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[The Editor8 of The Biochemical Journal accept no reeponibiity for the Reports of the Proceedings of the Society] 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 20P 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. 21P 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 22P 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.