Purification and characterization of cathepsin B from monkey skeletal muscle

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.     

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.     

Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins

The 73-kDa protease (73K protease) was purified from a clinical isolate of Serratia marcescens kums 3958. The purified protease appeared homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. The protease is active in a broad pH range with maximum activity at pH 7.5-8.0. The protease appeared to be a thiol protease, since it was inhibited by sulfhydryl reactive compounds such as p-chloromercuribenzoic acid, fluorescein mercuric acetate (FMA), iodoacetamide, and N-ethylmaleimide, and the protease activity was enhanced by various reducing agents such as cysteine, glutathione, 2-mercaptoethanol, and dithiothreitol. The protease contained 2 mol of free sulfhydryl residues per mol of protease. When the protease was reacted with FMA, a maximum of 2 mol of FMA per mol of enzyme was found reacted, based on fluorescence quenching in which the enzyme inactivation was paralleled linearly with the loss of both SH groups. This indicates possible equal involvement of the two thiol groups for the enzyme activity. The inactivation of the protease by FMA was partially restored by a dialysis in the presence of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by alpha 2-macroglobulin. The protease bound stoichiometrically to alpha 2-macroglobulin with 1:1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of alpha 2-macroglobulin. The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and alpha 1-protease inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

A unique trypsin-like protease associated with plasma membranes of rat liver

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.     

A neutral thiol protease secreted from newly excysted metacercariae of trematode parasite Paragonimus westermani: purification and characterization

1. A neutral thiol protease was purified from the culture filtrate of newly excysted metacercariae of Paragonimus westermani to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a monomeric form with mol. wt 22,000. 2. It expressed activity on t-butyloxycarbonyl-valyl-leucyl-lysyl-4-methyl-coumaryl-7-amide in the presence of cysteine at an optimal pH of 7.5, and also the activity was significantly affected by thiol protease inhibitors, indicating that the enzyme belongs to a neutral thiol protease family. 3. The enzyme hydrolyzed protein substrates, azocoll, casein and fluorescein isothiocyanate-labeled collagen, and showed low specificity toward hemoglobin, but no activity with elastin Congo Red and bovine serum albumin. 4. Catalytic property on fluorogenic substrates demonstrated that the enzyme cleaved preferentially the carboxylic side of the basic residue in N-substituted peptides.     

Purification of a neutral thiol protease from Paragonimus westermani metacercariae by single-step chromatography and preparation of antibodies

A neutral thiol protease from extracts of larvae of the mammalian digenean parasite Paragonimus westermani metacercariae was purified by single-step chromatography on Ultrogel AcA-54, measuring its activity on t-butyloxycarbonyl-valyl-leucyl-lysyl-4-methylcoumaryl-7-amide as a substrate. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate and size exclusion-high-performance liquid chromatography analysis of the enzyme indicated that the fraction obtained by gel filtration was homogeneous. Antibodies against the purified protease were raised in rabbits by immunizing with micro quantities of the enzyme protein. The antibodies revealed a single precipitin line against the enzyme on double immunodiffusion analysis.     

Purification and properties of a neutral thiol protease from larval trematode parasite Paragonimus westermani metacercariae

1. A neutral thiol protease was isolated from the extract of larvae of the mammalian trematode parasite, Paragonimus westermani metacercariae, by arginine-Sepharose, Ultrogel AcA-54 and DEAE-toyopearl column chromatography, measuring its activity by the hydrolysis of Boc-Val-Leu-Lys-MCA as a substrate. 2. The molecular weight of the purified enzyme was estimated to be 22,000 as a single polypeptide by SDS-polyacrylamide gel electrophoresis and was estimated to be 20,000 by size exclusion high-performance liquid chromatography. 3. The activity was suppressed by antipain, E-64, leupeptin, chymostatin, N-tosyl-L-lysine chloromethyl ketone, but was not affected by metallo protease inhibitors or serine protease inhibitors. 4. Studies on the substrate specificity showed that the enzyme hydrolyzed Boc-Val-Leu-Lys-MCA, Z-Phe-Arg-MCA, fluorescein isothiocyanate-labeled collagen, azocoll and casein. 5. The enzyme was found to hydrolyze peptide bonds of oxidized insulin B chain preferentially at the carboxy side of hydrophobic and basic amino acids.     

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.     

A novel serine protease (IRCM-serine protease 1) from porcine neurointermediate and anterior pituitary lobes. Isolation, polypeptide chain structure, inhibitor sensitivity, and substrate specificity with fluorogenic peptide substrates

A novel serine protease, which we have called IRCM-serine protease 1, was purified from both porcine neurointermediate and anterior pituitary lobes. The enzyme was inhibited by soybean trypsin inhibitor, pancreatic trypsin inhibitor, benzamidine, phenylmethyl-sulfonyl fluoride, and thiol reagents including HgCl2, p-chloromercuribenzoate, and 5,5'-dithiobis-(2-nitrobenzoic acid) and was resistant to lima bean trypsin inhibitor, alpha 2-macroglobulin, alpha 1-antitrypsin, and C1-esterase inhibitor. IRCM-serine protease 1 displayed "trypsin-like" specificity toward a number of tripeptide coumarin-containing substrates, with kcat/km values ranging from 10(4) to 10(6) M-1 S-1. The best substrate was benzyloxycarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide with a kcat/Km value of 2.27 X 10(6) M-1 S-1. IRCM-serine protease 1, Mr = 169,000-190,000 determined by gradient gel electrophoresis and gel filtration, respectively, appears to be a homologous dimer. The monomeric subunits of the enzyme are composed of an Mr = 38,000 polypeptide chain which is modifiable by 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl, disulfide-linked to another polypeptide resulting in a subunit molecular weight of 88,000.     

Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase. Characterization as a high molecular weight cysteine proteinase

A nonlysosomal alkaline protease which degrades the oxidatively modified form of Escherichia coli glutamine synthetase has been purified to apparent homogeneity from rat and mouse liver acetone powders. Its molecular weight was determined to be 300,000 by Sephacryl S-300 gel filtration but results of further studies using high pressure liquid chromatography gel filtration suggest a value of 650,000. Examination of the subunit structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple bands of molecular weights between 22,000 and 34,000. The alkaline protease was inhibited by thiol reagents. Phenylmethylsulfonyl fluoride, aprotinin, leupeptin, antipain, and chymostatin partially inhibited the protease. The inhibition by phenylmethylsulfonyl fluoride was prevented by dithiothreitol, and alpha 1-antitrypsin and soybean trypsin inhibitor did not inhibit. No inhibition was observed with metalloprotease inhibitors. The alkaline protease is active over a broad range of pH with optimum activity for the degradation of oxidized glutamine synthetase around pH 9.0. Its activity is not stimulated by MgATP. A study of the products of insulin B chain degradation demonstrated major cleavage sites at Gln13-Ala14, Leu15-Tyr16, Cys(SO3H)19-Gly20, Gln4-His5, and Leu17-Val18. Based on its endopeptidase activity and its inhibitor specificity, the alkaline protease should be classified as a cysteine proteinase. It appears to be distinct from previously described proteinases and is likely involved in nonlysosomal mechanisms of intracellular protein turnover.     

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.     

Investigation of the heterogeneity of rhesus monkey alpha 1-antitrypsin

Rhesus monkey alpha 1-antitrypsin (n = 144) was examined for heterogeneity by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis. In contrast to other studies, no heterogeneity of Rhesus monkey alpha 1-antitrypsin could be documented using specific antisera. Rhesus monkey alpha 1-antitrypsin contained a reactive thiol. The pIs of the major isoforms of Rhesus monkey alpha 1-antitrypsin were 4.63, 4.69, 4.84 and 4.86 at 4 degrees C. No deficiency state of Rhesus monkey alpha 1-antitrypsin was detected. The six protease inhibitors in Rhesus monkey sera cross-reacted with antisera to the six human protease inhibitors.     

Purification and partial characterization of a Nocardia brasiliensis extracellular protease.

Nocardia brasiliensis possess proteolytic activities that can be readily detected in a variety of media. In a modified formulation of a growth medium originally used for Streptomyces aureofaciens, N. brasiliensis was found to secrete proteolytic enzymes, one of which was capable of hydrolyzing casein. This enzyme was purified to homogeneity from cell-free culture filtrates of N. brasiliensis. The purification procedure included ion-exchange chromatography on carboxymethyl-Sepharose, gel filtration on Sephadex G-100, and affinity chromatography, using a hemoglobin-Sepharose resin. The molecular weight of the N. brasiliensis protease was found to be 25,000 by gel filtration and 35,000 by sodium dodecyl sulfate-discontinuous gel electrophoresis. The enzyme is inhibited by o-phenanthroline and 8-hydroxyquinoline-5-sulfonic acid but is not affected by EDTA. Average values for its kinetic parameters were 0.288 mumol of hemoglobin solubilized per min per mg of enzyme for Vmax and 0.76 mM for Km, using hemoglobin as the substrate.     

Isolation, purification and properties of aortic elastase.

An elastase from pig aorta has been partially purified and characterised; it exhibits immunological cross reaction with pig pancreatic elastase. Its proteolytic (k-elastin gel and polymeric elastin substrates) and esterolytic (N-succinoyl-trialanine paranitroanilide) activities as well as its degree of inhibition by serum protease inhibitors (alpha1-antitrypsin and alpha2-macro-globulin) differ sensibly from those of pancreatic elastase [14,16].     

Purification and characterization of a calcium-activated neutral protease from monkey brain and its action on neuropeptides

A calcium-activated neutral protease was purified from Japanese monkey brain by ammonium sulfate fractionation and sequential column chromatographies monitored by assay of caseinolytic activity. The purified enzyme gave a single protein band on non-denaturing polyacrylamide gel electrophoresis, and consisted of two subunits with molecular weights of 74,000 and 20,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme required millimolar order calcium ions for activation, and was optimally active at pH 7.5-8.0. Upon incubation with various neuropeptides as substrates, the enzyme preferentially cleaved the peptide bonds with Arg, Lys, or Tyr at the P1 position and an amino acid residue with a bulky aliphatic side chain, such as Leu, Val, or Ile, at the P2 position. The hydrolytic activity toward neuropeptides as well as casein was strongly inhibited by various thiol protease inhibitors. These results suggested that the brain calcium-activated neutral protease may participate in the degradation of neuropeptides in vivo.     

Purification of Bacteroides amylophilus protease

A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-alpha-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.     

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.     

Novel functions of human alpha(1)-protease inhibitor after S-nitrosylation: inhibition of cysteine protease and antibacterial activity

alpha(1)-Protease inhibitor (alpha(1)PI), the most abundant serine protease inhibitor found in human plasma (at 30-60 microM), is a glycoprotein (53 kDa) having a single cysteine residue at position 232 (Cys(232)). We have found that Cys(232) of human alpha(1)PI was readily S-nitrosylated by nitric oxide (NO) without affecting inhibitory activity to trypsin or elastase. S-nitrosylated alpha(1)PI (S-NO-alpha(1)PI) not only retained inhibitory activity against these serine proteases, but also gained thiol protease inhibitory activity against a Streptococcus pyogenes protease; the parental alpha(1)PI did not have this activity. Furthermore, S-NO-alpha(1)PI exhibited bacteriostatic activity against Salmonella typhimurium at concentrations of 0.1-10 microM, which were 20- to 3000-fold stronger than those of the other NO-generating compounds or S-nitroso compounds such as S-nitrosoalbumin and S-nitrosoglutathione. NO appears to be transferred into the bacterial cells from S-NO-alpha(1)PI via transnitrosylation, as evidenced by electron spin resonance spectroscopy with an NO spin trap. Thus, we conclude that S-NO-alpha(1)PI may be generated from the reaction between alpha(1)PI and NO under inflammatory conditions, in which production of both is known to increase. As a result, new functions, i.e., antibacterial and thiol protease inhibitory activities of alpha(1)PI, were generated.     

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.     

Characterization of a protein C activator from Agkistrodon contortrix contortrix venom

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.