the cell wall. The
influence of some enzyme inhibitors and aldosterone on this magnesium movement has been investigated.
Segments of guinea-pig small intestine were cut
longitudinally and opened out. They were suspended in bicarbonate saline (Krebs & Hensleit,
1932) containing 0.5% glucose together with
tracer amounts of 28Mg2+ and the substance under
test. The solution was maintained at 380 and was
continuously oxygenated. After 10 min. the
segments were washed and bathed in fresh bicarbonate saline for 2 hr. The 28Mg2+ liberated
during this time was taken as an indication of the
amount which had entered the tissue during the
initial 10 min. period. In this way it was shown
that sodium cyanide (22 mM), sodium fluoroacetate
(10 mM) and iodoacetic acid (1 5 mM) all greatly
reduced the uptake of 28Mg2+. Aldosterone
(0-3,UM) was approximately half as effective in this
respect.
In another series of experiments segments of
small intestine were bathed in bicarbonate saline
containing 28Mg2+ for 2 hr. The efflux of this isotope
was then studied by measuring the amount released
during eight serial 10 min. bathing periods. In one
of the periods, normally the fourth, the substance
under test was added to the bicarbonate saline.
Cyanide produced an inmediate increase in efflux
of 2SMg2+. Fluoracetate and iodoacetic acid produced a less-marked increase in efflux, which was
shown after a lag period of 20 miin. Aldosterone
had no effect on the efflux.
Since both fluoroacetate and iodoacetic acid increased the efflux of 28Mg2+ but decreased its influx
into the intestinal wall, it may be concluded that
the normal intracellular accumulation of magnesium
is dependent, at least in part, on energy derived
from the oxidation of glucose. Hanna & Maclntyre
(1960) showed that aldosterone produced a reduction in concentration of intracellular magnesium.
Our results suggest that aldosterone exerts this
effect by limiting the uptake of magnesium by the
cell, rather than by increasing its efflux.
Krebs, H. A. & Hensleit, K. (1932). Hoppe-Seyl. Z. 210,
33.
Hanna, S. & MacIntyre, I. (1960). Lancet, ii, 348.
*
Vitamealo Research Fellow.
b-2
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PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Measurement of the True Chloride Content
of Biological Tissues and Fluids
By E. CoTLovE (introduced by E. J. KING).
(National Inetitute of Health, Bethe8da, Maryland,
Cotlove, E. (1961). In Standard Mdethod, of Clinical
Chemistry, vol. 3, p. 81. Ed. by Seligson, D. New York:
Academic Press Inc.
Cotlove, E. & Green, N. D. (1958). Fed. Proc. 17, 30.
Cotlove, E., Trantham, H. V. & Bowman, R. L. (1958).
J. Lab. clin. Med. 51, 461.
U.S.A.)
Shenk, W. D. (1954). Arch. Biochem. Biophy8. 49, 138.
The chloride space has been widely utilized to Sunderman, F. W. & Williams, P. (1933). J. biol. Chem. 102,
estimate the extracellular volume of tissue samples
279.
and the partition of other substances between
extracellular and intracellular fluids. Although the
partition of tissue chloride itself has been disputed,
even more uncertain has been the validity of
Activation of Ox-Liver L-Glutamate Demeasurement of the total chloride of tissues. The
by Phenylmercuric Acetate and
hydrogenase
results of different analytical methods, even on
portions of the same tissue sample, have often Adenosine 5'-Diphosphate
differed substantially (Sunderman & Williams, By A. S. MILDVAN* and G. D. GpxvniL. (Bio1933; Shenk, 1954; Cheek & West, 1955).
chemi8try Department, Agricultural Research Council
A 3CI isotope-dilution method which provides an Institute of Animal Phy8iology, Babraham, Camadequate standard of reference for evaluation of bridge)
other methods has been developed (Cotlove &
The effects on glutamate dehydrogenase of
Green, 1958). The present method involves comacetate (PMA) (Hellerman, Schelphenylmercuric
plete isotopic exchange in a solution formed by hot
alkaline digestion of the sample, and determination lenberg & Reiss, 1958) and ADP (Frieden, 1959a, b)
of the constant value of the diluted specific activity have been compared using pyrophosphate buffer at
in purified solutions obtained by successive stages 250. With 20 mM-L-glutamate and 0-6 mM-NAD,
of alkaline dry ashing, oxidation and reduction, ADP (0.63 mM) accelerates from pH 7-0 to 9-8,
and distillationi. By means of this isotope-dilution but PMA (0.5 m-mole/g. of protein) inhibits below
method, the true chloride contents of biological pH 7-9 and accelerates above. Both raise the
samples were measured with a coefficient of optimal pH. At pH 8-5 ADP and PMA in the
variation of ± 0.6%. From the values obtained, above amounts increase the activity by 330 and
it appears that reported results of tissue chloride 150% respectively. Activation by PMA is a slow
content, particularly of muscle and liver, have second-order process (rate constant at pH 8-5,
1-0 mm-' sec.-'), first-order with respect to PMA
often been too high.
and
non-activated enzyme; protection by glutSimplified, non-isotopic methods of chloride
analysis have been developed and validated by amate (20 mM) or NAD (0- 6 mm) was negligible.
direct comparison with isotope-dilution results. Activation by ADP is rapid. With NAD greater than
The most reliable of the simplified methods, 0-2 mD, PMA (2 m-moles/g. of protein) and ADP
suitable for wet or dried tissues, involves hot (1-3 mM) which respectively triple and quadruple
alkaline digestion followed by precipitation of V,,a., decrease the Km for NAD slightly, but raise
protein with zinc hydroxide and removal of inter- the K. for glutamate 20-fold and 7-fold. With
fering sulphydryl groups in the supematant by 0-6 mm-NAD, ADP activated at all glutamate conoxidation with alkaline perborate. Accurate centrations tested, and the double-reciprocal plots
results also were obtained in almost all cases by with and without ADP were parallel ('uncomadequate extraction of fat-free dried tissue with petitive' behaviour). PMA, however, activated
0-75 N-nitric acid or with water. In the analysis of above 4-3 mm-glutamate and inhibited below, the
biological fluids, direct dilution and titration gave plots intersecting at one real glutamate concentraaccurate results. The titration of chloride with tion and velocity. One activator counteracts the
silver ion was performed throughout with an other. Thus, PMA slowly inhibits ADP-activated
automatic, coulometric-amperometric method (Cot- enzyme by a second-order reaction (rate constant
love, Trantham & Bowman, 1958; Cotlove, 1961) at pH 8-5, 1-6 mm-' sec.-'), first-order with respect
which enabled simple, rapid and highly reproducible to PMA and activated enzyme. Conversely, with
titration of less than l,uequiv. of chloride in PMA-activated enzyme, ADP is an inhibitor
biological fluids without prior removal of protein, partially competitive (Dixon & Webb, 1958) with
or in appropriately prepared solutions of tissue both substrates. PMA, like ADP, reverses the
inhibition by 1,10-phenanthroline, diethylstilboextracts or digests.
estrol and L-thyroxine.
* Postdoctoral Fellow, Division of General Medical
Cheek, D. B. & West, C. D. (1955). J. din. Invet. 34,
1944.
Sciences, United States Public Health Service.
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Activation of the enzyme by ADP correlates with
association of subunits (Frieden, 1959a, b). Kinetic
and ultracentrifuge experiments (200) at similar
enzyme concentrations revealed that 28,.moles of
PMA/g. of protein, which caused 30% acceleration, lowered S20 from 18s by 12%. In tris buffer,
pH 8 1, at 60, with NADH or ADP, PMA produced greater reductioIns in S (e.g. 29%). During
starch-gel electrophoresis of the enzvme with ADP
present, PMA caused appearance or intensification
of a faster-moving component, detected by protein
stain or enzymic activity. Retarded sedimentation with increased mobiJity in starch gel indicates
that PMA dissociates the enzyme, and strict
correlation between dissociation and loss of activity
(Frieden, 1959a, b; Yielding & Tomkins, 1960)
becomes unlikely.
Dixon, M. & Webb, E. C. (1958). In Enzyme8, p. 174.
London: Longmans, Green and Co. Ltd.
Frieden, C. (1959 a). J. biol. Chem. 234, 809.
Frieden, C. (1959b). J. biol. Chem. 234, 815.
Hellerman, L., Schellenberg, K. A. & Reiss, 0. K. (1958).
J. biol. Chem. 233, 1468.
Yielding, K. L. & Tomkins, G. M. (1960). Proc. nat. Acad.
Sci., Wash., 46, 1483.
Serum Phosphatases and Glycolytic
Enzymes in Cancer of the Breast
By DIANA M. CAMPBELL and E. J. KING. (Department of Chemical Pathology, Postgraduate Medical
Sci9ol of London, London, W. 12)
The blood-serum levels of non-specific acid and
alkaline phosphatase and the specific phosphatase
5-nucleotidase have been studied in serial specimens from patients with cancer of the breast. The
results, together with those for phosphoglucose isomerase and aldolase, have been correlated with
clinical data to assess the usefulness of enzymes as
an index of response to treatment, and as an indication of the location of metastases. Total acid
phosphatase (phenyl phosphate) is raised in
patients with oesteoclastic bone diseases (Woodard,
1959). Raised alkaline phosphatase occurs in
osteoblastic bone disease, and also in hepatobiliary disease: there is usually a clear distinction
between these conditions, but in metastatic carcinoma of the breast the origin of a raised enzyme
level in the blood serum may be less obvious.
5-Nucleotidase, however, is normal in bone disease
and raised in hepatobiliary disease (Dixon &
Purdom, 1954; Young, 1958).
The glycolytic enzymes (phosphoglucomutase,
phosphoglucose isomerase and aldolase) are widely
distributed in the tissues, which makes it im-
23P
possible to draw valid conclusions concerning
disease in a particular organ from the serum levels
of these enzymes. Nevertheless, their determination in blood serum is useful, since fluctuations in
their levels reflect the clinical condition, and serial
studies are helpful in following progress. Their
serum levels parallel each other, but the isomerase
provides the most sensitive index of the clinical
status.
Parallel estimations of acid and alkaline phosphatases, 5-nucleotidase (by the method of
Campbell, 1962) and isomerase are particularly
useful in diagnosis, in helping to locate metastases,
and in studying the effect of supression of cancer of
the breast, e.g. by pituitary ablation with radioactive yttrium. The isomerase, markedly raised
before treatment, falls during remission, but rises
again if relapse occurs. The acid phosphatase, if
greatly raised, probably indicates skeletal metastases with strong osteoclastic activity; raised
alkaline phosphatase implies marked osteoblastic
activity. A very high isomerase without greatly
raised phosphatases strongly suggests secondaries
in the liver. The 5-nucleotidase furniishes useful
confirmation.
Campbell, D. M. (1962). Biochem. J. 82, 34P.
Dixon, T. F. & Purdom, M. (1954). J. clin. Path. 7, 341.
Woodard, H. Q. (1959). Amer. J. Med. 27, 902.
Young, I. (1958). Ann. N.Y. Acad. Sci. 75, 357.
The Estimation of Glycerol in Plasma
H. HAGEN. (Department of Physiology,
JEAN
By
University of Manitoba, Winnipeg, Canada)
The commonly used method of glycerol estimation involves periodate oxidation (e.g. Korn, 1955).
Since glucose is also oxidized by periodate this
procedure is unsuitable for the estimation of
glycerol in blood. An enzymic method for the
estimation of plasma glycerol is described here, in
which the reduction of DPN by glycerol in the
presence of glycerol dehydrogenase is measured
spectrophotometrically. Glucose does not interfere. Glycerol dehydrogenase was partially purified
from Aerobacter aerogenes by the method of Burton
(1955). The yield of enzyme is highest when the A.
aerogenes is cultured under relatively anaerobic conditions (Lin, Levin & Magasanik, 1960).
The reaction mixture contains 50 mM-glycine
buffer, pH 9-5, 5 mM-DPN, and neutralized, protein-free plasma sample containing 2-10,um-moles
of glycerol, in a total volume of 1 0 ml. The reaction
is allowed to proceed for 30 min. and the absorption at 340 mp compared with that of a series of
glycerol standards. Appropriate controls are
24P
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
necessary to correct for enzyme blank and for
material in the plasma samples absorbing at
340 m,u. Glucose, lactate, malate, P-hydroxybutyrate, succinate, glutamate, oc-glycerophosphate and oc-oxoglutarate do not interfere with the
estimation of glycerol. Some reduction of DPN
does occur with relatively large amounts of
ethanol and with glyceraldehyde 3-phosphate.
However, no glyceraldehyde 3-phosphate can be
detected in plasma samples by assay with crystalline glyceraldehyde 3-phosphate dehydrogenase.
Although ethanol has been reported in plasma
(McManus, Contag & Olson, 1960) the amounts
present are insufficient to interfere with the glycerol
determination. Normal rabbit plasma has been
found to contain from 8 to 16,umoles of glycerol per
100 ml.
Glycerol kinase has previously been used for the
estimation of glycerol by Boltralik & Noll (1960)
and by Wieland (1957). The glycerol dehydrogenase
method is more sensitive than the former method
and about as sensitive as the latter, but is simpler
and less expensive.
Boltralik, J. J. & Noll, H. (1960). Analyt. Biochem. 1, 269.
Burton, R. M. (1955). In Method8 in Enzymology, vol. 1,
p. 397. Ed. by Colowick, S. P. & Kaplan, N. 0. New
York: Academic Press Inc.
Korn, E. D. (1955). J. biol. Chem. 215, 1.
Lin, E. C. C., Levin, A. P. & Magasanik, B. (1960). J. biol.
Chem. 235, 1824.
McManus, I. R., Contag, A. 0. & Olson, R. E. (1960).
Science, 131, 102.
Wieland, 0. (1957). Biochem. Z. 329, 313.
Control of Cystathionine Formation in
Escherichia coli by Methionine
By R. J. ROWBURY. (Microbiology Unit, Department of Biochemistry, University of Oxford)
The synthesis of amino acids in bacteria is often
controlled by the end product in one or both of two
ways: (a) it represses the formation of enzymes
required for its own synthesis, (b) it inhibits the
activity of such an enzyme (usually one early on
the pathway to the metabolite).
Since the formation of both homocysteine
methylase and cystathionase by E. coli is repressed
if organisms are grown with methionine (Rowbury
& Woods, 1961a, b), a similar study has been
made with a system forming cystathionine from
homoserine and cysteine (Rowbury, 1961) believed
to be a stage in the pathway of methionine synthesis. With extracts of organisms (strain 26/18)
grown with 3 mM-DL-methionine, cystathionine
formation was reduced to about 15%. Such synthesis requires at least two enzymes (Rowbury,
1961); the first (I, present in strain 7/9) converts
homoserine and succinate, in the presence of ATP
and glucose into an intermediate, which together
with cysteine and the second enzyme (II, present in
strain 2/2) yields cystathionine (strains 7/9 and 2/2
are methionine auxotrophs which respond also to
cystathionine). Growth of organisms with 3 mmDL-methionine results in a decrease of the activities
of both enzymes to about 15%. Thus the formation of all four enzymes so far known to be required for the overall synthesis of methionine from
homoserine and cysteine is repressed by methionine.
Cystathionine formation (from 5 mm-homoserine
and 3-3 mM-cysteine) by the joint action of enzymes
I and II (present in an extract of strain 26/18
grown without methionine) is inhibited 50% by
15 mM-DL-methionine. The activity of enzyme I
(from strain 7/9) has been measured by following
the disappearance of homoserine (initially 1-66 mM)
and is decreased to a half by 0 6 mM-DL-methionine.
Methionine is therefore an inhibitor not only of the
synthesis of the first enzyme peculiar to its own
synthetic path, but also of the activity of this
enzyme when formed.
Rowbury, R. J. (1961). Biochem. J. 81, 42P.
Rowbury, R. J. & Woods, D. D. (1961 a). J. gen. Microbiol.
24, 129.
Rowbury, R. J. & Woods, D. D. (1961b). Biochem. J. 79,
36P.
Anaerobic Metabolism in Musca domestica, the Housefly
By J. P. HESLOP, G. M. PRICE and J. W. RAY.
(Agricultural Re8earch Council, Pest Infes8tation
Laboratory, Slough, Bucks.)
Houseflies appear to suffer no lasting damage
from several hours' exposure to oxygen-free
nitrogen. After various periods of anoxia groups of
male flies were ground with 0 4N-perchloric acid
under liquid nitrogen and extracted at 00. The
following estimations were made: oc-glycerophosphate by a modification of an enzymic method
(Bublitz & Kennedy, 1954); glycerol (Lambert &
Neish, 1950), orthophosphate as by Martin & Doty
(1949), arginine phosphate as orthophosphate
released by 1 min. hydrolysis at 1000 in 0-IN-HCI,
total acid-soluble phosphorus, phospholipid phosphorus [acid-insoluble, sol. in chloroform-methanol
(1:1, v/v)], and residual phosphorus after 3 hr.
digestion in perchloric acid, and adenosine triphosphate (ATP) as by Strehler & Totter (1952).
Glycogen was estimated by the anthrone method
on flies extracted with 30% KOH (Hassid &
Abraham, 1957).
PROCEEDINGOS OF THE BIOCHEMICAL SOCIETY
During 3 hr. anoxia orthophosphate increased
from 4 to 13,umoles/g. In the first 30 min. there
was substantial depletion of arginine phosphate and
ATP while glycerophosphate rose from 2 to
9,umoles/g. but no further change in the levels of
these three metabolites occurred up to 3 hr. The
levels of acid-soluble, phospholipid and residual
phosphorus and of glycerol did not change during
3 hr. anoxia. The amount of glycogen consumed in
the second 30 min. of anoxia equalled that used in
the first 30 min. The changes found in glycerophosphate, orthophosphate and ATP agreed well with
the results of Winteringham (1960) on drowned
houseflies.
If the conversion of dihydroxyacetone phosphaten
into glycerophosphate in anoxic flies (for references
see Gilmour, 1961) were the principal reaction
yielding DPN and thereby permitting glycolysis to
proceed, further metabolism of glycerophosphate
after 30 min. anoxia would be implied, since the
level of glycerophosphate stops rising at that time
even though glycolysis continues. The most
probable metabolite would be glycerol but this
does not accumulate, and its further oxidation in
these conditions is unlikely. Price (1961) showed
that oc-alanine accumulates in anoxic houseflies up
to 2 hr. and suggested a route for its formation in
which DPNH is re-oxidized and the pyruvate
formed by glycolysis is removed. Both mechanisms
for DPN regeneration may operate in anoxic ffies
but the pathway leading to glycerophosphate
ceases to be important after 30 min. More than
three-quarters of the glycerophosphate increase in
the fly is localized in the thorax where most of the
insect's muscle is situated.
Bublitz, C. & Kennedy, P. (1954). J. biol. Chem. 211, 951.
Gilmour, D. (1961). In Biochemistry of Insects, p. 75.
London: Academic Press Inc.
Hassid, W. Z. & Abraham, S. (1957). In Methods in
Enzymology, vol. 3, p. 35. Ed. by Colowick, S. P. &
Kaplan, N. 0. New York: Academic Press Inc.
Lambert, M. & Neish, A. C. (1950). Canad. J. Res. 28B, 83.
Martin, J. B. & Doty, D. M. (1949). Analyt. Chem. 21, 965.
Price, G. M. (1961). Biochem. J. 81, 15P.
Strehler, B. L. & Totter, J. R. (1952). Arch. Biochem.
Biophys. 40, 28.
Winteringham, F. P. W. (1960). Biochem. J. 75, 38.
The Recovery of the Housefly Musca
domestica from Anoxia
By J. W. RAY and J. P. HESLOP. (Agricultural
Research Council, Pest Infestation Laboratory,
Slough, Buck8.)
The housefly is able to survive for several hours
in an atmosphere of nitrogen. Under these conditions the level of oc-glycerophosphate and ortho-
25P
phosphate rose and the level of adenosine triphosphate (ATP) and arginine phosphate fell substantially (Heslop, Price & Ray, 1962). Thoracic levels of
these intermediates have now been studied in flies
recovering from a 60 min. period of anoxia (for
methods see Heslop et al. 1962). Recovery times of
0, 1, 5, 10, 15, 20, 30, 40, 60, 90 and 120 min. and
19 hr. have been studied. The symptoms during
recovery were: 0-5 min. no movement; 5-15 min.
gut movement in abdomen; 10-20 min. leg movements; 20-30 min. flies walk about.
After 60 min. anoxia the level of glycerophosphate was 8 times that in the normal fly thorax, but
dropped to 3 times the normal value in the first
5 min. in air. At 90 min. the level was still twice
that in the normal fly. The orthophosphate level
was 21 times normal after the 60 min. period of
anoxia, and on returning the flies to air fell rapidly
during the first 5 min. to half the normal level.
Thereafter it rose slowly and by 30 min. had risen
to just above the normal level but soon returned
to it. The level of ATP rose sharply in the first
minute of recovery to about half that found in the
normal fly and remained constant for the next
90 min. By 19 hr. the levels of both ATP and
glycerophosphate had returned to those found in the
normal fly thorax.
After 5 min. in air, when the levels of ATP and
glycerophosphate had ceased to change rapidly, no
movements could be observed in the intact fly.
Very little further change had occurred in the level
of these two intermediates at 30 min. when the
flies were very active. The recovery of activity in
the flies coincided with the return of the arginine
phosphate level to normal but was not correlated
with the level of ATP or glycerophosphate.
Heslop, J. P., Price, G. M. & Ray, J. W. (1962). Biochem. J.
82,
24r.
Adenosine Triphosphate-Degrading Enzymes of Adrenal Medulla Mitochondria
By P. HAGEN. (Department of Biochemi8try,
Univeraity of Manitoba, Winnipeg, Canada)
In adrenal medulla cells adrenaline and noradrenaline are stored within chromaffin granules as
salts of adenine nucleotides (Blaschko, Hagen &
Hagen, 1957; Burack, Weiner & Hagen, 1960). It
has been reported that the chromaffin granules
contain adenosine triphosphatase (ATPase), which
functions in the release of the catecholamines
(Hillarp, 1958). On the other hand, Fortier, Leduc
& D'Iorio (1959) have claimed that the ATPase is
associated with the mitochondria and not with the
chromaffin granules.
In confirmnation of both claims ATPase activity
has been found in the 'large-granule' (11 000g)
26r
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
sediment isolated from ox adrenal medulla by
differential centrifugation. On further fractionation of this sediment by density-gradient centrifugation the ATPase was found predominantly in
the mitochondrial fraction as reported by Fortier
et al. (1959).
Much greater ATPase activity was found in the
microsomes. The mitochondrial ATPase differs
from the microsomal enzyme in its Michaelis
constant and in the influence of pH on its activity.
The mitochondrial enzyme liberates orthophosphate from either adenosine triphosphate (ATP) or
inosine triphosphate (ITP) at a rate about 10 times
that at which it hydrolyses adenylic acid (AMP) or
P-glycerophosphate. Attempts to separate these
activities have been unsuccessful. The enzyme(s) cannot be made soluble by digitonin or by sonication.
The products of the reaction with ITP are
orthophosphate and inosine diphosphate (IDP).
With ATP they are orthophosphate, adenosine
diphosphate (ADP) and AMP, or, on prolonged
incubation, orthophosphate, AMP, adenosine and
inosine. The AMP is probably produced from ADP
by the action of a heat-stable adenylate kinase
found to be present in the mitochondria. The inosine
is probably the product of an adenosine deaminase
also present in the mitochondria, probably as a contaminant from a highly active supernatant enzyme.
That the mitochondrial ATPase is specific for
the triphosphates and that the further breakdown
of ADP is catalysed by the other enzymes in the
mitochondria is suggested by the inability of the
mitochondrial preparation further to degrade IDP.
The presence of the mitochondrial adenylate
kinase can be demonstrated after inactivation of
the other enzymes by heating.
The finding that the 'large-granule' ATPase can
be virtually completely separated from the chromaffin granules raises doubts concerning any role
of this enzyme in the release of adrenaline and
noradrenaline from the chromaffin granules.
pentahydric bile alcohols, occurring as sulphates.
Both substances probably have the cholestane
carbon skeleton. Infrared comparisons indicated
that the nucleus in both alcohols is the same. The
infrared spectra of purified cyprinol and ranol
show a strong resemblance to those of derivatives
of allocholic acid. The acid obtained by chromic
oxidation of ranol (Haslewood, 1952) and cyprinol
has now been found to be 3,7,12-trioxoallocholanic
acid (dehydroallocholic acid). Hence, ranol and
cyprinol contain the nucleus and the side chain of
allooholic acid. Allo bile alcohols (polyhydroxycholestanes) have probably given rise during
evolution to the allocholic acid already found in
some fishes, reptiles and birds: it may be expected
that C27 allo bile acids will also be discovered.
Professor T. Kazuno and Miss T. Masui (Hiroshima
University School of Medicine, Japan) have independently concluded that bile of the frog Rana
catesbiana contains an alcohol having the allocholic
acid nucleus and carbon side chain.
Haslewood, G. A. D. (1952). Biochem. J. 51, 139.
Sterol Biosynthesis in Neoplastic Cells
By IRENE Y. GoRz and G. POPJAK. (Medical
Re8earch Council, Experimental Radiopathology
Research Unit, Hammersmith Hospital, London,
W. 12)
In contrast to embryonic tissues (Popjak &
Beeckmans, 1950; PopjAk, 1954), ascites tumour
cells are unable to utilize acetate for sterol synthesis (Popjfik & Berthet, unpublished results).
This agrees with observations made by others on a
variety of tuxnours (Busch, 1953; Busch & Baltrush, 1954). A possible relation between this
inability to convert acetate into lipid and some
characteristic aspect of malignancy, such as
Blaschko, H., Hagen, J. M. & Hagen, P. (1957). J. Phyiol. invasiveness, prompted further investigations.
189, 316.
The tumours used were either the mouse Ehrlich
Burack, R., Weiner, N. & Hagen, P. (1960). J. Pharmacol. ascites carcinoma or a rat ascites tumour derived
130, 245.
from a benzopyrene-induced sarcoma (RD 3).
Fortier, A., Leduc, J. & D'Iorio, A. (1959). Rev. canad. Procedures were standardized for harvesting the
Biol. 18, 110.
cells, and for making whole-cell suspensions and
Eilarp, N. A. (1958). Actda phyiol. 8cand. 42, 144.
ultrasonically disrupted preparations. Incubations
with radioactive precursors, isolation of products
and determination of their radioactivity were
The Natural Occurrence of allo Bile modelled on methods devised in this laboratory for
Alcohols
studies of sterol biosynthesis in liver systems.
The inability of tumour cells to convert 14C]By T. BRIGGS and G. A. D. HASLEWOOD. (Guy'8
acetate
into fatty acids and cholesterol was again
Ho8pital Medical School, London, S.E. 1)
confirmed, the metabolic block involved lying
Ranol from the frog Rana temporaria, and beyond the acetyl-CoA stage of the syntheses. The
from fishes of the family Cyprinidae are supposition (Medes, Thomas & Weinhouse, 1953)
~cyprinol
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
that tumour cells obtain their lipids preformed
from the host was experimentally confirmed.
Next a further demarkation of the enzymic or
co-factor deficiencies of sterol synthesis in these
cells was attempted, by using mevalonic acid as
substrate, which is known to be synthesized from
acetyl-CoA. Incorporation of [2-14C]mevalonic
acid by whole and disrupted tumour cells into
allyl pyrophosphates (squalene precursors), into
squalene, 3,B-hydroxysterols and terpenoid acids
(dimethylacrylic, geranoic, and farnesoic) was
demonstrated. The disrupted cell preparations were
less active than whole cells, especially in the formation of sterols. The whole chain of reactions from
mevalonate (but not from acetate) to sterol could
be activated in preparations of disrupted cells by
a simultaneous inhibition with NaF of pyrophosphatases present in the tumour cells and by addition of ATP and TPNH. The nature of the controls
operating against efficient production of sterols was
thus clarified: (a) the pyrophosphatases deplete the
available substrates for squalene synthesis by an
irreversible hydrolysis of the allyl pyrophosphates
to the inert free allylic alcohols (Christophe &
Popjak, 1961); (b) the amount of ATP available is
insufficient for optimum formation of allyl pyrophosphates; and (c) a low concentration of endogenous TPNH does not meet the requirement for
the steps of squalene and sterol formation.
Busch, H. (1953). Cancer Res. 18, 789.
Busch, H. & Baltrush, H. A. (1954). Cancer Re8. 14, 448.
Christophe, J. & Popjik, G. (1961). J. Lipid Re8. 2, 244.
Medes, G., Thomas, A. & Weinhouse, S. (1953). Cancer
Re8. 13, 27.
Popjik, G. & Beeckmans, M. L. (1950). Biochem. J. 46,
547.
Popjak, G. (1954). Cold Spr. Harb. Sym. quant. Biol. 19,
200.
Rapid Metabolism of '4C-Labelled Oestrogen in the Rat
By R. E. OAXWY, SHniUA S. EccLss and S. R.
STITCH. (Medical Research C(ouncil, Radiobiological
Re8earch Unit, Harwell, Di&ot, Berkc.)
Wotiz, Ziskind & Ringler (1958) infused rats
with [16-14C]oestrone and characterized oestradiol-17 P extracted from the plasma. Incubation in
vitro of oestradiol-17# with rat erythrocytes leads
to formation of oestrone (Lunaas & Velle, 1960;
Portius & Repke, 1960). Evidence from paper
chromatography indicated that oestrone, oestradiol-17x and oestradiol-17P were present in rat
urine (Ketz, Witt & Mitzner, 1961).
We have measured the relative proportions of
phenolic metabolites extracted from rat blood
27P
collected 2, 7 and 15 min. after injection of
[16-14C]oestradiol- 17,B.
Female rats (Wistar origin, 200-230 g.), in
oestrus, were anaesthetized and [16-14C]oestradiol17UP (chromatographically purified, 0-5,c, equivalent to 11,ug. in 0-4 ml. of 0.9% saline containing
0-01 ml. of ethanol) was injected into the femoral
vein. The rats were killed 2, 7 and 15 min. after
injection. Blood (samples of 3-6 ml.), collected
immediately by heart puncture, was diluted with
water (100 ml., containing 1 mg. each of oestrone,
oestradiol-17P and oestriol) and extracted with
ether. Approximately 90-98% of the injected
radioactivity could not be recovered, indicating
a rapid loss of the injected oestradiol-17fl. The
ether layer was extracted with N-NaOH and
oestrogens were separated by partition chromatography (Oakey, 1961). Each eluted fraction was
assayed for radioactivity (Stitch, 1959) and for
carrier by photofluorimetry. Of radioactivity in
the phenolic fraction, 52-63% after metabolism
and 72-81% from controls, was coincident with
carriers. This discrepancy may indicate formation,
in metabolic experiments, of compounds not eluted
under our conditions.
The radioactivity eluted coincident with carrier
oestrogen was corrected for loss during processing.
Small, but increasing, proportions of radioactivity,
relative to oestradiol-17, recovered, were eluted
coincident with oestrone. The proportions eluted
with oestriol were variable, after the same intervals. Although most of the injected oestradiol-17P
was not recovered, the results do indicate a rapid
metabolism to other oestrogens.
Despite prior purification of precursor [16-14C]oestradiol-17, (Bauld, 1955), further chromatography (Oakey, 1961) revealed small amounts of
radioactivity eluted in the oestrone and oestriol
regions. Also, control experiments indicated that
as much as 5% of radioactivity added to boiled
blood appeared as artifact with polarity similar to
oestriol. These observations emphasize the need for
rigorous controls in experiments involving the
extraction of minute amounts of oestrogen from
blood or urine.
We thank Dr D. R. Lucas for performing the injections
and blood collections.
Bauld, W. C. (1955). Biochem. J. 59, 294.
Ketz, H. A., Witt, H. & Mitzner, M. (1961). Biochem. Z.
334, 73.
Lunaas, T. & Velle, W. (1960). Acta phyeiol. Scand. 50,
Suppl. 175, 95.
Oakey, R. E. (1961). Biochem. J. 81, 13P.
Portius, H. J. & Repke, K. (1960). Arch. exp. Path.
Pharmak. 239, 144.
Stitch, S. R. (1959). Biochem. J. 73, 287.
Wotiz, H. H., Ziskind, B. S. & Ringler, I. (1958). J. biol.
Chem. 231, 593.
28P
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Estimation of Penicillinase in Single
Bacterial Cells
By J. F. COLLINS (introduced by M. R. POLLOCK).
(National In8titute for Medical Re8earch, Mill Hill,
London, N.W. 7)
The basis of this microspectrophotometric
method is the measurements of the rate of penicillin destruction in microdrops containing single
cells. Penicillin is destroyed by penicillinase to give
penicilloic acid, and the pH of the reaction mixture
decreases. Microdrops containing 2% (w/v)
bromocresol purple (pK 6.15) and 20 000 i.u. of
benzylpenicillin/ml. at pH 7-5 change in colour
from purple to yellow as penicillin is destroyed,
and the total rate of destruction of penicillin in the
drop is estimated from the rate of increase of light
transmission at 5900A, the peak absorption of the
purple form of the indicator, and the calculated
volume of the drop.
The microdrops are prepared by spraying a
mixture of cells and test solution from a drawn-out
capillary tube into a film of silicone oil on a coverslip made water-repellent by treatment with
dimethyldichlorosilane. Another water-repellent
coverslip is placed over the oil film. The drops rise
in the oil, which is slightly denser than water,
until they rest against the coverslip without wetting
it, thereby retaining their spherical shape. When
the oil forms a thin film (about 30, thick) between
the coverslips, the upper cover will not slide easily,
and the drops touching it show no tendency to
move. The drops vary in diameter (1-30iu) and in
the number of cells they contain; since they rest
against the top coverslip, a drop of diameter
7-15, containing a single cell can be found
quickly.
The transmission is measured through the central
area of the drop, using a field aperture to limit the
illuminated area on the slide to a circle of about
6,u diameter. The -images of the drop and field
aperture are projected on a screen and centred over
a small aperture equivalent to 1 u diameter on the
preparation. The light passes through the centre of
the drop and through the smaller aperture on to a
photomultiplier, and the output is recorded
continuously.
The distribution of penicillinase has been
measured in uninduced log-phase cells of Bacillu8
8ubtili8 749 (Kushner, 1960). These contain an
average of 100 molecules of enzyme/cell, but the
individual cells contain amounts spread widely
about the average value. The method can be
adapted to measure other enzymes which will
produce a colour change measurable in these
microdrops.
Long-chain Fatty Acid Synthesis by
Isolated Plant Leaves
By A. T. JAMES. (National In.stitute for Medical
Re8earch, Mill Hill, London, N.W. 7)
Uptake of [2-14C]acetate by isolated plant leaves
(especially Ricinu8 communi8) through the stem or
by chopped leaves in phosphate buffer (pH 7.0)
leads to the synthesis of the C14, C16, and C18
saturated acids and also oleic, linoleic and linolenic
acids. Labelled octanoic, decanoic and dodecanoic
acids give rise to labelled oleic acid, the position of
the label suggesting an in toto incorporation.
Labelled palmitic and stearic acids do not produce
any labelled unsaturated acids. Labelled oleic
acid produces labelled linoleic acid, the label
position being preserved. The evidence is similar
to that presented by Scheuerbrandt, Goldfine,
Baronowsky & Bloch (1961) for biosynthesis of unsaturated acids in an anaerobic micro-organism
(Clo8tridium butyricum), except that the latter
system cannot utilize dodecanoic acid.
Scheuerbrandt, G., Goldfine, H., Baronowsky, P. E. &
Bloch, K. (1961). J. biol. Chem. 236, PC70.
Kushner, D. J. (1960). J. gen. Microbiol. 23, 381.
tion, 1961.
Synthesis of Oleic Acid and Palmitic Acid
from Acetate by Lettuce Chloroplast Preparations
By P. K. STuMPF* and A. T. JAMES. (National
In8titute for Medical Re8earch, Mill Hill, London,
N.W. 7)
Chloroplast preparations have been obtained
from lettuce-leaf homogenates (Whatley, Allen,
Trebst & Arnon, 1960). Incorporation of acetate
into long-chain fatty acids by these preparations
has been examined under both dark and light conditions. When acetate is added to a reaction mixture
which is maintained in the dark under aerobic
conditions, ATP, CoA, Mn2+, Mg2+, CO2 and TPN
are required for maximum incorporation (4-10% in
2 hr. at room temperature at pH 7-4). Approximately 57% of the 14C is located in palmitic acid
and 38% in oleic acid. When exposed to light, the
reaction mixture incorporated [2-_4C]acetate again
into palmitic and oleic acids in approximately the
same ratio. Exposure to light increased acetate
incorporation approximately 1-7-2-4 times in
contrast to incubation in absence of light. We
favour the postulate that the process of photosynthetic phosphorylation provides conditions for
the synthesis of fatty acid, namely the continuing
formation of ATP, 02 and TPNH.
Whatley, F. R., Allen, M. B., Trebst, H. V. & Arnon, D. I.
(1960). Plant Phy8iol. 35, 188.
* Senior Postdoctoral Fellow, National Science Founda-
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Immunological Studies on Two Forms of
Circulating Insulin
By N. SAMAAN, W. J. DEMPSTER, R. FRASER and
D. STILLMAN (introduced by E. J. KING). (Departments of Medicine and Experimental Surgery, Po8tgraduate Medical School, London, W. 12)
Previous published studies, and later experiments in dogs, suggest two forms of circulating
insulin-like activity. One form, 'typical' insulin, is
inhibited by guinea-pig antiserum in the in vitro
tests; the other form, 'atypical' insulin, is not so
inhibited by antiserum.
Insulin-like activity has been measured in vitro,
using the rat epididymal fat-pad assay, which is
based on the stimulating effect of insulin on the
production by this tissue of both 14CO2 and 14C_
labelled fat from [1-14C]glucose in the incubating
medium as described by Slater, Samaan, Fraser &
Stillman (1961). 14CO2 was absorbed hyamine
hydroxide and 14C-labelled fat was extracted from
the fat tissue by an ethanol-light petroleum
mixture. The radioactivity of both 14CO2 and 14C_
labelled fat was counted in a Packard Tricarb
liquid scintillation spectrometer.
The antiserum to insulin was prepared by injection of commercial bovine insulin into guinea pigs.
Each set of samples in the bioassay runs included
at least one insulin standard. A standard log-dose
response curve has been established for both commercial pancreatic bovine insulin and for serum.
In dogs, simultaneous venous samples from
pancreatic, hepatic and peripheral venous sites
revealed equal concentration of 'atypical' insulinlike activity, but a large excess of 'typical' insulinlike activity in pancreatic venous blood.
Infusion of pancreatic insulin into the portal
vein was found to lead to a rise of the 'atypical'
insulin-like activity concentration in the hepatic
vein, but a similar infusion into liverless animals
was followed by no such rise.
These experiments suggest that insulin is
secreted by the pancreas in the 'typical' form and
that some of this is changed during its circulation
to the periphery, possibly in the liver, into the
'atypical' form of insulin.
Slater, J. D. H., Samaan, N., Fraser, R. & Stillman. D.
(1961). Brit. med. J. i, 1712.
The Effect of Phenolic Acids on Cerebral
Enzymes
By JOCELYN M. HICKS, I. D. P. WOOTTON and D. S.
YOUNG. (Department of Chemical Pathology, Postgraduate Medical School of London, London, W. 12)
Many phenolic acids have been identified as
abnormal constituents in the plasma of patients
29P
with acute renal failure (Hicks, Young & Wootton,
unpublished results). Since most of the signs of
clinical uraemia can be attributed to depression of
cerebral function, the effect of these compounds on
the metabolism of the central nervous system has
been studied. The following enzymes in rat-brain
homogenates have been investigated: glutamic
acid decarboxylase, dihydroxyphenylalanine decarboxylase, 5-hydroxytryptophan decarboxylase,
5-nucleotidase, amine oxidase, lactic dehydrogenase, glutamic oxaloacetic and glutamic pyruvic
transaminases. The decarboxylases were estimated
manometrically while colorimetric or spectrophotometric methods were used for the remaining enzymes.
The effect of phenolic acids on the respiration
of guinea-pig-brain slices was also investigated.
In any one enzyme system, the molar concentration of the substances tested was the same; this
concentration was adjusted so that a considerable
inhibition was produced by at least one of the
substances. Cinnamic acid and its hydroxylated
derivatives were found to inhibit respiration of
brain slices to the greatest extent, but phenylpropionic, benzoic and hippuric acids were equally
toxic towards glutamic acid decarboxylase; substituted cinnamic acids were less toxic and benzoic
acid less toxic still. Dihydroxyphenylalanine decarboxylase was considerably inhibited by 2- and
3-hydroxy derivatives of benzoic, phenylacetic
and cinnamic acids; by contrast the 4-hydroxy
derivatives were comparatively non-toxic. Aminobenzoic acids were more inhibitory to 5-nucleotidase
than any of the other compounds studied.
It is considered that the action of these acids may
be important in the abnormality of cerebral function manifested by patients with clinical uraemia.
Multiple Forms of Flavoprotein Oxidoreductases from Heart-Muscle Particles
By M. R. ATKINSON, M. DIxoN and JANET M.
THORNBER. (Department of Biochemistry, University of Cambridge)
NADH2-lipoamide oxidoreductase (EC 1. 6. 4. 3;
lipoamide dehydrogenase, Straub's 'diaphorase'),
prepared from pig-heart Keilin-Hartree particles,
is a flavoprotein homogeneous by ultracentrifuge
and Tiselius electrophoresis (Savage, 1957). However, electrophoresis in starch gel at pH 8-6 with
a discontinuous citrate-borate buffer (Poulik,
1957) separates it into six flavoproteins (I-VI, in
descending order of mobility), of which I and II are
minor components. These all possess the characteristic fluorescence and ability to react with lipoamide which distinguish this enzyme from other
flavoproteins, as well as diaphorase activity. How-
30r
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
ever, they are not all equally active; furthermore,
the ratio of lipoamide dehydrogenase to diaphorase
activity varies from one component to another.
Preparations made by the original method of
Straub, or by the method of Massey, Gibson &
Veeger (1960), either at room temperature or at
0-2o, as well as one kindly supplied by Dr Massey,
all showed the same components, though with some
variation of relative amounts. They were all present
in a preparation made from a single pig heart,
showing that the different forms were not merely
due to differences between individual animals. On
the other hand, a preparation from ox heart at 0-2°
showed only three zones, corresponding in mobility
to I, II and III, although one preparation made at
200 showed also components resembling V and VI.
'Fingerprinting' of a trypsin hydrolysate of the
mixed components by paper electrophoresis at
pH 5-6 and 3*5 showed about forty peptides, and
the high yield of the individual peptides suggests
that the components have largely the same amino
acid sequence.
The enzyme when present in the x-oxoglutaratedehydrogenase complex (Sanadi, Littlefield &
Bock, 1952; Massey, 1960) remains at the origin on
electrophoresis in starch gels, but in gels containing
3*5 M-urea it shows multiple zones with the same
mobilities as the free enzyme.
Samples of NADH2-cytochrome c oxidoreductase (EC 1. 6.2. 1) with specific activities similar to
those of the purest preparations of Mahler, Sarkar,
Vernon & Alberty (1952) were prepared from pigand ox-heart particles. These were separated by
starch-gel electrophoresis into a number of active
components. The possibility that this multiplicity of
mitochondrial oxidoreductases is of genetical origin,
or that it arises as an artifact during the preparation of Keilin-Hartree particles, is being examined.
Mahler, H. R., Sarkar, N. K., Vernon, L. P. & Alberty,
R. A. (1952). J. biol. Chem. 199, 585.
Massey, V. (1960). Biochim. biophy8. Ada, 88, 447.
Massey, V., Gibson, Q. H. & Veeger, C. (1960). Biochem. J.
(1961) have reported its amino acid composition.
In this investigation the amino acid sequence in the
haem region of the molecule has been determined.
P8eudomonas8fuore8cers P 6009/1 (obtained from
Dr N. 0. Kaplan) was grown on a yeast extractcitrate-nitrate medium in unstirred liquid culture,
the cells collected by centrifuging, and made into an
acetone powder. The acetone powder was extracted
at pH 6-5 and 450, and the soluble fraction chromatographed on carboxymethylcellulose at pH 4-0
(ammonium acetate buffer) followed by chromatography on diethylaminoethylcellulose in tris-HOl
buffer, pH 8-7. The non-crystalline preparation
had a purity of 1-0 on the index of Horio et al.
77, 341.
Poulik, M. D. (1957). Nature, Lond., 180, 1477.
Sanadi, D. R., Littlefield, J. W. & Bock, R. M. (1952).
J. biol. Chem. 197, 851.
Savage, N. (1957). Biochem. J. 67, 146.
The glutamic acid residue is the N-terminus of the
protein.
The sequence of amino acids around the haem
group has been studied in the cytochromes c from
several species of organism (see Tuppy, 1959).
P8eudomona8 cytochromeuL resembles the other
c-type cytochromes in containing the sequence
... Cys. X. Y. Cys. His. .. but differs completely in
the rest of the sequence.
The Amino Acid Sequence near the Haem
Group of Pseudomonas Cytochrome551
By R. P. A3mBTR.* (Department of Biochemistry,
University of Cambridge)
Horio et al. (1960) prepared crystalline Pseudomonaw cytochrome,1, and Coval, Horio & Kamen
* Member of the external staff of the Medioal Research
Council.
(1960).
The amino acid composition of the protein wasn
found to be glycine 6, alanine 11, valine 7, leucine
4, isoleucine 3, aspartic acid 8, glutamic acid 8,
serine 3, threonine 2, half cystine 2, methionine 2,
proline 5, tyrosine 1, tryptophan 1, phenylalanine
2, lysine 8, histidine 1 and arginine 1; this was in
fair agreement with the analysis of Coval et al.
(1961) except in lysine content, as Coval et al. (1961)
found only 4 lysine residues per molecule.
The protein was digested with trypsin, chymotrypsin, pepsin and subtilisin, and in each case the
haem-containing portion was separated from the
rest of the digest by adsorption onto carboxymethylcellulose. The haem was removed, and the
fraction oxidized with performic acid. The amino
acid sequences of the resultant cysteic acid peptides
were determined. The peptides from the non-haem
fraction of the digest were investigated, and the
position of the haem group relative to the Nterminus of the protein determined. Evidence wag
presented to show that the amino acid sequence
near the haem group of the protein is
Glu .Asp .Pro. Glu .Val . Leu . Phe . Lys . AspNH2.
Lys . Gly . Cys . Val. Ala . Cys . His. Ala. Asp.
I-HacmJ|
Ileu .Thr.Ly.Met...
Coval, M. L., Horio, T. & Kamen, M. D. (1961). Biochim.
biophy8. Acta, 51, 246.
Horio, T., Higashi, T., Sasagawa, M., Kusai, K., Nakai, M.
& Okunuki, K. (1960). Biochem. J. 77, 194.
Tuppy, H. (1959). In Sulfur in Proteins, p. 141. Ed. by
Benesch, R. et al. New York: Academic Press Inc.
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Studies on the Mechanism of Antigenic
Stimulation
By R. W. DuTTON and JENNIFER EADY.* (Depart-ment of Chemical Pathology, Po8tgraduate Medical
School of London, London, W. 12)
Spleen cells from immunized rabbits were incubated for 48 hr. in the presence or absence of the
immunizing antigen. DNA synthesis was measured
in the second 24 hr. period by the incorporation of
(14C]thymidine into DNA. At the end of incubation
the cells were washed and dissolved in 0-4 N-NaOH.
The DNA was recovered by precipitation with
salmine (McIntire & Sproull, 1957). The total radioactivity in the DNA has been taken as a measure of
DNA synthesis. Previous experiments have shown
that the addition of antigen to the incubation
medium stimulates DNA synthesis several-fold,
that the stimulation is antigen-specific and that the
increased DNA synthesis is paralleled by an increase in the number of cells taking up tritiated
thymidine into their nuclei (Dutton & Pearce,
1962).
The experiments to be described have shown
that the transfusion of antiserum into an unimmunized spleen cell donor 3 days prior to the
in vitro measurement of DNA synthesis did not
'sensitize' the cells to stimulation by the corresponding antigen. The same was true if the spleen
cells were pre-incubated in antisera for 60 min. at
5° immediately prior to the experiment. In the
course of these experiments it was shown that the
stimulation of DNA synthesis was not dependent
on any thermolabile component of the serum used
in the incubation medium.
The experiments do not support the hypothesis
that the stimulation of DNA synthesis is a consequence of the reaction of antigen with traces of
antibody which have persisted from the earlier
response, and which may be present on the surface
of some of the cells.
From other considerations it would seem more
likely that the antigen stimulates a specific population of cells which are present as a result of previous
exposure to the antigen. Further experiments are
in progress to identify the cell population responding to antigenic stimulation.
McIntire, F. C. & Sproull, M. F. (1957). Proc. Soc. exp.
Biol., N.Y., 95, 458.
Dutton, R. W. & Pearce, J. (1962). Nature, Lond., (in the
Press).
*
Nee Jennifer Pearce.
31P
The Metabolism of p-Nitrobenzyl chloride
in Locusts
By A. J. COHEN and J. N. SmaTH. (Department of
Biochemi8try, St Mary's Hospital, Medical School,
London, W. 2)
Injected doses of p-nitrobenzyl chloride were excreted by locusts (Schistocerca gregaria) as pnitrobenzylglutathione, p-nitrobenzylcysteine and
p-nitrobenzoic acid. These metabolites were identified by chromatography, ionophoresis and hydrolysis of material eluted from paper chromatograms.
Approximately one-third of the dose was excreted
as sulphur-containing metabolites, but the relative
amounts of glutathione and cysteine metabolites in
different experiments varied, and appeared to
depend to some extent on the rate of elimination of
the excreta. p-Nitrobenzylglutathione was slowly
converted into p-nitrobenzylcysteine, which was
in turn slowly destroyed. p-Nitrobenzylmercapturic acid could not be detected in excreta.
Homogenates of locust fat body or malpighian
tubules enzymically converted p-nitrobenzyl chloride into p-nitrobenzylglutathione and this reaction
was considerably enhanced in the presence of additional glutathione. Similar enzymic reactions were
found in experiments with blowfly larvae and with
cockroaches.
p-Nitrobenzylglutathione was rapidly converted
into p-nitrobenzylcysteine by malpighian tube or
gut homogenates.
The enzymic formnation of p-nitrobenzylglutathione in locust fat-body homogenates was inhibited by compounds known to form similar
compounds in other species (e.g. benzyl chloride,
butyl bromide, bromsulfonphthalein) but addition
of Gammexane to the reaction mixture caused no
inhibition of the reaction.
We are indebted to the Anti-Locust Research Centre for
supplies of locusts and financial support.
The Effect of Vitamin A on the Stability
of the Erythrocyte Membrane
By J. A. Lucy and J. T. DINTLE. (Strangeway8
Research Laboratory, Cambridge)
On the basis of experiments in which the intracellular enzymes of cartilage were released by
hypotonic treatment (Lucy, Dingle & Foil, 1961),
it was proposed by Fell, Lucy & Dingle (1961) that
the effects of excess vitamin A on embryonic
chick cartilage cultivated in vitro (Fell & Melianby,
1952; Dingle, Lucy & Fell, 1961) could be explained
on the hypothesis that the vitamin alters the
permeability of intracellular particles; this view
32P
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
was supported by the fact that vitamin A releases
a proteolytic enzyme from rat-liver lysosomes
(Dingle & Lucy, 1961; Dingle, 1961). In the
present experiments, it has been found that
vitamin A also alters the permeability of the cell
membrane of the erythrocyte of rabbit, pig, ox and
human.
Incubation at 370 of rabbit erythrocytes (1 ml.
of packed cells in 40 ml. of 0.9% NaCI) with
vitamin A (10l,ug./ml.) causes a rapid lysis of the
cells. The rate of loss of haemoglobin is linear with
time during the period studied (1 hr.); potassium
is released more rapidly than haemoglobin.
Haemolysis is inhibited by the presence of serum.
It has been found that the concentration of vitamin
required for 50% haemolysis is proportional to the
density of the cell suspension. Loss of haemoglobin
has a temperature dependence similar to that of
the release of protease from lysosomes, but different from that of haemolysis by lysolecithin or
polyethylene glycol. The molecular structural
requirements for activity in haemolysis are the
same as for release of lysosomal protease (Fell,
Dingle & Webb, 1962), and are similar to those for
preventing vitamin A deficiency; for example, the
hydrogenated and anhydro forms of the vitamin
and related terpenes show little activity.
It is suggested that, under suitable conditions,
vitamin A may act in essentially the same way on
the membranes of many different types of cells
and intracellular particles. Furthermore, in view
of the specific structural requirements for activity
both on living tissues and on lysosomal and erythrocyte membranes, it is thought that the mode of
action of excess of vitamin A may be directly
related to its normal action as a vitamin.
Dingle, J. T. (1961). Biochem. J. 79, 509.
Dingle, J. T. & Lucy, J. A. (1961). Biochem. J. 78, 11P.
Dingle, J. T., Lucy, J. A. & Fell, H. B. (1961). Biochem. J.
79, 497.
Fell, H. B., Dingle, J. T. & Webb, M. (1962). Biochem. J.
(in the Press).
Fell, H. B., Lucy, J. A. & Dingle, J. T. (1961). Biochem. J.
78, 11P.
Fell, H. B. & Mellanby, E. (1952). J. Phy8iol. 116, 320.
Lucy, J. A., Dingle, J. T. & Fell, H. B. (1961). Biochem. J.
79, 500.
Labile Constitutents of Rat Tissues
By R. A. DALE.* (Department of Chemical Pathology, Po8tgraduate Medical School of London,
London, W. 12)
The measurement of the concentration of labile
constituents of animal tissues is unsatisfactory.
* Saltwell Fellow, Royal College of Physicians.
This is partly because suitable methods of analysis
are not available, but it is especially because of the
disturbance produced in the animal before or
during the sampling of the tissue. The estimation of
many constituents of tissues by enzymic methods
and the introduction of rapid freezing of tissues by
the use of precooled metal tongs (Eranko, 1954;
Wollenberger, Krause & Wahler, 1958; Hohorst,
Kreutz & Bucher, 1959), have reduced the magnitude of these difficulties.
In this communication the application of these
methods to the estimation of dihydroxyacetone
phosphate, fructose diphosphate and pyruvate in
rat liver and muscle is described and it is emphasized that the conditions required to obtain
satisfactory results are critical. A summary of the
parameters investigated and a qualitative account
of the results are as follows.
(a) Type of anae8the8ia. Higher concentrations of,
substrates are obtained with ether than with
nembutal.
(b) Induction of anae8thesia. Rapid induction
results in higher yields of substrates.
(c) Trauma. Stunning, guillotining or freezing
the whole rat without anaesthesia produce increases or decreases in, and altered ratios of the
substrates examined.
(d) Rate of freezing. Rapid freezing gives the
highest yields of substrates and is best achieved by
using the tongs designed by Eranko (1954).
(e) Timing of enzyme destruction. This must be
accomplished while the tissue is still frozen and it
is carried out by powdering the frozen tissue,
followed by immediate immersion of the powder in
perchloric acid. Failure to observe this precaution
results in an altered ratio of dihydroxyacetone
phosphate and fructose diphosphate.
This work was supported by grants from the British
Empire Cancer Campaign and the Peel Trust.
Eranko, 0. (1954). Acta Anat., Basel, 22, 331.
Hohorst, H. J., Kreutz, F. H. & Bucher,T. (1959). Biochem.
Z. 332, 18.
Wollenberger, A., Krause, E. G. & Wahler, B. E. (1958).
NaturwiMeenschaften, 45, 294.
The Decomposition of Sialic Acid in Acid
By R. A. GIBBONS. (National In8titute for Re8earch
in Dairying, Shinfield, Reading, Berk8.)
The rate at which sialic acid is destroyed in
0. 1 N-H2SO4 at 800 has been found to be kinetically
complex, irrespective of the analytical method
used to follow the decomposition. Four methods
have been employed; (1) Ehrlich's reagent (Werner
& Odin, 1952). (2) resorcinol reagent (Svennerholm,
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
1957), (3) resorcinol reagent after column separation (Svennerholm, 1958), and (4) thiobarbituric
acid reagent after periodate oxidation (Warren,
1959). Methods 3 and 4 agree well, but give curves
quite different from methods 1 and 2, which agree
somewhat less well. The shape of these curves is
only slightly affected by quite large variations in
initial concentration of sialic acid. Accordingly,
bimolecular reactions are unlikely, and it is
probable that the decomposition occurs via an
intermediate reaction which is, in part at least,
reversible. The following reaction sequence appears
to be the simplest which will account for the
observed decomposition curves.
k,
k3
k4
B=Sialic acid -C.D.
k2
It can be shown that if methods 3 and 4 detect
sialic acid specifically, whereas reactions 1 and 2
detect in addition the decomposition products at
least as far as C, curves of the observed form will
result.
The thiobarbituric acid method of analysis is
both simple and economical. It is, therefore, the
most convenient for measuring the rate and extent
of hydrolytic cleavage of sialic acid from mucoids
33P
and other substances containing this compound.
In order to interpret such hydrolysis curves an
expression for the rate of destruction of sialic acid
estimated in this way is necessary. Writing x for
the amount of sialic acid, and using reaction
constants as in the reaction scheme above, this is
(d
+ (ki+ k2+k3) d +kk3} x
=
0 (Kynch, 1955).
The solution of this equation is algebraically unwieldy, but it does describe the destruction of
sialic acid with time effectively. The interpretation
of hydrolysis curves is, however, thereby considerably complicated.
N-Glycollylneuraminic acid behaves in the
same way as the N-acetyl compound. There is some
evidence that component B may be the lactone of
sialic acid.
Kynch, G. J. (1955). Mathematic8 for the Chemi8t. London:
Butterworths Ltd.
Svennerholm, L. (1957). Biochim. biophys. Ada, 24,
604.
Svennerholm. L. (1958). Acta chem. scand. 12, 547.
Warren, L. (1959). J. biol. chem. 234, 1971.
Wemer, I. & Odin, L. (1952). Acta Soc. Med. Up8alien, 57,
230.
DEMONSTRATIONS
Estimation of Non-Specific Alkaline Phosphatase and Specific Glucose 6-Phosphatase
By DIANA M. CAMPBELL (introduced by E. J.
KING). (Department of Chemical Pathology, Postgraduate Medical School, London, W. 12)
OC-D-Glucose 1-phosphate may be used for the
estimation of non-specific alkaline phosphatase,
and enzyme activity measured in terms of liberated inorganic phosphate or glucose.
10 mM-Glucose 1-phosphate (1 ml.) is incubated
at 370 with 0- IM-carbonate-bicarbonate buffer
pH 9-2 (1 rnl.). Addition of enzyme solution or
plasma (0.1 ml.) starts the reaction, which is
allowed to proceed for 15 min. 10% (w/v) Sulphosalicylic acid (0-4 ml.) is used to stop the reaction.
In the control (for non-enzymic hydrolysis and for
the inorganic phosphorus content of enzyme solution and substrate) addition of the enzyme is made
at the end of the incubation period.
Glucose 1-phosphate is acid-labile so the protein
precipitant must be added simultaneously to test
and control, and the solutions filtered immediately.
Inorganic phosphate is estimated in a coppercontaining acetate buffer, pH 4 (Delsal & Manhouri, 1958). At this pH the amount of acid
hydrolysis is at a minimum. The filtrate (1 ml.) for
tests and control, and the standard (1 ml., lO,ug.
of phosphorus) are quickly transferred to tubes
containing 3 ml. of buffer. The colour reagents,
05 ml. of 5% (w/v) ammonium molybdate and
0 5 ml. of rhodol-sulphite reagent [2% (w/v)
p-methylaminophenol sulphate in 5% (w/v)
sodium sulphite] are then added. Haste is unnecessary at this stage, because the solutions are
buffered at pH 4.
Glucose may be determined by the glucose
oxidase method of Huggett & Nixon (1957). There
is good correlation between measurement of
phosphate and of glucose.
The extent of hydrolysis is directly proportional
to time during the 15 min. incubation period and
up to 30 min.
The estimation of glucose 6-phosphatase is made
at the optimum pH (pH 6.5). OlM-Citrate buffer
at this pH replaces the carbonate-bicarbonate
buffer in the previous method. The substrate is
OOlM-glucose 6-phosphate. Since there is increasing deviation from linearity if the incubation is
continued for more than 10 min. at 370, because
of the extreme heat-lability of this enzyme, the
time of hydrolysis is restricted to 10 min.
Delsal, J. L. & Manhouri, H. (1958). BuU. Soc. Chim. biol.,
Paris, 40, 1623.
Huggett, A. St G. & Nixon, D. A. (1957). Lancet, ii, 368.
34r
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Determination of 5-Nucleotidase in Blood
Serum
By DiA M. CAMPBELL (introduced by E. J.
KING). (Department of Chemical Pathology, PoMtgraduate Medical School, London, W. 12)
5-Nucleotidase catalyses the dephosphorylation
of nucleotides that have the phosphate group
attached to C-5 of the ribose radical. The enzyme
has optimum activity at pH 7-5, and at this pH
there is a significant hydrolysis of the substrate by
non-specific alkaline phosphatase, for which a
correction must be applied. The method involves
two parallel enzyme activity determinations with
adenosine 5-phosphate as substrate. In one the
presence of nickel specifically inhibits 5-nucleotidase (Ahmed & Reis, 1958), and therefore
estimates the hydrolysis of the substrate by nonspecific alkaline phosphatase. In the second the
absence of nickel allows the estimation of total
phosphatase activity. The difference in activity (in
terms of inorganic phosphate liberated) gives the
5-nucleotidase activity. The reaction mixtures
contain mm-Mn2+, which activates 5-nucleotidase. In the determination of total enzyme
activity, blood serum (0.2 ml.) is incubated at 370
with 0-1 ml. of 20 mm-manganese sulphate and
1-5 ml. of 40 mm-veronal buffer, pH 7-5. Addition
of 0-2 ml. of 10 mM-adenosine 5-phosphate starts
the reaction, which is allowed to proceed for 30 min.
Trichloroacetic acid (10% w/v; 2 ml.) is added to
stop the reaction and precipitate the protein.
After centrifuging the mixture, 2 ml. of supernatant
is taken for the estimation of inorganic phosphate.
For the separate estimation of alkaline phosphatase activity 0- lx-nickel chloride (0.2 ml.) is
included in the reaction mixture and the amount of
buffer is reduced to 1-3 ml., to maintain a final
volume of 2 rml. Inorganic phosphate may be
estimated by the method of Fiske & Subbarow
(1925) or by the method of Delsal & Manhouri
(1958).
Results are expressed in terms of jumoles of
substrate utilized/min./l. of serum (i.u./l.). A
normal range of 2-15 units has been established for
this method. The relation between enzyme concentration and amount of hydrolysis is linear over
a range 1-12 times the normal serum activity. For
values above this the estimation must be repeated
with a shorter incubation time. The extent of
hydrolysis is directly proportional to time over the
30 min. reaction period and remains so up to
120 min. After this there is some deviation from
linearity.
Raised levels of 5-nucleotidase occur in the
serum of patients with hepatobiliary disease.
Ahmed, Z. & Reis, J. L. (1958). Biochem. J. 69, 386.
Desal, J. L. & Manhouri, H. (1958). Bull. Soc. Chim. Biol.
40, 1623.
Fiske, C. H. & Subbarow, Y. (1925). J. biol. Chem. 66,375.