Download Characteristics of a thermostable protease from Desulfurococcus, an

64 1
621st MEETING, LONDON
soluble protein fractions were diluted, adjusted to p H 5 and
applied to columns of CM-Sepharose. These were eluted
with 1 M-ammonium bicarbonate, and the protein peaks
collected and dialysed against SSC buffer (1 5 mM-trisodium
citrate/ 150 mM-NaC1, pH 7.0).
Analysis of the protein distribution in these fractions
by cationic-PAGE and SDS/PAGE indicated that the
Desulfurococcus strains contained a variety of cationic proteins, while both the number and yield of proteins was significantly lower for T . celer and strain NZ-T.
Partially purified protein preparations from D . mucosus
and D. mohilis produced significant increases in the T,
of both parent and NZ-T DNA preparations (purified by
standard procedures; Maniatis, 1982). A stoichiometry of
10 : 1 ( p g of protein/pg of DNA) or greater was required for
significant duplex stabilization (e.g. T,, at 0 : 1, 86.5; T,, at
5 : 1,87.0; T,, at 10 : 1, 89.3). Incidentally, the T, of the ‘fully
complexed’ DNA is very close to the upper growth temperature of the organism, suggesting that duplex stability may be
one of the factors controlling the growth limits of these
organisms.
Agarose electrophoresis of the protein/DNA mixtures
(Fig. 1) demonstrated that the DNA takes the form of
complexes of very low mobility. However, these complexes
were apparently soluble (not removed by centrifugation at
40000g for 5 min). At stoichiometries of 15 : 1 and above,
little DNA remained uncomplexed. To a first approximation [assuming an average base-pair molecular mass of
660 daltons and an average protein molecular mass of 45000
daltons (the molecular mass of the major band on SDS/
PAGE)], this corresponds to a ratio of about 30 base-pairs
per protein molecule.
The complexes were also highly resistant to cleavage
by restriction enzymes. The susceptibility to restriction
appeared to be closely related to the fraction of the DNA
remaining in the free state. It is not suggested that this
accurately reflects the protein-DNA interaction in vivo.
However, the data demonstrate that basic proteins capable
of binding to DNA and increasing the duplex melting
temperature can be extracted from these thermophilic
archaebacteria. Confirmation of the role of these proteins
may be derived from further purification of individual protein components and the identification of identical proteins
in native DNA.
I thank the Society for General Microbiology for financial assistance
Green, G. R., Searcy, D. G. & Delange, R. J. (1983) Biochim.
Biophys. Acta 741, 251-257
Kikuchi, A. & Asai, K . (1984) Nature (London) 309,677481
Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning,
Cold Spring Harbour Laboratory, Cold Spring Harbour, NY
Oshima, T. (1982) J . B i d . Chem. 257, 9913-9914
Searcy, D. G . (1975) Biochim. Biophys. Act0 395, 535-547
Stetter, K. 0.& Zillig, W. (1985) in The Bacteria, (Woese, C . & Wolfe,
R. S., eds.), vol. VIII, pp. 87-160, Academic Press, London
Received 28 November 1986
Characteristics of a thermostable protease from Desulfurococcus, an extreme thermophile
growing at 88OC
DON A. COWAN,* KATHLEEN A. SMOLENSKIf
and ROY M. DANIEL?
*Department of Biochemistry, University College London,
Cower Streef, London W C l E 6BT, U . K . , and
t Thermophile Research Group, University of Waikato,
Hamilton. New Zealand
An extremely thermophilic anaerobic coccus isolated from
a New Zealand acid thermal pool (Jasperse-Herst, 1984)
was identified as a strain of the genus Desulfurococcus.
Under optimal growth conditions (peptone medium containing cystine at pH 6.2 and in the presence of 0.1%
casein), a low level of proteolytic activity was detectable in
the culture supernatant. On concentration and purification by CM-Sepharose ion-exchange chromatography,
hydroxylapatite adsorption chromatography and gel permeation chromatography, a single protein possessing high
levels of proteolytic activity was obtained. Activity was
completely lost after incubation with either phenylmethanesulphonyl fluoride or di-isopropyl fluorophosphate,
suggesting the presence of an active-site serine residue.
Using SDS/PAGE, the protease was found to be monomeric with an apparent molecular mass of 52
2 kDa. A
single band of protein was evident which, after removal of
the SDS by washing in 0.1% Triton X-100, was shown to be
active by overlaying with a casein-containing agarose gel.
The molecular mass estimated by either low-pressure gel
permeation chromatography on Sepharose CL-6B (eluted
with 10m~-TrIs/C1/200rn~-NaC1,
pH 7.5), or by highperformance gel permeation chromatography on a TSK
G3000 SW column (eluted with 300 mM-ammonium acetate,
*
Abbreviation used: PAGE, polyacrylamide-gel electrophoresis.
Vol. I5
pH 6.5) was between 10kDa and 13kDa. If the ionic
strength of the eluent was reduced (in order to minimize
protein-matrix hydrophobic interactions), the elution of the
protease was further retarded and protein recovery was very
low. Estimations of the sedimentation coefficient by sucrose gradient centrifugation yielded molecular mass values
confirming the SDSjPAGE data. Using cytochrome c and
ovalbumin as standards, the sedimentation ratio method
of Martin & Ames (1961) yielded values of 43 kDa and
53.9 kDa respectively for the molecular mass of the Desulfurococcus protease.
A wide variety of protein substrates were hydrolysed,
with an ‘optimal’ activity at approx. 98°C. Tri- and tetrapeptide p-nitroanilide substrates were hydrolysed, although
a limited range of smaller substrates of the form X-AA-pnitroanilide (X = blocking group; AA = amino acid) were
not. Serum thymus factor (Glu-Ala-Lys-Ser-Gly-Gly-GlySer-Asn) was apparently cleaved at Ser-Gln. Angiotensin I1
(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe)
was hydrolysed at TyrIle, while oxidized ,&insulin was cleaved at the Ala,,-Leu,,
and Tyr,,-Leu,, peptide bonds. This pattern of hydrolysis
indicated a relatively high degree of hydrolytic specificity
(compared with many microbial extracellular proteolytic
enzymes) and suggests a possible preference for non-polar
residues on the amino side of the cleavage point.
Desulfurococcus protease was found to be highly thermostable in aqueous buffered solution (Table 1). Thermostability was not significantly reduced by the addition of
10 mM-EDTA. Thus unlike a number of other thermostable
microbial extracellular proteases (Ohta, 1967; Voordouw
et af., 1974; Cowan & Daniel, 1982), molecular stability
appears to be an intrinsic property of the protein and is not
enhanced by calcium chelation.
Denaturation at high temperatures was enhanced by
642
BIOCHEMICAL SOClETY TRANSACTIONS
Table 1. Thermostability of Desulfurococcus protease
Thermostability profiles were determined using solutions of
Desulfurococcus protease at 0.1LO.2pgml in l00m~-Na,HPO,/
NaHzPO4 buffer, pH 7.5 at the temperature indicated. Samples
were removed at intervals and activity determined using a
standard assay (Cowan & Daniel, 1982).
Incubation
temperature
("C)
95
I05
115
95
95
95
95
95
95
Incubation
conditions
+
+
+
+
+
+
Buffer only
Buffer only
Buffer only
IOmM-Dithiothreitol
6~-Urea
4 M-Guanidine/HCI
0.1% SDS
1% Triton X-100
IO~M-N~SCN
Half-life
(min)
Kinetics of
activity loss
75-90
8
5 0.5
61
24
3
I12
280
78
First order
First order
First order
Comp I ex
Second order
Second order
First order
First order
First order
the addition of detergents or chaotropic reagents (Table I),
although the rate of activity loss was consistent with a
high degree of molecular stability. This and other evidence
suggests that the enzyme is capable of rapid renaturation
after removal of detergents.
We thank Robyn Clemens for technical assistance and the Development Finance Corporation of New Zealand for financial support.
Cowan, D. A. & Daniel, R. M. (1982) Biochim. Biophys. Acfa 705,
293-305
Jasperse-Herst, P. M. (1984) M. Phil. Thesis, University of Waikato,
Hamilton, New Zealand
Martin, R. G. & Ames, B. N. (1961) J . Biol. Chem. 235, 1372-1379
Ohta, Y. (1967) J . Biol Chem. 242, 509-515
Voordouw, G., Gaucher, G. M. & Roche. R. S. (1974) Biochem.
Biophys. Res. Commun. 58, 8-12
Received 28 November 1986
Ontogenesis of acid proteases in human foetal brain regions
G . MAHAJAN,* S. MANDAL,* N. J. PATELt
and A. K. SINHA?
*Indian Institute of Chemical Biology, Calcutta 32,
India, and t Department of Molecular Endocrinology,
Middlesex Hospital Medical School, Mortimer Street,
London W I , U . K .
Although several proteinases with widely differing pH
optima have been reported in the developing mammalian
central nervous system, the major portion of the activity
(90%) in the rat central nervous system can be attributed
to cathepsin D (Mark & Lajtha, 1963, 1965), where it is
localized in myelin, purified synaptosomes and mitochondrial fractions. Ontogenically, cathepsin D activity has
been shown to coincide with gliogenesis and myelination
(Baseman, 1973). In this communication, we present similar
results from the developing human brain.
Human foetuses from between 12 and 20 weeks of gestation were obtained from patients admitted to the SSKM
Hospital Calcutta, India, for the termination of pregnancy
under the 'Family Welfare Programme' of the government
of India. Only those foetuses were used where both parents
had agreed to their use in experimentation and were free
of obvious neurological disorders. The foetuses were
removed by hysterotomy and the brains dissected out and
frozen as soon as possible. Fresh or frozen samples were
homogenized in distilled water at 4OC to give 10% homogenate (w/v). Cathepsin D activity was measured in 75 mMformate/acetate/phosphate buffer at a pH of 3.5 using aciddenatured haemoglobin as substrate. After an incubation of
60 min at 37°C the reaction was terminated by the addition
of 100p1 of 25% (w/v) trichloroacetic acid. The supernatant
obtained after centrifugation was neutralized by the addition of 0.5 mM-KOH and the liberated tyrosine measured by
the method of Lowry et al. (1951). Results were expressed as
nmol of tyrosine equivalent/mg of protein per h.
Cathepsin D activity in the cerebral cortex cerebellum and
mid-brain shows a biphasic developmental pattern with an
initial peak at 33g body weight with another broad peak
covering 180-216 g body weight. The spinal cord, however,
not only showed a continuous increase in activity with body
weight, but also exhibited far higher activity at all points
considered.
Several acid hydrolases have been reported in the devel-
oping human foetal brain and attempts have been made to
correlate the appearance of these with specific structural
ontogenisis. (Sinha & Sinha, 1980). Cathepsin D activity in
the rat brain has been shown to be preferentially localized in
cortical neurons (Sinha & Rose, 1972) which together with
the observation that spinal cord neurons mature before
those of the cortex and mid-brain may indicate that the
peaks of activity represent neuronal maturation phases. The
high activity in the spinal cord may also reflect the acquisition of glial cells and myelinogenisis, since in the postnatal
rat brain Snyder & Whittaker (1983) have shown that
maximal cathepsin D activity coincides with the phase
of maximal myelination and glial proliferation. Detailed
histological investigations are currently underway to correlate the appearance of cell types and subcellular compartments with the peak activity periods of cathepsin D.
/\
b
300
n
33
78
129
180
216
264
Foetal body weight (g)
Fig. I . Distribution of acid proteinase in developing human
foetal brain regions
Proteolytic activity in the cortex (A),mid-brain (m), cerebellum
(0)medulla (A) and spinal cord ( 0 )was assayed at pH 3.5 with
acid-denatured haemoglobin as substrate, and was expressed as
nmol of tyrosine equivalent/mg of protein per h. Foetal body
weight values are the mean of three values with a variation of
less than 10g from each other. Activities are the means of three
such experiments analysed in duplicate.
1987