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At least 19 recordsLinked to original sources
PubMed1976

Purification and properties of two protease inhibitors from rat skin inhibiting papain and other SH-proteases.

Two papain inhibitors, I1 and I2, from rat skin extract were purified by affinity chromatography on KSCN-modified papain-agarose gel and by gel filtration on Sephadex G-100. I1 had a molecular weight of 74 000, a pI of 4.6, and it contained 4% of carbohydrates. I1 inhibited papain, ficin, bromelain, rat skin benzoylarginine-2-naphthylamide hydrolase, and to a minor extent, rat skin cathepsin C and bovine trypsin. Bovine chymotrypsin or rat skin cathepsin D were not inhibited and benzoylarginine-2-naphthylamide hydrolase was inhibited only at alkaline pH. An inhibitor corresponding to I1 was present in various rat tissues and also in serum. A similar inhibitor was present in the skin of cat, rabbit, guinea pig, and man. I2 had a molecular weight of 13 400, a pI of 4.9 and it contained no carbohydrates. I2 inhibited all thiol proteases tested, but not trypsin, chymotrypsin, or rat skin cathepsin D. I2 formed an equimolar complex with papain and benzoylarginine-2-naphthylamide hydrolase. I2 was present in rat skin, muscle, lung, and small intestine, but not in kidney, liver, or serum. A similar inhibitor was found in skin extracts of cat, rabbit, guinea pig, and man.

Animals↗
PubMed1976

Alpha-N-Benzoylarginine-2-naphthylamide hydrolase (cathepsin BI?) from rat skin. III. Substrate specificity, modifier characteristics, and transformation of the enzyme at acidic pH.

Some properties of rat skin benzoylarginine-2-naphthylamide hydrolase types I (preparations I and AI) and II (preparations II and NII) were studied. Both types were activated by dithiothreitol and EDTA, but responded differently to 1 mM KCN, when benzoylarginine-2-naphthylamide (BANA) was used as a substrate: type I was inhibited, while type II was activated. When leucine-2-naphthylamide was used as a substrate, both types were activated by KCN. Thiol proteinase inhibiting substances, like heavy metals, iodoacetic acid, 4-chloromercuribenzoic acid, and tosyllysine chloromethylketone, inhibited the enzymes. Diisopropylfluorophosphate, phenylmethylsulfonyfluoride, 4-aminobenzamidine, and high-molecular-weight trypsin inhibitors were without effect. The substrate specificity of rat skin BANA hydrolase resembled that of an amino acid naphthylamidase, naphthylamides of methionine, lysine, arginine, and alanine being hydrolyzed most rapidly. The rate of hydrolysis of BANA was only 11% of that of methionine naphthylamide. Amino acid esters with a free alpha-amino group were also good substrates. The transformation of type II to type I at acidic pH was studied. During the transformation amino acids or peptides were formed and probably some inhibitor present in type II was destroyed proteolytically.

Animals↗
PubMed1981

Localization and characterization of N-ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus-transformed nil hamster cells.

Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 X g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 X g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedure. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90-100 degrees C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.

Aminopeptidases↗
PubMed1979

Proteinase binding and enhancing factor from human skin.

Fresh human skin extract made in salt solution after a prior buffer extraction was shown to enhance the hydrolysis of N-alpha-benzoyl-DL-arginine beta-naphthylamide (BANA) by trypsin. This trypsin enhancing effect was further shown to be both stabilizing and activating. After chromatography on Sephadex G-100, the trypsin binding factor was found in fractions of void volume. Protease binding took place in physiological and hypotonic but not in hypertonic NaCl-solutions (0.5 mol/l). The proteinase binding factor was further purified by trypsin-Sepharose 4 B affinity chromatography. It was found to bind also chymotrypsin and elastase and to be thermostable (100 degrees C for 20 min), precipitable at acidic pH (3.5), and by acetone and ammonium sulphate (60% saturation). The bound proteinases were found to preserve their hydrolytic activity towards protein substrates. Bound trypsin and chymotrypsin could completely be inhibited by soybean trypsin inhibitor. The binding factor did not react with anti-human-alfa2-macroglobulin antiserum from rabbit.

Benzoylarginine-2-Naphthylamide↗
PubMed1976

Human skin proteases. Separation and characterization of two acid proteases resembling cathepsin B1 and cathepsin D and of an inhibitor of cathepsin B1.

Two acid proteases, one hydrolysing hemoglobin and the other hydrolysing benzoyl arginine naphthyamide (BANA), were separated and partially purified from human skin buffer extract. The acid protease hydrolysing hemoglobin was purified about 190 fold by Sephadex G-100 gel filtration and DEAE-cellulose chromatography. It hydrolysed hemoglobin at pH 3.5, casein at pH 5.8 and skin protein substrate at pH 6.0. It did not markedly hydrolyse synthetic protease substrates. The molecular size of this protease was 38000. The protease was insensitive to common protease modifiers and closely resembles cathepsin D purified from other organs. The BANA-hydrolysing acid protease was purified about 760 fold by Sephadex G-100 gel filtration and affinity chromatography on organomercurial Sepharose 4B gel. It preferentially hydrolysed BAEE, BANA and BAA with an optimum at pH 5.8. The hydrolysis of BAPA, LeuNA and protein substrates was very low. This acid protease was found to be highly dependent on reducing agents, as DTT, and chelating agents, as EDTA, and was inhibited by pCMB and TLCK. The molecular size of the enzyme was 28000. This protease closely resembles cathepsin B1 purified from other organs. Human skin was also shown to contain a low activity of benzoyl arginine amide (BAA) hydrolysing acid protease with a molecular size of about 50000 and resembling cathepsin B2. Human skin contained an inhibitor with a molecular size of about 13000 against human skin cathepsin B1. This inhibitor did not inhibit trypsin, chymotrypsin or skin proteases other than cathepsin B1.

Benzoylarginine-2-Naphthylamide↗
PubMed1986

Isolation and characterization of cathepsin B from bovine brain.

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75-5.2 with the major band at pH 5.1-5.2, all of which were capable of degrading N alpha-carbobenzoxy-L-arginyl-L-arginine 4-methoxy-beta-naphthylamide. The cathepsin B activity against N alpha-benzoyl-DL-arginine beta-naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.

Animals↗
PubMed1985

Purification and properties of rabbit liver cathepsin M and cathepsin B.

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.

Amino Acids↗
PubMed1984

Characterization of a trypsin-like protease from the bacterium Bacteroides gingivalis isolated from human dental plaque.

A trypsin-like, membrane-bound protease from Bacteroides gingivalis was solubilized by Triton X-100 and partially purified by a combination of DEAE-Sepharose and aminophenylmercuric Sepharose chromatography, by taking advantage of the thiol group on the enzyme. The purified enzyme hydrolysed the synthetic substrates benzoyl-L-arginine-p-nitroanilide (L-BAPA), benzoyl-D,L-arginine-beta-naphthylamide (BANA) and tosyl-L-arginine methyl ester, as well as bovine serum albumin and ovalbumin, but not tosyl-L-lysine methyl ester. The enzyme activity was enhanced by SH-reagents and was inhibited to different degrees by SH-inhibitors, chelators and microbial low-molecular-weight inhibitors such as leupeptin, antipain and chymostatin. These microbial inhibitors could be of practical use as ligands for affinity chromatography for further purification. The possible involvement of the protease in periodontal diseases is also discussed.

Bacteroides↗
PubMed1975

Fractionation of the rat liver enzymes that hydrolyze benzoyl-arginine-2-naphthylamide.

1. The enzyme activity in the particulate fraction from rat liver that hydrolyzes alpha-N-benzoyl-DL-arginine-2-naphthylamide (Bz-Arg-NNap) has been separated into two approximately equal components by chromatography on DEAE-cellulose. One component (peak II) is completely retained by the column at low ionic strength while the other component (peak I) passes through. 2. In contrast to the enzyme in peak I, the enzyme in peak II is extremely sensitive to inhibition by leupeptin, it will hydrolyze carbobenzoxy-alanylarginylarginyl-4-methoxy-2-naphthylamine, and it will inactivate aldolase. 3. There appears to be also a minor high molecular weight component of the alpha-N-benzoyl-DL-arginyl-2-naphthylamine-hydrolyzing activity that is retained by the DEAE-cellulose but which has properties similar to those of the peak I enzyme.

Animals↗
PubMed1975

pH effects in plasmin-catalysed hydrolysis of alpha-N-benzoyl-L-arginine compounds.

The steady-state kinetics of plasmin (EC 3.4.21.7) catalysed reactions with some alpha-N-benzoyl-L-arginine compounds is investigated in the pH range 5.8--9.0. The results are interpreted in terms of a three-step mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. Alpha-N-Benzoyl-L-arginine methyl ester and ethyl ester show the same pH behaviour. The kinetic parameter kc/Km is influenced by two groups with pK values of 6.5 and 8.4, respectively. kc is affected only by the group with pK equal to 6.5 and Km only by the group with pK equal to 8.4. It is suggested that the group with pK equal to 6.5 is the 1-chloro-3-tosyl-amido-7-amino-2-heptanone-sensitive histidine residue in the active site and that the group with pK equal to 8.4 is perhaps the alpha-amino group of the N-terminus in analogy to trypsin and chymotrypsin. alpha-N-Benzoyl-L-arginine amide is not hydrolysed by plasmin, but proves to be a competitive inhibitor, Ki = 12.8 +/- 1.8 mM, pH = 7.8. Also the product alpha-N-benzoyl-L-arginine is a competitive inhibitor, Ki = 26 +/- 3.1 mM, pH = 7.8. Estimates of individual rate constants are compared with similar trypsin data.

Arginine↗
PubMed1979

Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum.

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.

Animals↗
PubMed1984

The combined action of two enzymes in human serum can mimic the activity of cathepsin B.

Synthetic substrates are often used to measure the activity of proteolytic enzymes. We have investigated the activities which cleave synthetic substrates such as alpha-N-benzyloxycarbonyl-Arg-Arg-beta-naphthylamide, for which the lysosomal proteinase cathepsin B has a high affinity, in sera from normal individuals, pregnant women and patients with breast cancer. As reported by other workers, activities against these substrates were elevated during pregnancy. Naphthylamine release, however, was shown to be the result of the combined action of two enzymes. The substrate is first cleaved by an endopeptidase to yield alpha-N-benzyloxycarbonyl-Arg and the aminopeptidase substrate Arg-beta-naphthylamide, which is then cleaved by serum aminopeptidases, particularly oxytocinase. A similar mechanism of cleavage was also found in the sera of breast cancer patients, where the endopeptidase catalyzing the first reaction was characterized as plasma kallikrein and the second reaction was carried out by serum leucine aminopeptidase. In no serum sample was there evidence for true cathepsin B activity.

Aminopeptidases↗
PubMed1989

Giardia lamblia: characterization of proteinase activity in trophozoites.

The proteinase activity of Giardia lamblia trophozoites, Portland 1 strain, was characterized with respect to substrate specificities and inhibitor sensitivities. Proteinase activity with urea-denatured hemoglobin (UDH), alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA), and alpha-N-benzoyl-argininamide (BAA) as substrates exhibited pH optima of 5.8, 3.8, and 5.0, respectively. For BANA, the apparent Km was 0.20 mM and the Vmax was 2.56 microM. For BAA, the apparent Km was 4.0 mM and the Vmax was 8.69 microM. Dithiothreitol (DTT, 5 mM) enhanced proteinase activity threefold for UDH, fourfold for BAA, and fivefold for BANA. Iodoacetamide, L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), each at 1 mM, inhibited proteinase activity by greater than 90% with BANA and BAA. Iodoacetamide inhibited proteinase activity by 35% with UDH; TPCK and TLCK inhibited activity greater than 70% with UDH. Activity on BAA was inhibited by 91% with Zn2+ and activity on UDH was inhibited by 30% with Cu2+. Virtually complete inhibition of proteinase activity on BANA and BAA was obtained with leupeptin and chymostatin at 1 microgram/ml. Pepstatin A, chelators, and other heavy metals had no apparent effect on proteinase activity. Two polypeptide bands (ca. 105 and 40 kDa) indicative of proteinase activity were visualized by sodium dodecyl sulfate-gelatin polyacrylamide gel electrophoresis. The 105 kDa band was visible over the pH range of 4 to 7, but with greater intensity from pH 5 to 7. The 40 kDa band, while present at pH 5, was most intense at pH 6 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Tryptic- and chymotryptic-like proteinases in early and late preimplantation mouse blastocysts.

Neutral tryptic- and chymotryptic-like enzyme activities have been identified in extracts of early and late mouse blastocyts. The enzyme activities are distinguishable my means of: (a) reactivity with specific naphthylamide derivatives; (b) reactivity with tosyl-L-lysine-chloromethyl ketone or L-tosylamido-2-phenylethyl-chloromethyl ketone, respectively; (c) electrophoretic mobilities; and (d) specific activity profiles of late vs. early blastocysts.

Animals↗