Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Some properties (molecular weight, pI, temperature stability, action of selected inhibitors, substrate specificity and pH-activity dependence) of two not yet known cathepsins from rat liver lysosomes are compared with the properties of the known cathepsin B1. Cathepsin L is a thiolproteinase, has a molecular weight of 23--24000 and a pI of 5,8--6,1. By disc electrophoresis and isoelectric focusing there appear several protein bands which all have enzymatic activity. Leupeptin behaves as a strong inhibitor. The pH-optimum for digestion of proteins is close to 5,0. Cathepsin L does not hydrolyse esters and splits synthetic low molecular substrates only to a low degree. Cathepsin L stored in presence of glutathion and EDTA in liquid nitrogen kept its activity for some months. Cathepsin H is an aminopeptidase as well as an endopeptidase. An enzyme with these bifunctional properties was detected up to now only in E. coli but not in animal cells. Cathepsin H is a thiol-enzyme with a molecular weight of 28000 and a pI of 7,1. Strong inhibitors are leucyl-chlormethan and SH-blocking substances. Leupeptin shows only a weak inhibitory effect to this enzyme compared to its action on cathepsins L and B1. The pH-optimum for hydrolysis of all substrates is 6.0. Cathepsin H splits proteins, amino acid derivatives and selected N-protected amino acid derivatives. Cathepsin H compared to cathepsin L and B1 is quite temperature stable.
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PMID:[Intracellular protein breakdown. VII. Cathepsin L and H; two new proteinases from rat liver lysosomes]. 0 66

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex. 2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8-6.1 ascertained by analytical isoelectric focusing. 3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6-7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30-40% at pH 7.0. 5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor. 6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.
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PMID:Cathepsin L. A new proteinase from rat-liver lysosomes. 1 35

The activation of procollagenase and prostromelysin by mechanisms that might be functional in vivo has been investigated. Studies with cell monolayers plated onto collagen films have indicated key roles for plasmin and TIMP in these processes. Prostromelysin activation could be rapidly effected by fibroblast monolayers in the presence of plasminogen, with identical kinetics to plasminogen-streptokinase generated plasmin. Procollagenase activation by plasmin was shown to be poor, although an M(r) shift of 11,000 occurred. Activation was enhanced ten-fold by the presence of active stromelysin even at a very low molar ratio. A tumour cell line secreting procollagenase but not stromelysin was found to be dependent upon the addition of both stromelysin and plasminogen to effect degradation of collagen films. Biochemical studies of metalloproteinase activation were carried out using other purified proteinases synthesized by connective tissue cells including endopeptidase 24.11, endopeptidase-2, cathepsin B and cathepsin L. None was a particularly effective activator relative to plasmin, but cathepsin B was shown to activate stromelysin. By use of both cell model systems and biochemical studies of purified enzymes we have found that the role of plasmin as the major metalloproteinase activator in normal connective tissue cells remains unchallenged.
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PMID:Physiological mechanisms for metalloproteinase activation. 148 31

Phe5(4-nitro)-bradykinin has been identified as a good synthetic substrate to study the kinetics and mechanism of action of the metalloendopeptidase meprin. No convenient substrate for kinetic analysis of the enzyme had been previously described. HPLC analyses indicated that meprin cleaved bradykinin and nitrobradykinin between Phe5 (or Phe5(NO2)) and Ser6. Reaction rates for bradykinin were determined by quantitative HPLC analyses, whereas rates for nitrobradykinin were measured by continuous monitoring of the spectral change that occurs at 310 nm when the Phe(NO2)-Ser bond is hydrolyzed. For nitrobradykinin and unmodified bradykinin, respectively, Km values were 281 and 425 microM, kcat values were 28 and 22 s-1, and kcat/Km values were 9.7 x 10(4) and 5.1 x 10(4)M-1. The two products of bradykinin hydrolysis were not substrates for the enzyme, but they were inhibitors. The initial rates of hydrolysis of nitrobradykinin increased linearly with enzyme concentration (0.09-2.2 micrograms/ml), and increased linearly with temperature in the range from 15 to 55 degrees C. Hydrolysis of the substrate was optimal at alkaline pH values. The cysteine endopeptidases papain and cathepsin L and the metalloproteases thermolysin, angiotensin-converting enzyme, and neutral endopeptidase (EC 3.4.24.11) also cleaved nitrobradykinin, but at different peptide bonds than meprin. The single cleavage of nitrobradykinin at the Phe(NO2)-Ser bond and the concomitant spectral shift that occurs at alkaline pH makes this a particularly suitable substrate for meprin.
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PMID:Phe5(4-nitro)-bradykinin: a chromogenic substrate for assay and kinetics of the metalloendopeptidase meprin. 196 66

Limited proteolysis of T-kininogen by heterologous and homologous endopeptidases (bovine trypsin, human leukocyte elastase, rat submaxillary gland endopeptidase k, and rat mast cell chymase) produced similar fragmentation. Amino-terminal sequence analysis of whole T-kininogen lysates and purified proteolytic fragments identified four susceptible regions which contained all the preferential cleavage sites for these proteinases. Two of these susceptible regions were close to the junction between heavy chain cystatin-like domains, the third was in the kinin-containing region, and the fourth was close to the carboxyl terminus of the T-kininogen light chain. There was only one primary site for each proteinase in the kinin-containing region, which explains why catalytic amounts of these proteinases did not release immunoreactive kinin from this kininogen. However, preferential cleavage of T-kininogen close to the junction between cystatin-like domains released fragments which, provided they included cystatin-like domains 2 and/or 3, strongly inhibited papain and cathepsin L. The fragments were inhibitory even when parts of the amino-terminal ends of the domains were lacking. The highly conserved glycyl residue, thought to be involved in the inhibitory reactive site of cystatin-like inhibitors, was not required in purified domain 3 for inhibition of cathepsin L.
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PMID:Limited proteolysis of T-kininogen (thiostatin). Release of comparable fragments by different endopeptidases. 264 33

Previous attempts to liberate T kinin from T kininogen [Moreau et al. (1986) Eur. J. Biochem. 159, 341-346; Gutman et al. (1988) Eur. J. Biochem. 171, 577-582] have shown that complete fragmentation of the precursor molecule into inhibitory peptides was achieved before any vasoactive peptide was released, suggesting a possible physiological significance for this phenomenon. In this study, cysteine-proteinase-inhibiting properties of rat T kininogen and of its proteolytic fragments issuing from trypsin and submaxillary gland endopeptidase k hydrolysis, have been investigated using rat lysosomal cathepsins B, H and L, papain and bovine calpains I and II. All three lysosomal cathepsins were inhibited by T kininogen but tighter interactions were observed with cathepsin L and papain. Though higher Ki values were obtained for cathepsins B and H, rate constants for association were found to have high and almost similar values (in the 10(6) M-1 s-1 range) whatever the enzyme used. Proteolytic fragments also inhibited cathepsin L and papain very strongly and even better than the entire molecule for some of them, but no significant inhibition of cathepsins B and H was observed. Bovine calpains were not inhibited by T kininogen nor by its proteolytic fragments. From the results of this kinetic analysis, which indicates that both the association and the dissociation of lysosomal cysteine proteinases with T kininogen may occur rapidly, an hypothesis has been put forward on the possible in vivo functioning of T kininogen as a proteinase inhibitor.
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PMID:Cysteine-proteinase-inhibiting function of T kininogen and of its proteolytic fragments. 335 89

The distribution of proteases potentially reactive with peptide sequences containing pairs of basic amino acids or single basic amino acids was studied in subcellular fractions of a transplantable rat insulinoma using the affinity probes 125I-Tyr-Ala-Lys- ArgCH2Cl and 125I-Tyr-Ala-norleucine- ArgCH2Cl . Both probes labeled predominantly proteins of Mr = 39,000, 31,500, and 25,000. The Mr = 25,000 component appeared to be of lysosomal origin, while the Mr = 39,000 and 31,500 proteins were present in both the lysosomes and insulin granules. The Mr = 39,000 and 31,500 proteins were identified as precursor/product forms of the cysteine protease cathepsin B, while assays performed with fluorigenic peptide substrates suggested that the Mr = 25,000 protein was probably cathepsin L and/or H. The greater reactivity of the Mr = 39,000 form with the dibasic probe suggests that the relative proportions of the Mr = 39,000 and 31,500 forms of cathepsin B in different organelles may determine the extent to which the enzyme expresses activity as a specific (prohormone processing) endopeptidase or a more general (degradative) peptidase.
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PMID:Cathepsin B-related proteases in the insulin secretory granule. 632 60

The action of two earlier isolated highly purified spleen thiol proteinases on angiotensins I and II, bradykinin and kallidin was investigated. It was demonstrated that proteinase I which is apparently cathepsin L from bovine spleen brings about rapid inactivation of angiotensin II with a splitting of the Tyr-Ile bond and a formation of two tetrapeptides. Proteinases I also split angiotensin I. Proteinase I partially inactivates bradykinin and kallidin by splitting the Gly4-Phe5 bond. The activity of proteinase I toward angiotensin II is about 50 times higher than that toward bradykinin. The corresponding values of Km and V are 7.5 X 10(-5) M and 10.0 mumole/min/mg. The possible role of proteinase I in angiotensin II inactivation under physiological conditions is discussed. Proteinase II converts kallidin to bradykinin by splitting off the N-terminal lysine. Proteinase II causes partial inactivation of bradykinin by splitting of the Gly4-Phe5 and Phe5-Ser6 bonds of this peptide. Proteinase II possesses both aminopeptidase and endopeptidase activities and is therefore cathepsin H from spleen. Proteinase II does not split either angiotensin I or angiotensin II.
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PMID:[Action of two thiol proteinases from the spleen which are active in neutral media on vasoactive peptides]. 634 1

Plasma membranes (PM) were prepared from nuclear and microsomal fractions of rat liver as described in the preceding paper. Four different proteolytic activities were studied and found to be solubilized from PM fractions after detergent treatment: endopeptidase activity at neutral and acid pH, dipeptidyl-aminopeptidase IV, and (alanine-)aminopeptidase. Both PM preparations contain a serine-endopeptidase with optimal activity against azocasein at pH 7.6. After solubilization of PM derived from microsomal fractions (PM-m) and gel filtration this activity shows an apparent molecular mass of 220 +/- 20 kD. This membrane proteinase is different from other known serine proteinases of liver cells because of its large molecular mass and an activating effect of 1,10-phenanthroline. PM derived from microsomal fractions contain additional endopeptidase with a pH optimum of 5.2 that could be inhibited by 4-chloromercuribenzoate, leupeptin and Z-Phe-Ala-diazomethylketone. Using detergent solubilization and gel filtration this acid endopeptidase activity in PM-m can be resolved into three peaks with apparent molecular masses (detergent forms) of 180 +/- 10, 80 +/- 10 and 35 +/- 10 kD, the latter may be cathepsin L. In addition to the endopeptidases, PM-m were shown to contain also aminopeptidases degrading Ala-Pro-pNA and Ala-pNA. The former activity, dipeptidyl-aminopeptidase IV (DPP IV), has features similar to DPP IV from other microvillus membranes (inhibition by DIFP and PMSF, pH optimum at 7.7). The Ala-pNA degrading aminopeptidase is inhibited by chelating agents and some bivalent heavy metal ions, but is activated by Co++-ions. Both enzymes apparently were eluted as monomers (molecular mass 180-190 kD) after gel filtration in detergent containing buffers.
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PMID:Proteolytic activities in plasma membrane preparations from rat liver. 2. Partial purification and characterization of membrane bound endopeptidases, dipeptidyl-aminopeptidase IV and aminopeptidase. 636 Jan 64

Intraperitoneal administration of leupeptin to rats induced a hemoglobin-hydrolyzing protease which was most active at pH 3.5 and was insensitive to pepstatin in various tissues such as the liver, kidney, and muscle, as observed previously in adult rat hepatocytes in primary culture (Tanaka, K., Ikegaki, N., and Ichihara, A. (1979) Biochem. Biophys. Res. Commun. 91, 102-107). The induced acidic protease was purified about 600-fold in 30% yield from rat liver by conventional chromatographic techniques. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate and was a monomeric protein of Mr = 20,000. The enzyme appeared to be a glycoprotein because its induction was blocked by the addition of tunicamycin to cultures of hepatocytes and because the induced protease was absorbed on concanavalin A-Sepharose and eluted with methylglucoside. It seemed to be present in lysosomes and was fairly stable at various pH values and temperatures. It showed endopeptidase activity on various protein substrates, but scarcely hydrolyzed N-substituted derivatives of arginine. It did not hydrolyze esters, showed no aminopeptidase or carboxypeptidase activity, and did not inactivate glucose-6-phosphate dehydrogenase or aldolase. The enzyme appeared to be a thiol protease, since it was strongly inhibited by sulfhydryl-reactive compounds and N-( [N-(1-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine and was not inhibited by reagents specific for carboxyl-, serine-, or metalloproteases. This induced protease could be separated from cathepsins B, D, and H by chromatography. The enzyme was similar to cathepsin L in chromatographic behavior, Mr and pI, but differed from the latter in stability and in its inability to inactivate some enzymes. These results suggest that it differs from any known proteases found previously in rat liver.
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PMID:Purification and characterization of hemoglobin-hydrolyzing acidic thiol protease induced by leupeptin in rat liver. 637 Oct 12


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