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.     

Characterization of triacylglycerol hydrolase activities in isolated myocardial cells from rat heart.

Triacylglycerol (TG) hydrolase activities were characterized in myocytes isolated from rat hearts. Acid hydrolase activity with a pH optimum of 5 could be measured in myocyte homogenates, and the subcellular distribution suggested that this activity originated in lysosomes. Lipoprotein lipase (LPL) was also present in myocyte homogenates, as evidenced by TG hydrolase activity that was stimulated by serum and apolipoprotein CII, and inhibited by apolipoprotein CIII2, high ionic strength (NaCl and MgCl2, I = 1 M) and antibodies to LPL. Serum-independent neutral (pH 7.5) TG hydrolase activity was less sensitive to inhibition by 1 M-NaCl, by antibodies to LPL and by preincubation at 40 degrees C than was serum-stimulated hydrolase activity. Furthermore, there were modest but significant differences in the subcellular distribution of the serum-independent and serum-stimulated hydrolase activities. Hydrolase activities in myocyte homogenates could be solubilized by 7.2 mM-deoxycholate. Acid hydrolase activity was recovered in the unbound fraction after heparin-Sepharose chromatography, whereas LPL was bound to the affinity column and was eluted by 0.9-1.2 M-NaCl. Approximately one-third of the serum-independent TG hydrolase activity was not bound to the heparin-Sepharose affinity column. This unbound TG hydrolase activity had a pH optimum of 7 and was stimulated by 50 mM-MgCl2, but not by serum and was resistant to inhibition by high ionic strength (1 M-NaCl), to preincubation at 40 degrees C for 2 h, and by antibodies to LPL. It is concluded that, in addition to an acid lysosomal TG hydrolase and LPL, myocytes from rat heart contain a serum-independent TG hydrolase with unique characteristics.     

The possible existence of a heat-stable alkaline proteinase (HAP) in rat skeletal muscle

1. A unique caseinolytic activity was found in the crude extract from chicken and rat skeletal muscle. Hardly any activity was detected at physiological assay temperatures at pH 8.0 but did well at around 60 degrees C. 2. The activity partially purified from rat skeletal muscle showed optimum pH at around 8.0 at 60 degrees C. It hardly hydrolyzed casein below 50 degrees C, but in the presence of 5 M urea it showed relatively high activity at 30 degrees C. The activity was completely stable at 50 degrees C for 1 hr. 3. The activity seems to be contained in a high mol. wt (450,000) protein from the elution volume and is due to cysteine proteinase from the effect of inhibitors. 4. The above properties agreed with those of the heat-stable alkaline proteinase (HAP) of fish purified homogeneously by electrophoresis. This seems to suggest that HAP may also exist in rat skeletal muscle.     

Testing transport models and transport data by means of kinetic rejection criteria.

The proenzyme form of C1r catalytic domains was generated by limited proteolysis of native C1r with thermolysin in the presence of 4-nitrophenyl-4'-guanidinobenzoate. The final preparation, isolated by high-pressure gel permeation in the presence of 2 M-NaCl, was 70-75% proenzyme and consisted of a dimeric association of two gamma B domains, each resulting from cleavage of peptide bonds at positions 285 and 286 of C1r. Like native C1r, the isolated domains autoactivated upon incubation at 37 degrees C. Activation was inhibited by 4-nitrophenyl-4'-guanidinobenzoate but was nearly insensitive to di-isopropyl phosphorofluoridate; likewise, compared to pH 7.4, the rate of activation was decreased at pH 5.0, but was not modified at pH 10.0. In contrast, activation of the (gamma B)2 domains was totally insensitive to Ca2+. Activation of the catalytic domains, which was correlated with an irreversible increase of intrinsic fluorescence, comparable with that previously observed with native C1r [Villiers, Arlaud & Colomb (1983) Biochem. J. 215, 369-375], was reversibly inhibited at high ionic strength (2 M-NaCl), presumably through stabilization of a non-activatable conformational state. Detailed comparison of the properties of native C1r and its catalytic domains indicates that the latter contain all the structural elements that are necessary for intramolecular activation, but probably lack a regulatory mechanism associated with the N-terminal alpha beta region of C1r.     

Characterization of an alkaline proteinase of fish muscle

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.     

萌发绿豆中水解大豆胰蛋白酶抑制子蛋白酶的纯化及固定化

大豆是豆类植物中最早发现存在蛋白酶抑制子的 ,由于其存在影响了豆类的利用价值 ,因此研究人员一直在寻找着解决办法。采用加热处理方法不能彻底钝化豆类蛋白的蛋白酶抑制子活性 ,且豆类蛋白的含硫氨基酸主要存在于各类蛋白酶抑制子中 ,从豆类蛋白中除去抑制子蛋白将大大降低其营养效价。本研究的目的是试图寻找一种可在常温下降解豆类胰蛋白酶抑制子的蛋白酶 ,从而钝化豆类的胰蛋白酶抑制活性。在前期工作中 ,我们发现枯草杆菌蛋白酶 (Ｓｕｂ ｔｉｌｉｓｉｎ)可在在常温下降解花生及大豆胰蛋白酶抑制剂[1] ,近期我们的研究表明 ,Ａｌｃａ…     

Extracellular alkaline proteinase of Colletotrichum gloeosporioides

The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.     

Enzyme activities of cell-free extracts from mutant strains of lactic streptococci subjected to sublethal heating or freeze-thawing

Two mutant lactose-negative (Lac-), proteinase-negative (Prt-) strains of lactic streptococci, Streptococcus lactis 25Sp and S. cremoris KHA2, and their parents, S. lactis C2 and S. cremoris KH Lac+ Prt+, were grown in a suitable medium with the pH maintained at 6.5 by addition of NH4OH. Cells were harvested by centrifugation, resuspended, and then heated sublethally at 54 or 69 degrees C for 15 sec. Cells also were frozen and stored for 1 week at -20 or -100 degrees C. Cell-free extracts of cells heated at 54 degrees C had more proteinase and aminopeptidase activities than did a similar extract of cells heated at 69 degrees C. The greatest enzyme activities occurred in the cell-free extracts prepared from cells frozen and stored at -100 degrees C. Specific activities of proteinase and dipeptidase generally decreased in extracts of freeze-shocked cells compared to those in extracts of untreated cells. Enzyme activity of extracts also decreased in the presence of 5% NaCl at pH 5.0. Cell-free extracts at pH values of 5 to 8 were heated at 69 degrees C for 1.5, or 10 min. Heating them for 10 min caused a loss of dipeptidase activity which was most pronounced at pH 5.0 and least pronounced at pH 7.0.     

Submaxillary mucins. Intermolecular interactions and gel-forming potential of concentrated solutions.

The intermolecular interactions in concentrated solutions of pig submaxillary mucin (PSM) and sheep submaxillary mucin (SSM) were studied by mechanical spectroscopy. PSM and SSM were purified from detectable protein and nucleic acid by equilibrium centrifugation in a CsCl density gradient. PSM and SSM isolated in the presence of proteinase inhibitors showed distinct differences from preparations isolated in the presence of 0.2 M-NaCl alone, the latter having a carbohydrate and amino acid analysis similar to other preparations isolated by precipitation or ion-exchange techniques. Gel-filtration studies showed that preparations isolated in the presence of 0.2 M-NaCl alone were dissociated into smaller-sized glycoprotein units by 3.5 M-CsCl or 2.0 M-NaCl (SSM), pH 2.0 (PSM) or heating at 100 degrees C for 10 min (PSM and SSM). Preparations isolated in the presence of proteinase inhibitors were not dissociated by these treatments. Proteolysis fragmented all submaxillary mucin preparations into small glycopeptides of Mr 13,700 for PSM and of Mr 14,000 and 15,000 for SSM. PSM preparations when concentrated formed viscoelastic gels, as determined by mechanical spectroscopy. In contrast, SSM showed characteristics of a weak viscoelastic liquid under comparable conditions (coil overlap). PSM glycoprotein isolated in proteinase inhibitors formed weak viscoelastic gels at concentrations between 5 and 15 mg/ml. Preparations of PSM glycoprotein isolated in the presence of 0.2 M-NaCl (concentration 10-97 mg/ml) had the same overall mechanical gel structure as those preparations extracted in the presence of proteinase inhibitors. This gel structure was seen to collapse following proteolysis of both preparations or after acid treatment of the glycoprotein isolated in the presence of 0.2 M-NaCl, consistent with the breakdown in size of the polymeric glycoprotein. Treatment of PSM gel with 0.2 M-2-mercaptoethanol caused a surprising increase in gel strength, which was further markedly increased on removal of the reducing agent by dialysis. An association of reduced subunits of PSM was observed by gel filtration after removal of 0.2 M-2-mercaptoethanol. These results point to intermolecular disulphide exchange occurring on reduction of these PSM glycoprotein preparations. These results demonstrate that gel formation in PSM glycoprotein is similar to that for other gastrointestinal mucus glycoproteins from stomach to colon. Gel formation in PSM, as in other mucins, depends on polymerization of subunits.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteinase and proteinase-inhibitor activities of rat uterine myometrium during pregnancy and involution.

A supernatant fraction was prepared from rat uterine myometrium by homogenization, sonication and centrifugation. In this supernatant the protein concentration and the activities of an acid proteinase, an acid phosphatase and a proteinase inhibitor were measured. From the fibrous sediment, after washing with 0.5% Triton X-100 and with water, an actomyosin-containing solution was obtained by extraction with 0.6M-NaCl, and in this extract the protein concentration and a neutral proteinase activity were measured. The myometrial wet weight and the activities of the acid proteinase, acid phosphatase and proteinase inhibitor increased by factors of 3-15 during pregnancy and decreased to the same or a greater extent during involution. The amount of protein extracted with 0.6M-NaCl increased by a factor of only 2.3 and the neutral proteinase activity remained essentially constant during pregnancy and involution. The pH optimum of the neutral proteinase, and its pattern of activity compared with those of the lysosomal enzymes, show that the neutral proteinase is not of lysosomal origin. Actomyosin is degraded by the neutral proteinase activity in vitro. Since actomyosin is rapidly broken down only after parturition, the action of the neutral proteinase activity on actomyosin, if this occurs in vivo, must be regulated in some way. The proteinase-inhibitor activity measured in the first supernatant varied in a manner which suggested that it could be involved in this control.     

Purification and characterization of cathepsin L-like proteinase from goat brain

Cathepsin L-like proteinase was purified approximately 1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).     

Extracellular proteinase fromEnterococcus faecalis subsp.liquefaciens

An extracellular proteinase from Enterococcus faecalis subsp. liquefaciens has been purified 780-fold by a method including gel filtration on Sephadex G-50 and affinity chromatography with gramicidin J as ligand. Approximately 15% of the original enzyme activity was recovered. A purification of 14,800-fold, with 11.4% yield, may be reached using chromatofocusing as final step in the purification procedure. The molar mass of the enzyme has been estimated to be approximately 30 kDa by Sephadex gel filtration and approximately 26 kDa by SDS-PAGE. The isoelectric point has been found to be 4.6. Maximum enzyme activity of the proteinase has been observed at pH 7.5 and 45 degrees C. The enzyme hydrolyzed bovine serum albumin, alpha-lactoalbumin, beta-lactoglobulin, casein and pork myofibrillar and sarcoplasmic proteins. The extracellular proteinase was very stable; the enzyme maintained its activity in cell-free extracts over a very wide range of temperatures (-25 to 37 degrees C) for at least 2 months. At 12 degrees C, it was stable in the pH range of 5.5 to 8.0.     

Purification and properties of a novel latent proteinase showing myosin heavy chain-degrading activity from threadfin-bream muscle

A novel latent proteinase of which activity was induced by heating in the presence of NaCl was purified to homogeneity from threadfin-bream muscle by a combination of DEAE-cellulose, Con A-Sepharose, Arg-Sepharose, and Shim-pack HAC chromatographies. This proteinase was a glycoprotein having a monomeric subunit structure; Mr was estimated to be 77,000 on SDS-PAGE analysis. The proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA as well as myosin heavy chain in the presence of 2-4% NaCl at pH 7.0 and at 60 degrees C, optimally. The proteinase was classified as serine proteinase based on the effects of soybean trypsin inhibitor, leupeptin, and antipain.     

Purification of the Ca2+-dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca2+-dependent proteinase

A protein inhibitor of the Ca2+-dependent proteinase has been purified from bovine cardiac muscle by using the following steps in succession: salting out 17,600 X gmax supernatants from muscle homogenates in 50 mM Tris acetate, pH 7.5, 4 mM EDTA between 25 and 65% ammonium sulfate saturation; eluting between 25 and 120 mM KCl from a DEAE-cellulose column at pH 7.5; salting out between 30 and 60% ammonium sulfate saturation; Ultrogel-22 gel permeation chromatography at pH 7.5; heating to 80 degrees C followed by immediate cooling to 0 degree C; 6% agarose gel permeation chromatography in 4 M urea, pH 7.5; and elution from a phenyl-Sepharose hydrophobic column between 0.7 and 0.5 M ammonium sulfate. Approximately 1.16-1.69 mg of purified Ca2+-dependent proteinase inhibitor are obtained from 1 kg of bovine cardiac muscle, fresh weight. Bovine cardiac Ca2+-dependent proteinase inhibitor has an Mr of 115,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pI of 4.85-4.95, very little alpha-helical structure, a very low specific absorbance of 1.647 (A1% 280), and very low contents of histidine, tryptophan, phenylalanine, and tyrosine. Bovine cardiac Ca2+-dependent proteinase inhibitor probably contains a single polypeptide chain in nondenaturing solvents. One 115-kDa inhibitor polypeptide inactivates 10 110-kDa millimolar Ca2+-requiring proteinase (millimolar Ca2+-dependent proteinase) molecules in assays of purified proteins. Inhibition of millimolar proteinase by the proteinase inhibitor did not change in the pH range 6.2-8.6. The inhibitor requires Ca2+ to bind to millimolar Ca2+-dependent proteinase. The Ca2+ concentration required for one-half-maximum binding of millimolar Ca2+-dependent proteinase to the inhibitor was 0.53 mM, compared with a Ca2+ concentration of 0.92 mM required for one-half maximum activity of millimolar Ca2+-dependent proteinase in the absence of the proteinase inhibitor. Unless millimolar Ca2+-dependent proteinase is located subcellularly in a different place than the proteinase inhibitor or unless the proteinase/inhibitor interaction is regulated, millimolar proteinase could never be active in situ.     

A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.     

Isolation and partial characterization of a vitamin K-dependent carboxylase from bovine aortae.

Vitamin K-dependent carboxylase activity has been demonstrated in the crude microsomal fraction of the intima of bovine aortae. The procedure for the isolation of vessel wall carboxylase is a slight modification of the general preparation procedure for tissue microsomes. The highest activity of the non-hepatic enzyme was observed at 25 degrees C and hardly any NADH-dependent vitamin K reductase could be demonstrated. The optimal reaction conditions for both vessel wall as well as liver carboxylase were similar: 0.1 M-NaCl/0.05 M-Tris/HCl, pH 7.4, containing 8 mM-dithiothreitol, 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (CHAPS), 0.4 mM-vitamin K hydroquinone and 2 M-(NH4)2SO4. Warfarin inhibits the hepatic and non-hepatic carboxylase/reductase enzyme complex more or less to a similar degree. We have measured the apparent Km values for the following substrates: Phe-Leu-Glu-Glu-Leu ('FLEEL'), decarboxylated osteocalcin, decarboxylated fragment 13-29 from descarboxyprothrombin and decarboxylated sperm 4-carboxyglutamic acid-containing (Gla-)protein. The results obtained demonstrated that liver and vessel wall carboxylase may be regarded as isoenzymes with different substrate specificities. The newly discovered enzyme is the first vitamin K-dependent carboxylase which shows an absolute substrate specificity: FLEEL and decarboxylated osteocalcin were good substrates for vessel wall carboxylase, but decarboxylated fragment 13-29 and decarboxylated sperm Gla-protein were not carboxylated at all.     

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography. The major proteinase activities in the nonactivated extracts corresponded to relative molecular masses (Mr) of 51,000 to 56,000, while less distinct digestion occurred with relative molecular masses of 37,000 to 49,000. It was demonstrated that after a serial dilution of the sperm extract, the proteinase activity in as few as 6,000 sperm could readily be detected by the gelatin-SDS-PAGE methods. Time-course activation studies showed that the zymogen was completely converted to active proteinase in 45-60 min at pH 8.0 and 25 degrees C. This autoconversion process was markedly inhibited by calcium, sodium, and heparin. However, each of these compounds stimulated the proteolytic activity of acrosin. These studies demonstrate that the proacrosin-acrosin system can be investigated in extracts of nonpurified hamster epididymal sperm.     

Protease activities of rumen protozoa

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.     

Purification and properties of three endopeptidases from baker's yeast

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea. Urea also stimulated the enzyme activity by 30-50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type. A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)