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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