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)

Bence Jones proteins can be cleaved specifically by several types of endopeptidases into fragments corresponding to the amino-terminal, variant (VL) portion and to the carboxyl-terminal, constant (CL) portion of the light polypeptide chain. Two types of neutral proteases, designated elastase-like (ELP) and chymotrypsin-like (CLP), have been isolated and purified from human polymorphonuclear leukocytes. Because these proteases have defined proteolytic activity under physiologic conditions for several types of human proteins, we investigated their effect on human Bence Jones proteins. Incubation of kappa-type or lambda-type Bence Jones proteins with ELP or CLP under appropriate conditions resulted in cleavage of both types of light chains as evident by immunochemical and electrophoretic analyses. Treatment with ELP or CLP of one kappa Bence Jones protein resulted in the formation of a single component that had antigenic and electrophoretic properties similar to the VL fragment derived from pepsin digestion of the native protein. No component corresponding to the CL could be detected immunochemically or electrophoretically. Studies of isolated pepsin-labile (37 degrees C) and pepsin-stable (55 degrees C) CL fragments demonstrated the marked susceptibility of the carboxyl-terminal half of the light chain to proteolysis by the leukocyte-derived neutral proteases. Incubation with ELP of three other kappa Bence Jones proteins and three reduced-alkylated lambda Bence Jones proteins resulted, in each case, in the formation of a homogeneous component which was electrophoretically and immunochemically distinct from the pepsin-derived VL fragment. An identical component could also be formed by incubating a pepsin-derived VL fragment with ELP. In the ELP-treated samples, no CL-related material was detected electrophoretically or immunochemically with antisera possessing specificity for CL antigenic determinants present on the unfolded light polypeptide chain or on the isolated CL. The component formed by ELP or CLP treatment of certain Bence Jones proteins thus appears to be VL-related, but lacks the idiotypic antigenic determinant present on the native protein. In this respect, these neutral protease-derived light chain components are similar to the amyloid-like VL fragments generated in vitro from certain endopeptidase-treated Bence Jones proteins.
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PMID:Bence Jones proteins and light chains of immunoglobulins. XIII. Effect of elastase-like and chymotrypsin-like neutral proteases derived from human granulocytes on Bence Jones proteins. 6 Apr 45

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.
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PMID:Protease II from Escherichia coli. Purification and characterization. 24 Aug 39

Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000, suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea. In agreement with previous evidence of a chymotrypsin-like specificity, hydrolytic assays of various p-nitrophenyl esters of N-substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The Km(app) for N-benzoyloxycarbonyl-L-tyrosin-p-nitrophenyl ester at pH 7.5 In 100 mM sodium phosphate buffer at 25 degrees C was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N-acetyl-L-phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 degrees C, it cleaves the carboxymethylated B-chain of bovine insulin at four points: Phe25-Tyr26, Phe24-Phe25, Leu15-Tyr16 and Ser9-His10. From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.
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PMID:Protease I from Escherichia coli. Some physicochemical properties and substrate specificity. 79 43

1. We have fractionated the bradykinin inactivating activity of human urine by stepwise elution chromatography on DEAE-cellulose and recovered 95% of the inactivating activity and 29% of the protein (absorbance at A280 nm). 2. Seven of nine fractions which presented activity were also tested for angiotensin I and II inactivating activity, angiotensin converting activity and for the hydrolysis of hippuryl-His-Leu and hippuryl-Arg. Sites of hydrolysis in bradykinin were determined by HPLC of the hydrolysates and fragments were compared with authentic peptides. 3. Cleavage sites demonstrated for Fractions A through G were: Phe8-Arg9 (A and B), Phe5-Ser6 (C and F), Pro7-Phe8 (D), Gly4-Phe5 and Pro7-Phe8 (E) and Pro3-Gly4 (G). 4. The relative molecular weight of the bradykininase activity present in each fraction, determined by gel filtration, was: 16 kDa (A), 70 kDa (B), 60 kDa (C), 88 kDa (D), 230 kDa (E), 45 kDa (F) and 49 kDa (G). 5. Bradykinin inactivating activity was inhibited 50-100% by 3 mMEDTA (A, B, D, E and G), 1 mMM 2-mercaptoethanol (A, B, C and G), 0.1 microM Hg2+ (A, C and G), 0.1 mM PMSF (C and F), 1 mM TPCK (C and F), 1 mM Zn2+ (C), 60 microM BPP5a and 40 microM BPP9a (D), 0.1 microM phosphoramidon (E) and 3 mM sodium p-hydroxymercuribenzoate (G). 6. The properties of some of these bradykinin inactivating activities correspond to enzymes previously described in urine and tissues: carboxypeptidases (Fractions A and B), angiotensin I converting enzyme (Fraction D), neutral endopeptidase (Fraction E). However, the chymotrypsin-like activity of Fractions C and F and the prolylendopeptidase activity of Fraction G have not been described before in urine and they are being purified in order to obtain a more accurate characterization.
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PMID:Endopeptidase and carboxypeptidase activities in human urine which hydrolyze bradykinin. 134 17

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)-inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2-terminal end of the synthetic peptide Ala-Ala-Phe-p-nitroanilide (pNA) and was not inhibited by inhibitors of metallo-, cysteic-, and aspartic-proteinases, or by those of elastase-, trypsin- and chymotrypsin-like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly-Pro from Gly-Pro-pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N-exo-aminopeptidase activities specifically inhibited by bestatin, was also detected when Ala-, Leu-, Arg- and Lys-pNA were used a substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha and interferon-gamma was next examined. The results indicated that N-aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF-alpha degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17-kDa molecule TNF-alpha, and the concomitant release of biologically inactive fragments of less than or equal to 2 kDa. Together, these observations indicate new roles for both the DPP IV-like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF-alpha concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.
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PMID:Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha. 134 32

1. Lobster muscles contain a latent multicatalytic proteinase; heating at 60 degrees C for 1-2 min converts the latent form to a heat-activated form with enhanced proteolytic activity. Both forms have three endopeptidase activities, which are classified as the trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide bond hydrolyzing activities. 2. Sulfhydryl reagents (mersalyl acid, N-ethylmaleimide, hemin, iodoacetamide, and p-chloromercurisulfonic acid), benzamidine, and chloromethyl ketones inhibited all three activities of the heat-activated form. Leupeptin and antipain inhibited only the trypsin-like activity, while the chymotrypsin-like activity was the most sensitive to diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor. Pepstatin and L-trans-epoxysuccinylpeptides had little effect on the peptidase activities. 3. Sodium dodecyl sulfate and oleic acid preferentially activated the peptidylglutamyl-peptide hydrolyzing activity of the latent form, whereas N-ethylmaleimide stimulated both the trypsin-like and peptidylglutamyl-peptide hydrolases. These results suggest that the lobster enzyme is an atypical serine proteinase.
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PMID:Differential effects of oleic acid, sodium dodecyl sulfate, and protease inhibitors on the endopeptidase activities of the lobster multicatalytic proteinase. 176 21

Histochemical analysis of some lysosomal and sarcoplasmic proteolytic enzymes was assayed in human myocardial biopsies taken from 26 cardiopathic patients subjected to open heart operations, under extracorporeal circulation and protection with cardioplegic solution and hypothermia. The investigated myocardial proteases were: cathepsin B, cysteine aminopeptidase, acid gelatinases, trypsin-like endopeptidase, chymotrypsin-like endopeptidase and neutral gelatinases. The effects of surgical interventions appreciated by comparing the myocardium fragments harvested before, and at various intervals after aorta clamping (6-90 minutes) revealed disorders in the activity and compartmentalization of all the investigated proteases, whose histochemical reactions increased between 10 and 20 minutes after aorta clamping and manifested a lowering tendency with sarcoplasmic diffusion and extracellular release at longer periods than 20 minutes. The early activation of the neutral proteases and their sarcolemmal expression even before 10 minutes after aorta clamping, suggested the involvement of the nonlysosomal proteases in the first proteolytic events implied in the molecular membrane damage of the myocardial fibre. Sequential proteolytic cascades of abnormal neutral and acid proteases were emphasized as possible mediators and effectors of molecular and subcellular damages suffered by the myocardial fibers during the open heart operations, even under cardioplegic and hypothermic protection.
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PMID:Histochemical reactions of myocardial proteases during open heart surgery. 252 27

Parathyroid hormone (PTH) -degrading activity was studied using osteoblast-like UMR-106 cells. PTH-degrading activity was assessed by the amount of PTH fragments produced in the medium after exposure of intact human PTH-(1-84) to UMR-106 cells. PTH immunoreactivity recovered in trichloroacetic acid-soluble products of the medium and in fractions eluted from reverse-phase high-performance liquid chromatography (HPLC) was measured by radioimmunoassay using an antibody specific for the mid-region and C-terminus of PTH. In this study, intact UMR-106 cells but not extracellular enzymes cleaved human PTH(1-84) into fragments which were released into the medium (in a time- and temperature-dependent fashion). HPLC analysis of the PTH fragments depicted three immunoreactive peaks (peaks 1, 2 and 3) besides intact PTH, indicating a limited PTH-hydrolyzing activity of the cells. Furthermore, a 1000-fold molar excess of either hPTH-(3-34) or [Nle8,Nle18,Tyr34]hPTH-(3-34)amide inhibited PTH-degrading activity by 63% and 80% of control, respectively, whereas neither calcitonin, vasopressin nor growth hormone suppressed it. Additionally, HPLC analysis of the samples treated with [Nle8,Nle18,Tyr34]hPTH-(3-34)amide showed a reduction of the three peaks, suggesting an involvement of PTH receptor in the production of PTH fragments. This PTH-degrading activity was strongly inhibited by phenylmethylsulfonyl fluoride and chymostatin, but not by soybean trypsin inhibitor, elastatinal or inhibitors of cysteine, aspartic or metalloproteinases, indicating that it is due to a seryl chymotrypsin-like endopeptidase. Chymotrypsin-like activity seems to be solely responsible for PTH-degrading activity in intact UMR-106 cells, since all three PTH fragments were predominantly suppressed in the presence of chymostatin. Further analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by ultraviolet absorbance at 210 nm, three of which were measurable by PTH radioimmunoassay, each corresponding to the three PTH fragments produced by UMR-106 cells. To explore the cleavage sites of PTH further, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the view that the chymotrypsin-like enzyme in UMR-106 cells cleaved the hormone between residues 23-24 and 34-35, to produce, at least, hPTH-(24-84) and -(35-84). Our present study indicates that a chymotrypsin-like endopeptidase is solely responsible for limited hydrolysis of PTH by intact UMR-106 cells.
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PMID:Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the clonal osteogenic UMR-106 cell. 291 1

Cathepsin-D has been previously reported to cleave intact PTH into PTH-(1-34) and -(35-84) in membranous fractions of rat and bovine kidney. Whether PTH degradation occurs by intact kidney cells, however, has not been examined in detail. We have, therefore, examined this possibility using an opossum kidney (OK) cell line which possesses the characteristics of proximal renal tubules and responds to PTH. PTH radioimmunoreactivity recovered in trichloroacetic acid-soluble products and in fractions eluted from reverse phase HPLC was measured using an antibody directed to the midregion and C-terminus of PTH. In this study, intact OK cells, but not extracellular enzymes, cleaved human (h) PTH-(1-84) into three discrete fragments which were released into the medium in a time- and temperature-dependent fashion. Half-maximal velocity of PTH-degrading activity (PTHDA) was observed at 9 nM hPTH-(1-84). A 1000-fold molar excess of PTH antagonists [hPTH-(3-34) and [Tyr34]hPTH-(7-34)amide] markedly inhibited PTHDA, whereas ACTH, glucagon, or big gastrin did not suppress it, suggesting an involvement of the PTH receptor in PTHDA. This PTHDA was strongly inhibited by phenylmethylsulfonylfluoride and chymostatin, but not by trypsin inhibitor, elastatinal, or inhibitors of aspartic, cysteine, or metalloproteinases, suggesting that it is due to a seryl chymotrypsin-like endopeptidase. Analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by UV absorbance (210 nm), three of which were measurable by PTH RIA, and each corresponded to the three PTH fragments produced by OK cells. All three fragments were predominantly suppressed in the presence of chymostatin, suggesting that chymotrypsin-like activity is solely responsible for PTHDA in intact OK cells. To further explore the cleavage sites of PTH by chymotrypsin, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the conclusion that a chymotrypsin-like enzyme in OK cells cleaved the hormone between residues 23-24, and 34-35 to produce, at least, hPTH-(24-84) and -(35-84). Lysosomal blockers (chloroquine, ammonium chloride, or monensin) did not affect this PTHDA. Our present study indicates that chymotrypsin-like endopeptidase, but not other endopeptidase or lysosomal enzymes, is responsible for the limited hydrolysis of PTH by intact OK cells.
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PMID:Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the opossum kidney cell. 305 60

Rat liver cytosol has low hydrolytic activity against [3H]methylcasein at neutrality, but activity increases greatly on addition of various compounds such as poly-L-lysine, N-ethylmaleimide, and sodium dodecyl sulfate, suggesting that it contains latent proteolytic activity. The latent enzyme was found to be stabilized in the presence of 20% glycerol and to be activated by addition of poly-L-lysine. The latent enzyme was purified from a crude extract of rat liver to apparent homogeneity in the presence of 20% glycerol by conventional chromatographic techniques. The purified enzyme showed endoproteolytic activity toward various proteins when it was activated by the compounds listed above. It preferentially degraded N-substituted tripeptide substrates with a basic amino acid at the carboxyl terminus, as well as peptides containing neutral hydrophobic amino acids. It did not require activation for these peptidase activities, in contrast to its activity toward large proteins. Interestingly, a proteinase and a trypsin-like and a chymotrypsin-like peptidase activity could not be separated by customary chromatographic methods but were distinguishable by their sensitivities to various inhibitors, activators, and covalent modifiers, suggesting that the enzyme has three distinct active sites within a single protein. The enzyme seems to be a seryl endopeptidase showing maximal activity at neutral and weakly alkaline pH values. Thus, the enzyme is a unique protease with latent multifunctional catalytic sites. The distribution of the protease in soluble extracts of various rat tissues and cells was examined quantitatively by an enzyme immunoassay. The enzyme level was highest in liver and also in spleen, stomach, lung, small intestine, and kidney, but was low in heart, diaphragm, skeletal muscle, brain, and skin. The concentrations of enzyme in some established cell lines including hepatoma and rat kidney cells were comparable to that in normal liver hepatocytes. The enzyme was found mainly in the cytosol fraction, although a small amount was associated with microsomal membranes, suggesting that it is an extralysosomal protease. Immunohistochemical staining of the liver and skeletal muscles showed that the protease is distributed diffusely in panlobular hepatocytes with slight centrilobar predominance and is present in Kupffer cells, vascular endothelial cells, and bile duct epithelial cells in the liver and also diffusely in the intermyofibrillar spaces and vascular endothelial cells in skeletal muscle. The quantitative data obtained in the present study indicate the presence of the protease in the cytosol fraction of all rat tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution. 309 25


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