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)

A multicatalytic endopeptidase (ME) with three distinct activities, chymotrypsin-like, cucumisin-like, and trypsin-like, occurred in all rat tissues examined with highest activities in kidney, testes, liver, and spleen; they were assayed with benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide (Z-Gly-Gly-Leu-pNA), benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Z-Leu-Leu-Glu-2NA), and benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide (Z-Gly-Gly-Arg-2NA), respectively. All three activities were recovered from a single protein band on a polyacrylamide gel after electrophoresis of purified human kidney ME. The native enzyme had a Mr of 650,000, and it consisted of about 5,135 amino acid residues. After denaturation and electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels kidney ME dissociated into several low Mr components ranging from 23,000 to 33,000. Kidney ME had a pH optimum of 7.6-8.1 with Z-Gly-Gly-Leu-pNA, 7.3 with Z-Leu-Leu-Glu-2NA, and 9.8 with Z-Gly-Gly-Arg-2NA. SDS enhanced chymotrypsin- and cucumisin-like activities by two to three times whereas trypsin-like activity was not enhanced. The specificity constant (kappa cat/Km) of human kidney ME for Z-Gly-Gly-Leu-pNA was 6.7 X 10(3) M-1 S-1; Z-Gly-Gly-Leu-2NA was not hydrolyzed. The specificity constant for Z-Leu-Leu-Glu-2NA was similar to, and for Z-Gly-Gly-Arg-2NA was one half of that for Z-Gly-Gly-Leu-pNA. ME cleaves only the Phe5-Ser6 bond of bradykinin (BK); however, all three ME activities were inhibited by BK. Strong inhibition of ME by albumin suggests that ME is involved in cleavage of larger polypeptides. Antipain and leupeptin almost completely inactivated the trypsin-like activity whereas they had no significant effect on the other two activities. ME is not a metal-loenzyme nor is the serine residue essential for its activities; however, thiol groups are involved. Na+ and K+ inhibited all ME activities. Trypsin-like activity was more sensitive to divalent cations than the other two.
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PMID:A multicatalytic high-molecular-weight neutral endopeptidase from human kidney. 331 Sep 3

The activities of the lysosomal endopeptidase cathepsin B (cath B; CZB-Ala-Arg-Arg-MNA as substrate) and the lysosomal exopeptidase dipeptidylpeptidase II (DAP II; Lys-Ala-2NA as substrate) were fluorometrically determined in the renal homogenate of normal and experimental (castration followed by a 14-day treatment with estradiol and testosterone) rats of both sexes. In addition, methodological investigations of the renal homogenate were performed in order to differentiate cath B from other proteinases. These showed that cath-B activity was highest at around pH 6, was strongly inhibited by 4-hydroxymercuribenzoate and leupeptin, and was activated by dithiothreitol. Trypsin-like activities were not demonstrable under the used incubation conditions. The animal experiments showed that renal cath-B activities (1) were significantly higher in females than in males, (2) increased significantly in males and decreased significantly in females after castration (no significant difference between both sexes), (3) decreased in female and male castrates after treatment with testosterone and increased strongly after treatment with estradiol, and (4) showed an activity pattern similar to that of DAP II. The results are discussed in relation to the sex-dependent and sex-hormone-dependent proteinuria of rats. It is suggested that there is a correlation between protein catabolism in the kidney and proteinuria, i.e. high lysosomal proteinase activities correspond with low proteinuria.
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PMID:Renal cathepsin-B activities in rats after castration and treatment with sex hormones. 374 98

A mouse alpha-macroglobulin (AMG), a homologue of human alpha 2-macroglobulin (alpha 2 M), has been purified to homogeneity. In contrast to human and acute-phase rat alpha 2 M which contains subunits of about Mr 190 000, the mouse protein contains two major (Mr 163000 and 35000) and one minor (Mr 185000) subunits. Also unlike human alpha 2 M, which can be broken down into about 85000-dalton subunits when reacted with an endopeptidase, the native AMG is cleaved by trypsin into multiple components (Mr 86000, 63000, 61000 and 33000). Two-dimensional peptide map analysis of these various 125I-labeled subunit components reveals that the 185000- and 163000-dalton components are homologous proteins but only the 185000-dalton protein contains the 35000-dalton component. The 163000-dalton protein is cleaved by trypsin into 86000- and 63000-dalton components, and the 86-kDa component in turn can be broken down into 61000- and 33000-dalton fragments. Since the 35000-dalton component is serologically related to AMG but does not share any tryptic peptides with both the 163000- and 33000-dalton components, it is neither a copurified impurity nor a cleavage product of the major (163000-dalton) subunit. AMG, therefore, is composed of covalently linked subunits of Mr 163000 and 35000, and the 185000-dalton protein may be a variant subunit of AMG. Trypsin treatment of the [14C]methylamine-labeled AMG and alpha 2 M also sequentially generate subunit patterns indistinguishable from those of the unlabeled macroglobulins. The methylamine-sensitive site(s) of AMG is localized in the 63000-dalton peptide, which is rather resistant to trypsin digestion and to staining by Coomassie brillant blue. We conclude from this study that the mouse homologue has a subunit composition and primary structure distinctly different from those of human and rat alpha 2 M.
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PMID:Subunit and primary structure of a mouse alpha-macroglobulin, a human alpha 2-macroglobulin homologue. 617 45

An enzyme capable of converting putative opioid peptide intermediates to free enkephalin has been purified 300-fold from washed rat brain membranes. The action of this enzyme, an enkephalin-generating endopeptidase (EGE), was compared with the action of carboxypeptidase B after trypsin treatment on enkephalin precursor peptides present in rat striata. After Sephadex G-100 gel filtration of striatal material, fractions were radioimmunoassayed for enkephalin content using an antiserum specific for the carboxyl terminal of enkephalin. Additionally, aliquots of the column fractions were treated with either trypsin and carboxypeptidase B, trypsin and EGE, or EGE alone. The peak of enkephalin immunoreactivity increased with the enzymes' treatment indicating the conversion of the low molecular weight proenkephalin precursor peptides to enkephalin. Trypsin and EGE generated almost as much enkephalin as trypsin and carboxypeptidase B in the conditions of the experiment. Thus EGE is capable of processing precursors to enkephalin after the action of trypsin-like enzyme(s) in the brain. The gel filtration fractions containing enkephalin and its low molecular weight precursors were pooled and one-half treated with EGE. The contents were analyzed by HPLC and the increase in immunoreactivity co-eluted with enkephalin and Leu-enkephalin. Small peptides found to be the most potent competitive inhibitors of this enzyme are Met-Arg-Phe-Ala, and Met-Arg-Phe.
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PMID:Brain endopeptidase generates enkephalin from striatal precursors. 675 May 68

1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme--inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15--30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7--7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.
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PMID:The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan. 701 17

The hydrolytic cleavage of a cyanine (Cy3)-labeled angiotensin, catalyzed by various proteases, was studied by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). The end-labeled peptides and the Cy3 diacid internal standard were separated on a 20-microns x 27-cm capillary with LIF detection (emission, 580 nm) using a frequency-doubled solid-state diode laser emitting at 532 nm or a He-Ne laser emitting at 543 nm. Hydrolysis of the Cy3-labeled angiotensin I, catalyzed by proteinase K, is a sequential process beginning from the C-terminal of the peptide, instead of from random cleavages. Trypsin catalyzes a specific cleavage of Cy3-angiotensin I to Cy3-Asp-Arg as anticipated. Using a combination of endopeptidase and carboxypeptidases, the remnant of the labeled species was characterized by CE-LIF. The method provides a general tool for studying the mechanism of protease-catalyzed hydrolysis of peptide.
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PMID:Characterization of protease-catalyzed hydrolysis of cyanine-labeled angiotensin using capillary electrophoresis with laser-induced fluorescence detection. 776 1

A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1 alpha/alpha genotype (high meprin, expressing meprin-alpha and meprin-beta ) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-beta ). Western blotting with antisera specific to the meprin-alpha and the meprin-beta subunits established that the only form of meprin present in urine samples was derived from meprin-alpha. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N-terminus of the larger protein band begins with the pro-peptide sequence of the alpha-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N-terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X-I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms.
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PMID:Characterization of the soluble, secreted form of urinary meprin. 861 15

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm of Lactococcus lactis ssp. lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and alpha-chymotryptic hydrolysates of beta-casein (beta-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin released beta-CN f193-209, kinetic constant (Ki) of which for inhibition of PepO was 60 microM. This peptide also inhibited (Ki = 1700 microM) the 95-kDa aminopeptidase (PepN) from L. lactis ssp. lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one, beta-CN f69-97, was not degradable by PepO (Ki = 4.7 microM), while the other, beta-CN f141-163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide, beta-CN f69-84, which inhibited PepO with a Ki of 8.1 microM, was isolated from the alpha-chymotryptic hydrolysate. Peptides released from beta-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degraded beta-CN f193-209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (beta-CN f193-209, f69-97, f69-84, f141-163) were readily degraded by thermolysin.
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PMID:Peptides inhibitory to endopeptidase and aminopeptidase from Lactococcus lactis ssp. lactis MG1363, released from bovine beta-casein by chymosin, trypsin or chymotrypsin. 863 36

A novel assay method based on the endopeptidase activities of the botulinum neurotoxins has been developed and applied to the detection of botulinum type A and B toxins. An assay system developed for the detection of botulinum type B neurotoxin (BoNT/B) is based on the cleavage of a synthetic peptide substrate representing amino acid residues 60 to 94 of the intracellular target protein for the toxin, VAMP (vesicle-associated membrane protein, or synaptobrevin). In this assay system, immobilized VAMP (60-94) peptide substrate is cleaved by BoNT/B at the Gln-76-Phe-77 bond, leaving the C-terminal cleavage fragment on the solid phase. This fragment is then detected by the addition of an antibody-enzyme reagent which specifically recognizes the newly exposed N terminus of the cleavage product. The developed assay was specific to BoNT/B, showing no cross-reactivity with other clostridial neurotoxins, and had a sensitivity for BoNT/B of 0.6 to 4.5 ng/ml, which could be increased to 0.1 to 0.2 ng/ml by using an assay amplification system based on catalyzed reporter deposition. Trypsin treatment of BoNT/B samples, which converts the single-chain toxin to the active di-chain form, was found to increase the sensitivity of the endopeptidase assay from 5- to 10-fold. An endopeptidase assay for BoNT/A, based on the cleavage of a peptide substrate derived from the protein SNAP-25 (synaptosome-associated protein), was also developed and characterized.
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PMID:Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities. 881 85

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.
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PMID:Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion. 959 72


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