Studies of a calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization.

A calcium-activated neutral protease was purified 2,700-fold over the crude extract from chicken skeletal muscle. The purified protease migrated as a single band on polyacrylamide gel electrophoresis with or without SDS. Its molecular weight was 80,000 and pH optimum for activity was 7.7. The activity required strictly the presence of calcium (optimum concentration: 1.8 mM) or strontium (optimum concentration: 10 mM) ions. The protease was inhibited by leupeptin, which is known to be a strong inhibitor of papain, cathepsin B, trypsin, and plasmin.     

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.     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.     

Protease from bovine spleen with kininogenase activity]

A protease with kininogenase activity at pH 7.5 was isolated from bovine spleen extract by gel filtration and ion exchange chromatography. The protease was found in the fraction with molecular weight lower than 25.000 and was separated from the other neutral SH-dependent protease by chromatography on KM-cellulose. The kininogenase activity was inhibited by DFP and trasylol; soybean trypsin inhibitor had no effect. The protease did not split N-benzoyl-L-arginine ethyl ester and N-benzoyl-D, L-arginine p-nitroanilide.     

Isolation,partial purification,and some properties of protease I from a thermophilic mold Thermoascus aurantiacus var. levisporus

When the thermophilic mold Thermoascus aurantiacus var. levisporus was grown in a modified Czapek Dox medium containing casein the filtrate was found to contain proteolytic activity. The maximum production of activity occurred at 50 ° C in a medium containing 8% casein. The filtrate was subjected to ammonium sulfate fractionation and chromatography on DEAE-cellulose. Two proteases were separated. No further work was done on protease II. Protease I was further purified by gel filtration on Sephadex G 100–200. It showed a 40-fold purification with a final recovery of approximately 25%. It is a neutral protease with a pH optimum at 7.0. The optimal activity of the enzyme occurred in 0.02 M phosphate buffer but was completely inhibited at a concentration of 0.1 M. The optimum temperature for casein hydrolysis was found to be 55 ° C. The enzyme was inhibited by Hg⁺⁺ but was greatly stimulated by Cu⁺⁺ and mercaptoethanol. Metallo and sulfhydryl agents had no significant effect on enzyme activity.     

Identification of two new calcium dependent hydrolases in the human heart

We have identified and isolated two new calcium-activated neutral hydrolases from human ventricular muscles. The one is an esterase, of which molecular weight was 300,000, required millimolar concentration of Ca2+, hydrolyzed Ac-Tyr-OEt X H2O, optiaml pH at 7.0. The other is an amidase, of which molecular weight was 70,000, also required millimolar concentration of Ca2+, hydrolyzed a synthetic substrate for chymotrypsin, Suc-Leu-Leu-Val-Tyr-MCA, with optimal pH at 7.2. Both enzymes did not degrade casein or contractile proteins (myosin, actin, troponin and tropomyosin). Their activities were not inhibited by exogenous protease inhibitors, leupeptin, antipain, monoiodoacetic acid and chymostatin, while the amidase activity was blocked by the endogenous inhibitor against calcium-activated neutral protease (CANP). Thus, their characters are different from chymotrypsin or CANP and they seems to be new hydrolases in the human heart.     

Degradation of myelin basic protein by calcium-activated neutral protease (CANP) in human brain and inhibition by E-64 analogue

A calcium-activated neutral protease (CANP) was extracted from human brain and partially purified. The activity was measured using alkali-denatured casein (Hammersten) as a substrate. The optimum pH was around 7.0. The activity required the presence of calcium ions, maximum activity was obtained with over 5 mM calcium ions. TheK _m for the casein concentration was about 1.62 mg/ml. The activity of CANP was inhibited by one of the thiol protease inhibitors, E-64 analogue (E-64-a). The rate of inhibition was about 50% at an E-64-a concentration of 10^–5M. This CANP degraded selectively basic protein in myelin proteins and the degradation was inhibited by E-64-a or EGTA. The role of the brain CANP in the process of demyelination was suggested by this study.     

枯草芽孢杆菌ZC-7中性蛋白酶的分离纯化及酶学性质研究

枯草芽孢杆菌ZC-7的发酵液,经离心分离得到粗酶液,再经硫酸铵盐析、中空纤维膜除盐浓缩、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75柱层析等步骤获得电泳纯的中性蛋白酶。SDS-PAGE测得其分子量大约为42KDa。以酪蛋白为底物时,该酶的Km为5×10-3,Vmax为2.5×104ug/min,酶的最适作用pH为7.0,最适反应温度为55℃,在pH6.5～8.0, 40℃以下较稳定,对1mol/L H2O2具有一定的耐受性。EDTA、异丙醇和乙醇对该酶有抑制作用,Ca2+、Mg2+和Li+离子对其具有保护作用。     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

Partial characterization of steroid sulfohydrolase and steroid sulfotransferase activities in purified porcine Leydig cells

Subcellular fractions of purified pig Leydig cells from 7 different animals have been investigated with respect to their abilities to catalyze the sulfation of several steroids and the hydrolysis of the sulfated forms of these same steroids. Considerable estrone sulfate sulfohydrolase of pH optimum 7.5 and high apparent Km was found to be concentrated in the 105,000 g pellet but no evidence was obtained, in any subcellular fraction, for the presence of any activity toward the 3-sulfate of pregnenolone, dehydroepiandrosterone (DHA) or delta 5-androstene-3 beta,17 beta-diol (androstenediol). Cytosolic sulfotransferase activity toward estrone, pregnenolone, DHA and androstenediol was present in each animal. The activity toward these 4 substrates was eluted from a gel filtration column as a single peak of apparent molecular weight 43 KDa. Upon chromatofocusing, a sharp estrogen sulfotransferase peak of apparent pI 6.1 and pH optimum 9.5, was clearly separated from the neutral steroid sulfotransferase which eluted over a more acidic pH range in a manner suggestive of the presence of several isozymes. This latter, which exhibited a wide pH optimum range between 6 and 8.5, was most active toward androstenediol, and least active toward pregnenolone. The estrogen sulfotransferase exhibited Michaelis-Menten kinetics (apparent Km = 4 microM). The neutral steroid sulfotransferase activity increased in velocity with increasing androstenediol or DHA concentration up to 1 microM beyond which considerable substrate inhibition occurred. It appears from these data that neutral steroid sulfates synthesized in the pig Leydig cell are not subject to enzymic desulfation in the same cells.     

Isolation and partial characterization of trehalose 6-phosphate synthase aggregates from Selaginella lepidophylla plants

Trehalose 6-phosphate synthase was purified from Selaginella lepidophylla plants and three aggregates of the enzyme were found by molecular exclusion chromatography, ion exchange chromatography and electrophoresis. Molecular exclusion chromatography showed four activity peaks with molecular weights of 624, 434, 224 and 115 kDa. Ion exchange chromatography allowed three fractions to be separated with TPS activity which eluted at 0.35, 0.7 and 1 M KCl. Native PAGE of each pool had three protein bands with apparent M(r) 660, 440 and 200 kDa. Western blot results showed that anti-TPS antibody interacted with 115 and 67 kDa polypeptides; these polypeptides share peptide sequences as indicated by internal sequence data. The effects of pH and temperature on enzyme stability and activity were studied. For fractions eluted at 0.35 and 1.0 M KCl, the optimum pH is 5.5, while an optimum pH of 7.5 for 0.7 M fraction was found. The three fractions eluted from ion exchange chromatography were stable in a pH 5-11 range. Optimal temperatures were 25, 45 and 55 degrees C for 0.7, 0.35 and 1.0 M fractions, respectively. The 0.7 M KCl fraction showed highest stability in a temperature range of 25-60 degrees C, whereas the 0.35 M KCl fraction had the lowest in the same temperature range.     

Purification and characterization of two novel halotolerant extracellular proteases from Bacillus subtilis strain FP-133

Bacillus subtilis strain FP-133, isolated from a fermented fish paste, synthesized two novel halotolerant extracellular proteases (expro-I and expro-II), showing activity and stability at concentrations of 0-20% (w/v) NaCl. Each protease was purified to homogeneity and characterized. The purified expro-I was a non-alkaline serine protease with an optimum pH of 7.5, although most serine proteases from Bacillus strains act at the alkaline side. The molecular mass of expro-I was 29 kDa. The purified expro-II was a metalloprotease with a molecular mass of 34 kDa. It was activated by Fe(2+), which has never been reported as a bacterial protease activator. At a concentration of 7.5% (w/v) NaCl, both proteases preferred animal proteins to vegetable proteins as natural substrates. In addition, under saline conditions, expro-I and II showed high catalytic activity toward gelatin and casein respectively.     

Characterization of proteases of Toxoplasma gondii

The proteases of Toxoplasma gondii were purified partially and characterized for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteases working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion exchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotors. The protease working at pH 6.0 was inactivated by iodoacetamide with LD50 of 10(-3) M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD50 of 10(-5) M and was promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.     

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.     

Activation of fibroblast procollagenase by mast cell proteases.

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.     

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.     

Purification and properties of a thiol protease from rat liver nuclei

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30-40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.     

Novel protease bound with chromatins in normal and tumorous tissues of rats

A protease capable of hydrolyzing casein with optimum pH 10 (alkaline protease), perhaps functional in hydrolysis of non-histone proteins and Hl histone, was found to exist at the state bound with chromatins of various normal and tumorous tissues of rats, in addition to the protease capable of hydrolyzing histone with optimum pH 8 (neutral protease). Alkaline protease was not observed in other subcellular fractions than nuclear fraction. It had approximately 18,000 daltons, and was chymotrypsin-like as inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor and Chymostatin. Its contents were significantly high in rapidly proliferating cells; Yoshida sarcoma? Rhodamine sarcoma≥ AH 130≥ thymus> spleen? kidney≥ liver? brain.