Properties of delta5-3beta-hydroxysteroid oxidoreductase isolated from Streptomyces griseocarneus.

Delta5-3beta-hydroxysteroid oxidoreductase was extracted in magnesium-containing Tris buffer from sonicated Streptomyces griseocarneus cells. The enzyme was partially purified (150 X) by ion exchange chromatography and gel filtration following (NH4)2SO4 fractionation. Upon gel filtration on Sephadex G-75 to G-200, the greatest part of the activity gave a peak in the fractionation range. The enzyme obtained from the gel yielded small enzyme molecules on repeated chromatography. A molecular weight of 32 to 36 000 was calculated for the activity appearing in the fractionation range of Sephadex G-75 to G-200. The enzyme is highly specific for the irreversible oxidation of the 3beta-hydroxyl group in steroids with a trans-anellated A : B ring system with either C5 or C6 double bond. Delta5-3-ketosteroids are converted into delta5-3-ketosteroids at a high rate, but the isomerase activity cannot be separated from the oxidoreductase activity either by chromatography or by selective heat inactivation. NAD, NADP, FMN or FAD did not influence the activity, but the enzyme is inactive in the absence of molecular oxygen.     

Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp.

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3'-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.     

Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.     

Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz.NCIM 2711

A new extracellular protease having a prospective application in the food industry was isolated from Bacillus sUbtilis NCIM 2711 by (NH4)2SO4 precipitation from the cell broth. It was purified using DEAE-Cellulose and CM-Sephadex C-50 ion-exchange chromatography. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 7.0 and temperature 55 degrees C with Km 1.06 mg/ml. The enzyme was stable over a pH range 6.5-8.0 at 30 degrees C for 1 hr in presence of CaCl2 x 2H2O. At 55 degrees C, the enzyme retained 60% activity up to 15 min in presence of CaCl2 x 2H2O. EDTA and o-phenanthroline (OP) completely inhibited the enzyme activity while DFP, PMSF and iodoacetamide were ineffective. The enzyme was completely inhibited by Hg2+ and partially by Cd2+, Cu2+, Ni2+, Pb2+ and Fe2+. The OP inhibited enzyme could be reactivated by Zn2+ and Co2+ up to 75% and 69% respectively. It is a neutral metalloprotease showing a single band of 43 kDa on SDS-PAGE.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

Purification and properties of GTP-cyclohydrolase of the yeast Pichia guilliermondii

GTP-cyclohydrolase was isolated from the Fe-deficient cells of Pichia guilliermondii and purified 440-fold by treatment of extracts with streptomycin sulfate as well as by protein fractionation with (NH4)2SO4 at 25-45% saturation, gel filtration through Sephadex G-200 and DEAE-cellulose chromatography. The curves for the dependence of specific activity of GTP-cyclohydrolase on substrate and cofactor concentrations are non-hyperbolic; the values of [S]0.5 for GTP and Mg2+ are 2.2 X 10(-5) and 2 X 10(-4) M, respectively. The enzyme activity is inhibited by pyrophosphate ([I]0.5 = 5.8 X 10(-4) M), orthophosphate ([I]0.5 = 4.5 X 10(-3) M), heavy metal ions and chelating agents. The temperature optimum for the enzyme activity lies at 42-45 degrees C. The enzyme is labile at 4 degrees C but can well be stored at -15 degrees C. The pyrimidine product of the cyclohydrolase reaction, 2.5-diamino-6-oxy-4-ribosyl-aminopyrimidine-5'-phosphate, as well as pyrophosphate were purified from the reaction medium and identified.     

Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.     

Purification and Characterization of the polygalacturo nase from Tomato Fruits

The polygalacturonase (PG) isolated fron the pericarp of fully ripe tomato fruits was purified by (NH4)2SO4 precipitation, carboxyme-thylsepharose ion-exchang column and sephadex G-75 gel filtration. Specific activity of purified PG was 1.5 μmol galacturonic acid mg-1 protein min-1, which was 30 times as high as that of the crude extract with 1.7mol NaC1. When the elution separated by second sephadex G-75 column was analyzed by SDS-PAGE, only a single protein band was detected. It was shown by heat and pH experiments of the purified enzyme that the enzyme activity retained 50%, after treatment with heat at 50℃ for 10 min, and that the optimal pH was 4.6.     

Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.     

Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.     

Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase.

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7--0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.     

Purification of a derepressible arylsulfatase from Chlamydomonas reinhardti. Properties of the enzyme in intact cells and in purified state.

Arylsulfatase (aryl-sulfate sulfohdydrolase, EC 3.1.6.1) has been purified from SO4-2-minus-starved cells of Chlamydomonas reinhardti. The enzyme was isolated from acetone-powder extract by (NH4)2SO4 precipitation, Sephadex G-200 filtration and ion-exchange chromatography. Only one fraction of aryl-sulfatase was found. The final preparation was homogenous by the criteria of sedimentation, diffusion and polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of about 150 000, estimated by ultracentrifugation and gel filtration, and an isoelectric point of 9.0. The properties of the enzyme as investigated in intact cells and in the purified state were found to be very similar except for the temperature optimum. Imidazole strongly increased the enzyme by increasing the V, but reduced the affinity for the substrate. The enzyme activity was competitively inhibited by borate with a greater affinity for borate than for the substrate. The Chlamydomonas enzyme is a Type I arylsulfatase since it was inhibited by CN-minus, but not SO4-2-minus and phosphate.     

Purification and properties of a carboxymethylcellulase from phytopathogenic fungus Macrophomina phaseolina

From the culture filtrate of Macrophomina phaseolina, two forms of carboxymethylcellulase were separated by ion-exchange chromatography and designated as CMCase-I and CMCase-II. CMCase-I was purified following a four-step procedure involving gel filtration on Sephadex G-75, Con-A Sepharose 4B affinity chromatography, fast protein liquid chromatography on mono Q anion-exchanger and on Superose 12 gel filtration. The final preparation was homogeneous by SDS-PAGE, isoelectric focussing in thin layers of polyacrylamide gels and immunoelectrophoresis. The enzyme showed optimum activity at pH 5.5 and 65 degrees C, was stable to heating at 65 degrees C for 10 min, and retained 31% of original activity after heating at 80 degrees C for 10 min. The molecular weight of the enzyme was 3.5 x 10(4) Da. A Km of 0.25 mg/ml was determined using carboxymethyl-cellulose as the substrate.     

华丽曲霉Z58有机磷农药降解酶的纯化和性质

华丽曲霉(Aspergillus ornatus)Z58有机磷农药降解酶经硫酸铵分级沉淀、Sephadex G100凝胶过滤、DEAE52离子交换层析得到了分离纯化,用聚丙烯酰胺凝胶电泳(PAGE)鉴定为单一组分。凝胶过滤法测得分子量为67 000,提纯倍数为34.2,收率为17.8%。该酶的最适反应温度45℃,最适反应pH72,对热较稳定,并且能在pH6～10范围保持活性。重金属Cu2+对该酶具有明显的促进作用,而SDS对酶具有抑制作用。此酶对所试的有机磷农药都有较好降解作用。     

Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri.

N-Formylmethanofuran(CHO-MFR):tetrahydromethanopterin(H4MPT) formyltransferase (formyltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined. The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revealed that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65 degrees C, the Vmax was 2700 U/mg (1 U = 1 mumol/min), the Km for CHO-MFR was 50 microM and the Km for H4MPT was 100 microM. At 90 degrees C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65 degrees C. Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90 degrees C decreased in the order K2HPO4 = (NH4)2SO4 > KCl = NH4Cl = NaCl > Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.     

The effects of low temperature acclimation of winter rye on catalytic properties of its ribulose bisphosphate carboxylase-oxygenase.

A comparison was made of the kinetics of the carboxylation reaction of bicarbonate-magnesium-activated ribulose biphosphate carboxylase-oxygenase purified from cold-hardened and unhardened winter rye (Secale cereale L. cv. Puma). The activity of the (NH4)2SO4-precipitated enzyme from hardened plants was stable at -20 degrees C for a month, whereas the form from unhardened plants was reversibly cold inactivated. The KmCO2 of the unhardened form increased more rapidly with decreasing pH below 8.2, but the estimated pKa of chemical groups associated with the active site was not affected by the cold hardening. The temperature dependencies of the KmCO2 of the two forms of the enzyme crossed at 10 degrees C with the effect that the catalysis of carboxylation by ribulose biphosphate carboxylase-oxygenase from Puma rye was most efficient in the temperature range to which the plants had been adapted.     

A mitochondrial NADP+-dependent reductase related to the 4-aminobutyrate shunt. Purification, characterization, and mechanism

Succinic semialdehyde reductase, a NADP+-dependent enzyme, was purified from whole pig brain homogenates. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Succinic semialdehyde reductase (Mr 110,000) catalyzes the reduction of succinic semialdehyde to 4-hydroxybutyrate. The equilibrium constant of the reaction is Keq = 5.8 X 10(7) M-1 at pH 7 and 25 degrees C. The inhibition kinetic patterns obtained when 4-hydroxybutyrate or substrate analogs are used as inhibitors of the reaction catalyzed by the reductase are consistent with an ordered sequential mechanism, in which the coenzyme NADPH adds to the enzyme before the aldehyde substrate. A specific aldehyde reductase was also purified to homogeneity from brain mitochondria preparations. Its catalytic properties are identical to those of the enzyme isolated from whole brain homogenates. It is postulated that two enzymes, i.e. a NAD+-dependent dehydrogenase and a NADP+-dependent reductase, participate in the metabolism of succinic semialdehyde in the mitochondria matrix.     

Aspergillus oryzae acid proteinase. Purification and properties, and formation of pi-chymotrypsin.

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.     

Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.

Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.     

Purification of bull sperm hyaluronidase by concanavalin-A affinity chromatography.

A new method for obtaining highly purified hyaluronidase (hyaluronate glycanohydrolase EC 3.2.1.25) in high yield is described. Bull seminal plasma was fractionated with (NH4)2 SO4 and the 30 to 65% saturation fractions were applied to a DEAE-cellulose column. The first protein peak contained hyaluronidase, beta-N-acetylglucosaminidase and beta-glucuronidase. The latter two enzymes were separated by gel filtration on Sephadex G-200. The hyaluronidase was further purified by a Concanavalin-A Sepharose 4B affinity column. By gradient elution with alpha-methyl-D-glucoside a fraction which had a specific activity of 1998 units/mg protein (57 942 National Formulary Standard units/mg protein) was obtained. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 4.3. The purified hyaluronidase did not show any beta-glucuronidase or beta-N-acetylglucosaminidase activities. The percent yield of purified hyaluronidase calculated on the basis of total activity was ten times higher than by any pervious method [Yang, C.H. and Srivastava, P.N. (1975) J. Biol. Chem. 250, 79-83].