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.     

Characterization of neutral proteinases from human spleen]

Several proteinases hydrolyzing histone and caseine in neutral media were obtained by Sephadex G-100 fractionation of water and salt (1 M KCl) extracts of human spleen. The level of the activity of proteinases in the extracts was very low as a result of the presence of an inhibitor. Neutral proteinases were found in two protein fractions. The "high-molecular-weight-" proteinases were inhibited by DFP and therefore they were attributed to a group of serine proteinases. The "low-molecular-weight" fraction contained neutral SH-dependent proteinase(s) and DFP-inhibited enzymes. In this fraction, the kininogenase activity and the hydrolysis of Boc-1-ananine p-nitrophenyl ester, N-benzoyl-L-tryosine ethyl ester and N-benzoyl-DL-arginine p-nitroanilide were observed.     

Purification and partial characterization of an exocellular proteinase from Trichophyton rubrum

An exocellular proteinase produced by Trichophyton rubrum in a glucose-peptone broth was purified from lyophilized and dialysed culture filtrate of the dermatophyte by Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified enzyme was a homogeneous protein of molecular weight 34700 and it could hydrolyse azoalbumin, casein, bovine serum albumin, alpha-N-benzoyl-L-arginine ethyl ester and p-toluenesulfonyl-L-arginine methyl ester but not N-benzoyl-L-tyrosine ethyl ester, alpha-N-benzoyl-DL-arginine-p-nitroanilide and keratin. The enzyme showed an alkaline pH optimum and was not activated by divalent metal ions but inhibited strongly by phenylmethylsulfonylfluoride. Thus the enzyme was identified as an alkaline serine proteinase.     

A trypsin and chymotrypsin inhibitor from Taenia pisiformis

The somatic extract of mature T. pisiformis has been demonstrated to contain a potent inhibitor capable of inactivating the esterolysis of $\text{N-α-}\text{benzoyl-}\text{L}\text{-arginine ethyl ester}$ and $\text{N-}\text{benzoyl-}\text{L}\text{-tyrosine ethyl ester}$ by trypsin and chymotrypsin, respectively, of bovine, dog and rabbit origin, but not affecting the caseinolytic activity of subtilisin and elastase. The protease inhibitor, partially purified by trichloroacetic acid treatment, Sephadex G-100 column chromatography and affinity chromatography on CNBr-activated Sepharose 4B-bovine chymotrypsin conjugate, was soluble in 5% trichloroacetic acid, stable to heating at 100°C for up to 30 min, tolerated the pH range of 1.5–9.0, and was unaffected by 8 m-urea or 0.2 M-2-mercaptoethanol. The molecular weight of the inhibitor was estimated to be 7000–7200 by Sephadex G-100 chromatography. Activity determinations on crystalline bovine trypsin and chymotrypsin revealed that both inhibitory actions are located on the same or closely adjacent sites of the inhibitor molecule. Complex formation between the inhibitor and mammalian trypsin and chymotrypsin required 3–4 min for completion.     

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。     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

Purification and characterization of a myosin-cleaving protease from rat heart myofibrils.

A proteolytic enzyme, which causes the limited degradation of cardiac myosin, was purified from rat heart myofibrils. The purified enzyme (a myosin-cleaving protease) was apparently homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Autolysis of the purified enzyme was observed at neutral pH without high concentration of CaCl2. The molecular weight was estimated to be 26 000-27 000. The enzyme was active against casein, N-acetyl-L-tyrosine ethyl ester and N-glutaryl-L-phenylalanine-4-nitroanilide (Glu-Phe-NAn), but less active with N-benzoyl-DL-arginine-4-nitroanilide. Optimum pH values for the enzyme were 9.0 for casein and 8.4 for Glu-Phe-NAn. Caseinolytic activity of the enzyme was completely inhibited with phenylmethylsulfonyl fluoride and diisopropylphosphofluoride and partially inhibited with L-1-tosyl-L-phenylalanine chloromethyl ketone (Tos-PheCH2Cl) and soybean trypsin inhibitor. Tos-LysCH2Cl had no effect. Sulfhydryl reagents, metal-chelating agents and metal ions except for Zn2+ had little or no effect on the activity. Degradation of cardiac myosin with the enzyme produced two fragments having molecular weights of 130 000 and 94 000, accompanied by the disappearance of myosin heavy chain and light chain 2. Myosin degradation with the enzyme was more restrictive than with chymotrypsin.     

Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.     

Multiplicity of bovine liver GM1 ganglioside beta-galactosidase

The multiplicity of bovine liver acid beta-galactosidase was investigated. Acid beta-galactosidase activity was measured in the presence of glucono-delta-lactone, which inhibited the neutral beta-galactosidase activity but not the acid beta-galactosidase activity in bovine liver. Three forms of acid beta-galactosidase were separated by Sephadex G-200 gel filtration and the elution pattern of the 4-methylumbelliferyl-beta-galactosidase activity coincided with that of the GM1-beta-galactosidase activity. These forms were relatively stable under acidic conditions (pH 4.5), but the two high molecular weight forms were inclined to dissociate into the low molecular weight form under neutral conditions (pH 7.0). The three forms of the enzyme showed similar pH-optima and apparent Michaelis constants for GM1 ganglioside.     

The occurence of a neutral protease and its inhibitor in rat peritoneal macrophages.

The lysate of the glycogen-induced macrophages in rat peritoneal exudate was fractionated by centrifugation and extraction into a water extract, 1 M KCl extract and residue fractions. Approximately 50% of the neutral protease activity toward casein in the lysate was recovered in the KCl extract fraction, which was practically devoid of acid protease, cathepsin D. The pH optimum of the neutral protease toward casein and urea-denatured hemoglobin was pH 8.5. The activity was inhibited strongly by DFP or chymostatin and only partially by HgCl2 or PCMB. Addition of a salt to the reaction medium caused enhancement of the activity with an optimum concentration of 0.25 M: KCl, KBr, KI, NaCl, NaBr, NaI, and MgCl2 were all almost equally effective. When the enzyme preparation was filtered through a column of Sephadex G-75 gel in the presence of 1 M KCl, a larger molecular weight fraction at the void volume was obtained in addition to a smaller molecular weight fraction showing a caseinolytic activity insensitive to KCl concentration. The former was found to have a specific inhibitory effect on the latter activity.     

Studies on the Lipase of Thermophilic Fungus Humicola lanuginosa

A protease occurring in the endosperm fraction of germinating corn was purified by means of (NH₄)₂SO₄ fractionation, CM-celluIose chromatography, DEAE-cellulose chromatography, Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified protease was found to have a molecular weight of about 21,000 and an isoelectric point of pH 2.3 or lower. The optimum pH was found to lie at 3.0 when measured with denatured hemoglobin as substrate. The protease was generally activated by thiol compounds and completely inhibited by p-chloromercuribenzoic acid. Neither diisopropylphosphofluoridate nor diazoacetyl-dl-norleucine methyl ester affected the protease activity. Antipain greatly inhibited the protease action whereas pepstatin had no significant effect. These data indicate, in conclusion, that the protease possesses a unique property to be a sulfhydryl enzyme most active in an acidic region around pH 3.     

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.     

Partial characterization of a novel cathepsin L-like protease from Fasciola hepatica

A 30-kDa protease, purified previously from Fasciola hepatica, was sequenced and the first 15 N-terminal residues were found to be 100% homologous to a region in the protein Fcp1c, which was cloned and expressed from F. hepatica. This terminal region was also 53 and 54% identical to two other cathepsin L-like proteases isolated from the same source. The 30-kDa protease demonstrated a specificity different from humancathepsin L when assayed with novel peptidyl enediones of the type Z-Phe-Ala-CH&dbond;CH(2)-CO(2)R (where R = Me/Et/Bu(t)). The ethyl ester peptide was a more efficient inhibitor of the protease than the corresponding methyl ester. This is in contrast to bovine cathepsin B and human cathepsin L where both are more readily inhibited by the methyl, rather than the ethyl ester peptide. These differences in the inhibition of the novel parasite protease may allow it to be exploited as a chemotherapeutic target.     

Ligula intestinalis (Cestoda: Pseudophyllidae): Studies on the properties of proteolytic and protease inhibitor activities of plerocercoid larvae

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl-l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10^?5m-Ca²⁺, Mg²⁺ or Mn²⁺, it was inhibited by 10^?5m-Cu²⁺, but not by 10^?5m-Zn²⁺. 0.001% soybean trypsin inhibitor, 2 × 10^?3m-EDTA, 1 mm-tosyl-l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 mm-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘in vitro’ by the inhibitors isolated from this parasite.     

Neutral elastolytic proteinase from canine leucocytes. Purification and characterization.

1. A neutral proteinase (EC 3.4.-.-) with elastolytic activity was isolated from canine bloodstream leucocytes, and purified to apparent homogeneity by a two-step procedure consisting of DEAE-Sephadex chromatography and molecular sieving on Sephadex G-75. 2. The molecular weight of the enzyme was 23 500, and the absorbance (A1%1cm) at 282 nm was 6.1. Amino acid analysis showed high content of glycine, aspartic acid, and valine, and low proportion of methionine, lysine and histidine as well as the absence of tyrosine in the enzyme molecule. 3. The proteinase was active against several protein substrates as well as towards N-t-butyloxycarbonyl-L-alanine p-nitrophenyl ester, N-acetyl-L-alanyl-tyrosine ethyl ester. 4. The enzyme was inactivated by diisopropylfluorophosphate, N-acetyl-L-alanyl-L-alanyl-L-alanine chloromethyl ketone, and N-p-tosyl-L-phenylalanine chloromethyl ketone. Inhibition by some natural proteinase inhibitors was also noted.     

Isolation and some properties of an acid protease from Fasciola hepatica.

A new protease, detected in an extract of Fasciola hepatica, was isolated and partly purified. The pH optimum for the cleavage of denaturated haemoglobin by the enzyme is pH 3.0. This proteolytic activity is inhibited by diazoacetylnorleucine methyl ester, pepstatin, the pepsin inhibitor from Ascaris suum, and phenylalanine. The cathepsin D inhibitor from potatoes, EDTA, mercaptoethanol and the inorganic salts tested have no inhibitory effect. The cleavage of the B-chain of oxidized insulin by enzyme was studied and compared with the digestion of the same substrate by chicken and pig pepsin. The protease from Fasciola hepatica belongs to the carboxyl group of proteases and probably plays an important role in helminth nutrition.     

Activation of a new plasma enzyme by antigen-antibody reaction.

The change of enzyme activity in immunized rabbit plasma after addition of the homologous antigen was examined. The activities of N alpha-tosyl-L-arginine methyl ester (TAMe) and N alpha-tosyl-L-lysine methyl ester (TLMe) hydrolysis increased about 15 to 18 days after immunization. This increase was especially marked before the maximal rise of antibody content, and is thought to be related to the IgM antibody not to the IgG antibody. Enzyme activation was strongly inhibited by chelation of Ca2+ with 5 mM disodium ethylenediamine tetraacetate (EDTA), but not by other protease inhibitors, such as epsilon amino-caproic acid (epsilon-ACA), bovine lung kallikrein inhibitor (Trasylol) or soybean trypsin inhibitor (SBTI).     

Molecular properties of the Factor V-activating enzyme from Russell's viper venom.

The protease from Russell's viper venom that activates Factor V was purified by gel filtration on Sephadex G-150 and ion exchange column chromatography on sulfopropyl (SP)-Sephadex C-50. The purified enzyme is a glycoprotein containing 6% carbohydrate. It migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 29,000. A minimum molecular weight of 27,200 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Val-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Pro-Ile. The specific activity of the Factor V activator toward tosyl-L-arginine methyl ester and D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide was 380 and 11 nmol/min/mg, respectively. The esterase and coagulant activities of the enzyme were readily inhibited by diisopropyl fluorophosphate. The enzyme was not inhibited by bovine antithrombin III in the presence or absence of heparin. The amino acid and carbohydrate compositions of the enzyme are also reported.     

Purification and some properties of a neutral protease from human leukocyte granules and its comparison with pancreatic elastase.

1. A cationic protease has been purified from the granule fraction of blood-donor leukocytes by a preparative method including precipitation by acetone and chromatography on Bio-Gel A 1.5 m, CM-Sephadex C-50 and Sephadex G-G-75. 2. The pH optimum against denatured bovine hemoglobin is 7.4. Gel chromatography indicated a molecular weight close to 23 000. 3. This neutral protease (EC 3.4.-.-) is able to split the synthetic esters Z-Ala-NPh and AcAla3OMe, its activity on the former substrate being 2.2 times greater than that of pancreatic elastase, on the latter the same. It differs crucially from pancreatic elastase in having small elastinolytic activity. 4. In cationic disk electrophoresis, neutral protease resolves into three protein bands with lower mobility than lysozyme: all bands exhibit esterolytic activity against 2-acetoxy-3-naphthoic acid o-toluidide, strongly suggesting that they represent isoenzymes. 5. The enzyme is completely inhibited by iPr2P-F, partially so by soybean trypsin inhibitor and Trasylol. Cysteine, EDTA and TosLysCH2Cl have no effect. 6. During chromatography on CM-Sephadex C-50 a more positively charged enzyme(s) was identified. This had hemoglobinolytic activity at pH 7.4 but only a small esterolytic effect on Z-Ala-NPh; it showed only traces of activity against AcAla3OMe.     

Purification and characterization of a coagulant protein from the venom of Russell's viper.

The coagulant protein from the venom of Russell's viper was purified by means of successive chromatography on Sephadex G-50, DEAE-cellulose and Sephadex G-200. The purified coagulant protein was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight was estimated to be about 100 000 by ultracentrifuge analysis and 130 000 by gel filtration. The coagulant protein contains 11.1% carbohydrate which includes 5.1% hexose (galactose: mannose = 1:1), 5% hexosamine (glucosamine), and 1% neuraminic acid (N-acetylneuraminic acid and N-glycolyneuraminic acid). The isoelectric point is pH 6.3. The results of both sodium dodecyl sulfate electrophoresis and gel filtration in 6 M guanidium chloride suggest that it consists of four polypeptide chains. The coagulant protein functions as an enzyme in activating blood coagulation factor X in the presence of Ca2+. N-a-p-Toluenesulfonyl-L-arginine methyl ester hydrolyzing activity in the preparation definitely decreased during purification and it suggests that the clotting activity is not associated with the esterase activity. The clotting activity is inhibited by diisopropyl phosphorofluoridate and by phenylmethylsulfonyl fluoride, suggesting that the coagulant protein is a serine protease. The optimum pH is between pH 7.0 and pH 8.0. At neutral pH the coagulant protein is stable below 50 degrees C, but is rapidly inactivated above 55 degrees C.