Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity

The specific inhibitor of calcium-dependent proteases was purified from soluble extracts of bovine heart. The protein had a molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gels and migrated on gel filtration chromatography with an apparent molecular weight of 250,000. The inhibitor specifically blocked the action of the two calcium-dependent proteases, CDP-I and CDP-II, but did not influence a variety of other proteases including trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. These latter enzymes extensively degraded the inhibitor to discrete lower molecular weight peptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration chromatography. Under the conditions studied, proteolysis of the inhibitor had little or no effect on its inhibitory activity; isolated peptides with molecular weights as low as 17,000 retained inhibitory function. A number of various-sized inhibitor fragments were isolated by gel filtration chromatography and by SDS-PAGE. These fragments were compared with the intact inhibitor for their ability to inhibit CDPs. As suggested previously by us and others, one molecule of intact inhibitor appears to inhibit up to five molecules of calcium-dependent protease. The inhibitor fragments of decreasing size inhibited correspondingly fewer molecules of protease. These results suggest that the inhibitor protein contains multiple functional domains and may explain some of the discrepancies in reported molecular weights for this protein.     

Studies on a Doubleheaded Protease Inhibitor from Phaseolus mungo

A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors.     

Purification and characterization of membrane-bound alkaline proteases from midgut tissue of the silkworm, Bombyx mori

Membrane-bound alkaline proteases from the midgut epithelia of the silkworm, Bombyx mori, were solubilized with 1% Lubrol-WX, at pH 11.2. They were purified by gel filtration on Sepharose 6B and Ultrogel AcA-202 columns and a preparative polyacrylamide gel electrophoresis. Two proteases, caseinolytic (6B3-Tc) and benzoyl-arginine-p-nitroanilide-lytic (6B3-Tb) were obtained. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis. These enzymes showed high pH optima, 11.2, and pI values, above 11, and were extremely stable over a wide range of pH. The Km values for 6B3-Tb and Tc were 0.476 mM and 2.5 mg/ml respectively. Hammarsten casein and mulberry leaf protein were rapidly hydrolyzed by Tc, whereas the hydrolytic activity of Tb for Azocoll was higher than that of Tc. The protease Tb was strongly inhibited by diisopropylfluorophosphate, p-chloromercuribenzoate, benzamidine, leupeptin, and soybean trypsin inhibitor; Tc was inhibited by diisopropylfluorophosphate, tosyl phenylalanine chloromethylketone and chymostatin, but not by tosyl lysine chloromethylketone, p-chloromercuribenzoate, or iodoacetamide. The molecular weights of the proteases were estimated to be 12,800 (Tb) and 13,300 (Tc) by Sephacryl S-300 gel filtration. The amino acid analyses showed that both proteases contain a large number of acidic amino acids but a relatively small number of basic amino acids.     

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Extracts from white croaker skeletal muscle showed two alkaline proteases and a trypsin inhibitor when they were chromatographed in DEAE-Sephacel. The activity against azocasein was maximal at pH 8.5 and 9.1 for proteases I and II, respectively. Both enzymes showed optimum activity at 60° C. The molecular masses were found to be 132 kDa for protease 1,363 kDa for protease II, and 65 kDa for the inhibitor. Protease I showed the characteristics of a trypsin-like enzyme, and protease II those of a SH-enzyme. These proteins may play important roles in mechanisms of cellular proteolysis.     

Purification and Properties of Proteases from a Marine-psychrophilic Bacterium

Protease which was found in the culture fluid of Pseudomonas sp. No. 548 was fractionated into four components with protease activity by a two step chromatography using DEAE-cellulose. Each protease was further purified by gel filtration on Sephadex G-100 and/or G-75. The protease of Ia was obtained in crystalline form and was shown to be homogeneous by analysis with electrophoresis, while the other three enzymes were also highly purified. The enzymatic properties of the proteases were investigated. All of the four enzymes were inactivated by ethylene diamine tetraacetate. Proteases Ia, Ib, and IIb were inactivated by diisopropylfluorophosphate. The optimum activity of protease Ia was shown to be at pH 10.0, and that of the other enzymes were at pH 7.0 to 8.0. The proteases of Ia, Ib, and IIb were stabilized by calcium ion. The effect of temperature, pH, and metal ions on the activity of the enzyme were also investigated.     

Trypanosoma cruzi: Isolation and characterization of aspartyl proteases

Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO₂)-Phe-Val-Leu-O₄MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (?68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (?80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation.     

Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues

Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.     

An endogenous inhibitor of thiol protease from adult lung fluke Paragonimus miyazakii

An endogenous inhibitor of thiol protease from adult Paragonimus miyazakii was found to occur during the gel filtration on Sephacryl S-300. The partially purified inhibitor was specific for thiol proteases such as ficin and papain. The inhibitor also suppressed tosyl-L-lysine alpha-naphthylester hydrolytic activity of Paragonimus thiol protease. The molecular weight of the inhibitor was found to be 430,000 by the gel filtration. This inhibitor was thermostable and resistant to trypsin and glycosidase digestions.     

Purification and characterization of high Ca2+-requiring neutral proteases from porcine and bovine brains

Porcine and bovine brain high Ca2+-requiring neutral proteases were purified to homogeneity by the same isolation procedures, and their properties were compared. A high degree of similarity existed between the two proteases. The purification procedures included ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose CL-4B, second DEAE-cellulose chromatography, second phenyl-Sepharose CL-4B chromatography, and gel filtration on Ultrogel AcA 34. Both purified enzymes were composed of Mr 75,000 and 29,000 subunits, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes required 250 microM Ca2+ for half-maximal activity and 700 microM Ca2+ for maximal activity. Sr2+ and Ba2+, but not Mg2+ or Mn2+, also activated both enzymes but not as effectively as Ca2+. Both enzymes displayed maximum activity at pH 7.5-8.0. Leupeptin, antipain, and trans-epoxysuccinyl-L-leucylagmatine inhibited both enzymes. Neurofilament triplet proteins and microtubule-associated proteins were extensively hydrolyzed by both proteases, but tubulin and actin were not hydrolyzed. The amino acid compositions of the two proteases were very similar. Antisera against bovine brain protease cross-reacted with porcine brain protease when examined by immunoelectrotransfer blot techniques.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Protease inhibitors from Streptomyces violascens

Summary Two protease inhibitors from the culture fluid of Streptomyces violascens U 10600 have been purified with a method including freeze-drying, methanol extraction, dialysis, and ultrafiltration. By gel filtration on Sephadex G-15 a separation in two active inhibitors, one of trypsin and one of chymotrypsin, was made.The inhibitors were stable at 100°C, pH 5, for 20 min and at 24°C between pH 1.8 to 9.7. Both inhibitors were dialysable. They had no bacteriostatic or fungistatic effects. The trypsin inhibitor inhibited also papain and proteases from Aspergillus oryzae, Alternaria tenuissima, Entomophthora coronata, and to some extent Gibberella fujikuroi, but not chymotrypsin, kallikrein, ficin, or pepsin. The chymotrypsin inhibitor inhibited also papain and proteases from Aspergillus oryzae, Alternaria tenuissima, and Gibberella fujikuroi, but not trypsin, kallikrein, ficin, pepsin, or protease from Entomophthora coronata.     

Isolation of a neutral histone-hydrolyzing protease from bovine spleen]

Neutral histone-hydrolyzing protease has been isolated by fractionation of bovine spleen extract. The low level of the protease activity in the extract may be due to the presence of an inhibitor. The enzyme activity was increased 100--1200-fold during ammonium sulfate fractionations, gel filtration on Sephadex G-100 and G-75, chromatography on CM- and DEAE-celluloses. The protease was detected in the fraction with a molecular weight lower than 25000. The enzyme was markedly activated by dithiothreitol and EDTA and inhibited by p-chloromercuribenzoate and iodoacetic acid. It was also inhibited by N-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanyl chloromethylketone, bovine blood serum and partially by soybean trypsin inhibitor DFP, trasylol and epsilon-amino caproic acid had no effect. Beside histone, the neutral protease hydrolyzed casein and gamma-globulin and fibrinogen in a low extent. The enzyme had no activity toward N-benzoyl-D,L-arginine p-nitroanilide, N-benzoyl-L-arginine ethyl ester and N-acetyl-L-tyrosine ethyl ester, collagen, elastin and fibrin. Some properties of the enzyme were similar to those of neutral SH-dependent proteases described by Hayashi and Lo Spalluto et al.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Multiple proteases from Streptomyces moderatus: I. Isolation and purification of five extracellular proteases

The presence of multiple proteases in the culture filtrate of Streptomyces moderatus was detected. After preliminary purification by ammonium sulfate precipitation and decolorization using DEAE-cellulose, the fractionation of various proteases was carried out using CM-trisacryl cation-exchange chromatography. By this procedure, four different protease fractions (Fr.) were separated (Fr. I, II, III, and IV). The first fraction was further separated into two different proteolytically active fractions (Fr. Ia and Fr. Ib) by DEAE-trisacryl anion-exchange chromatography. Fraction Ia was purified further by affinity chromatography on N-carbobenzoxy-d-phenylalanyl triethylenetetramine-Sepharose 4B. The second fraction (Fr. Ib) was purified by gel filtration on Ultrogel AcA 44. For the purification of the other protease fractions (Fr. II, III, and IV) single-step affinity chromatography methods were employed. Protease fractions II and III were purified by ϵ-aminocaproyl-4-(4-aminophenylazo)phenylarsonic acid Sepharose 4B and protease fraction IV was purified on ϵ-aminocaproyl trialanine-Sepharose 4B. All five proteases purified were found to be apparently homogeneous by gel electrophoretic methods.     

Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."     

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.     

Purification and characterization of two types of trypsin-like enzymes from sperm of the ascidian (Prochordata) Halocynthia roretzi. Evidence for the presence of spermosin, a novel acrosin-like enzyme

Two types of trypsin-like proteases, spermosin and acrosin, have been highly purified from spermatozoa of the ascidian (Prochordata) Halocynthia roretzi by a procedure including diethylaminoethylcellulose chromatography, Sephadex G-100 gel filtration, and soybean trypsin inhibitor-immobilized Sepharose 4B chromatography. Each purified preparation was judged to be homogeneous on the basis of chromatographic analysis and sodium dodecyl sulfate-gel electrophoresis. The molecular weights of spermosin and acrosin were estimated to be 27,000 and 32,000-34,000, respectively, by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the former was 6.5, while that of the latter was 5.5. Non-ionic detergents, e.g. Brij 35, showed marked stabilizing effects on the purified enzymes. Both of these enzymes had pH optima between 8.5 and 9.0, and their activities were enhanced by the addition of calcium chloride. The enzymes were inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, leupeptin, antipain, soybean trypsin inhibitor, aprotinin, ovomucoid, valyl-prolyl-arginyl-chloromethane, glycyl-valyl-arginyl-chloromethane, p-aminobenzamidine, benzamidine, zinc chloride, and mercuric chloride. Lima bean trypsin inhibitor and tosyl-lysyl-chloromethane strongly inhibited acrosin, but not spermosin. While the substrate specificity of acrosin was rather broad, that of spermosin was very narrow; the latter enzyme hydrolyzed only t-butyloxycarbonyl-valyl-prolyl-arginine 4-methylcoumaryl-7-amide among 12 peptidyl-arginine (or lysine) 4-methylcoumaryl-7-amides tested. Thus, the ascidian spermatozoa possess at least two proteases, acrosin and spermosin; the former shows the properties closely related to those of mammalian acrosin (EC 3.4.21.10), but the latter is a unique type of acrosin-like enzyme in respect to the substrate specificity and inhibitor susceptibility.     

棉铃虫幼虫中肠主要蛋白酶活性的鉴定

根据棉铃虫Helicoverpa armigera(Hubner)中肠酶液对蛋白酶专性底物在不同pH下的水解作用,棉铃虫中肠的3种丝氨酸蛋白酶得到鉴定。它们是：强碱性类胰蛋白酶,水 解a-N-苯甲酰-DL-精氨酸-p-硝基苯胺的最适pH在10.50以上;弱碱性类胰蛋白酶,水解p-甲苯磺酰-L-精氨酸甲酯的最适pH为8.50～9.00;类胰凝乳蛋白酶, 水解N一苯甲酰-L-酪氨酸乙酯的最适pH亦为8.50-9.00。中肠总蛋白酶活性用偶 氮酪蛋白测定,最适pH亦在10.50以上。Ca²⁺对昆虫蛋白酶无影响,Mg²⁺仅对弱碱性类胰蛋白酶有激活作用。对苯甲基磺酰氟和甲基磺酰-L-赖氨酸氯甲基酮对弱碱性类胰蛋白酶的抑制作用较强,而对强碱性类胰蛋白酶的抑制作用较弱。甲基磺酰-L苯丙氨酸氯甲基酮除能抑制类胰凝乳蛋白酶外,还能激活弱碱性类胰蛋白酶。对牛胰蛋白酶有强抑制作用的卵粘蛋白抑制剂对昆虫蛋白酶却无抑制作用。大豆胰蛋白酶抑制剂对该虫的3种丝氨酸蛋白酶均有强的抑制作用。     

Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes

1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and in-aminohexanoate (in-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells.     

Purification and Characterization of a Neutral Protease from Saccharomycopsis lipolytica

Saccharomycopsis lipolytica 37-1 produced two inducible extracellular proteases, one under neutral or alkaline growth conditions and the second under acid conditions. Secretion of the neutral protease was repressed in the presence of glycerol or glucose, both of which supported rapid growth of the organism. Ammonium ions also repressed the secretion of the enzyme. The neutral protease activity copurified with esterase activity during ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose, and gel filtration on Sephadex G-150. The molecular weight of the enzyme was estimated to be 42,000 by sucrose density gradient centrifugation and 38,500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme had a pH optimum of 6.8. Phenylmethylsulfonylfluoride inhibited both protease and esterase activities, indicating the presence of a serine residue in the active center. Protease, but not esterase, activity was sensitive to ethylenediaminetetraacetate and was significantly activated by divalent ions. Dithiothreitol inhibited both protease and esterase activities, indicating the presence of a critical disulfide bridge. The enzyme hydrolyzed casein (K(m) = 25.6 muM) and hemoglobin as well as the nitrophenyl esters of tyrosine (K(m) = 2.4 mM), glycine, tryptophan, and phenylalanine.