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

A simple method was developed for the isolation of the cationic endopeptidase from a crude extract prepared from the culture medium of Thermoactinomy ces vulgaris, consisting in chromatography on Sephadex G 75 and subsequent separation on CM-Sephadex in 50 mM Tris/HCl, pH 8, using a NaCl-gradient (0 - 0,2 M). This procedure results in a 4,2 fold increase of the elastolytic activity (substrate: N-acetyl-(L-ala)3methyl ester) of the enzyme. It moves as a single band in SDS-gel electrophoresis and crystallizes as needles up to 0,2 mm in length after standing at 5 degrees C in 50 mM Tris/HCl (pH 8,3).
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PMID:[Isolation, crystallization and partial characterization of a cationic protease from thermoactinomyces vulgaris]. 61 86

Induction of arthritis in rats with Freund's complete adjuvant was accompanied by a distinctive alteration of concanavalin A (Con-A) reactivity in their serum proteins in which the concentrations of selected Con-A reactive proteins were significantly higher when compared to healthy rats. To assess if the observed increase in Con-A reactivity of specific serum proteins reflects an increase in carbohydrate moieties in these proteins in addition to an increase in their protein concentrations, a heme binding serum glycoprotein, hemopexin, also an acute phase reactant, was selected as a marker protein. Hemopexin was purified to apparent homogeneity from pools of serum samples derived from rats with yeast induced inflammation, a monospecific polyclonal antibody was prepared and was used for immunoblot analysis. It was noted that the concentration of hemopexin increased in rats with adjuvant induced arthritis; however, its concentration fell to normal levels after administration with a newly synthesized drug, bindarit, (2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid, C19H20N2O3. Hemopexin was micropurified individually from healthy rats, adjuvant induced arthritic rats, and adjuvant arthritic rats treated with bindarit, cleaved with a Glu-C endopeptidase, Staphylococcus aureus protease V8, and the resultant peptide fragments resolved by SDS-PAGE and examined by silver staining, Coomassie blue staining, and lectin blots using Con-A. It was subsequently noted that hemopexin isolated from adjuvant induced arthritic rats showed a significant increase in Con-A reactivity in selected peptide fragments and that such an increase in glycosylation could be reversed to a pattern similar to healthy rats following treatment with bindarit.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Abnormal glycosylation of hemopexin in arthritic rats can be blocked by bindarit. 128 32

Neutral endopeptidase 24.11, also known as the common acute lymphoblastic leukemia antigen, is a zinc metallopeptidase involved in the inactivation of biologically active peptides, such as the enkephalins and atrial natriuretic peptide. The highly potent radiolabeled inhibitor 2-((3-[125I]iodo-4-hydroxy)phenylmethyl)-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)propyl]amino-4-oxobutanoic acid ([125I]RB104; Ki = 30 pM) has been developed for the enzyme. [125I]RB104 is highly specific, its Ki for another widely distributed zinc peptidase, angiotensin-converting enzyme, being 15 microM. In binding studies using rat brain slices, [125I]RB104 was shown to have a high affinity (Kd = 300 +/- 20 pM) and high specific binding at the Kd concentration (90%). With rat brain homogenates the Kd of [125I]RB104 was 26.8 +/- 0.9 pM, close to the kinetically derived Kd, 7.0 +/- 0.8 pM. Using the inhibitor, we have developed a simple, rapid, and quantitative technique to detect low nanogram quantities of the endopeptidase directly from tissue extracts after SDS/PAGE. The method has been used to show the presence of low quantities of the enzyme in rabbit bone marrow. Apart from its sensitivity, "inhibitor gel electrophoresis" using [125I]RB104 has the advantage over immunohistochemical methods of being able to label the enzyme in all tissues and species. It will therefore be of great value in determining the exact role of this important regulatory peptidase in a number of biological systems. Moreover, this one-step characterization of neutral endopeptidase 24.11 could be extended to other zinc metallopeptidases such as angiotensin-converting enzyme or collagenases, and inhibitors with affinities as high as RB104 could open the way to visualization of zinc metallopeptidases in different tissues by electron microscopy.
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PMID:Development of [125I]RB104, a potent inhibitor of neutral endopeptidase 24.11, and its use in detecting nanogram quantities of the enzyme by "inhibitor gel electrophoresis". 138 73

A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.
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PMID:Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580. 142 18

The primary structure of mouse interleukin-3 (IL-3) expressed by recombinant baculovirus-infected silkworm (Bombyx mori) larvae was analyzed by subjecting isolated IL-3 derived peptides to liquid secondary ion mass spectrometry. Two species of IL-3 were isolated from the silkworm hemolymph by reverse-phase high-pressure liquid chromatography. The major component has M(r)20-22 x 10(3) as determined by SDS-PAGE. Liquid secondary ion mass spectrometric analysis was carried out on the reduced tryptic and endopeptidase lysyl-C peptides of glycosylated and deglycosylated IL-3. These studies provided evidence that (1) Asn-16 is heterogeneously glycosylated with four different oligosaccharides, (2) Asn-86 is either nonglycosylated or has attached to it one oligosaccharide, (3) the N-glycosylation sites Asn-44 and Asn-51 are not glycosylated, and (4) there is no O-glycosylation. Liquid secondary ion mass spectrometric analysis of the unreduced tryptic peptides provided evidence for disulfide linkages between Cys-140 and Cys-79 or Cys-80 and between Cys-17 and Cys-79 or Cys-80. In comparison to the major component, a minor IL-3 species (M(r) 17-19 x 10(3) by SDS-PAGE) isolated from the hemolymph showed no difference with respect to the glycosylation pattern or the disulfide linkages, but it was cleaved between Ala-127 and Ser-128, and only a disulfide linkage between Cys-140 and Cys-79 or Cys-80 held the molecule together.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Determination of the glycosylation patterns, disulfide linkages, and protein heterogeneities of baculovirus-expressed mouse interleukin-3 by mass spectrometry. 144 2

A synthetic peptide corresponding to the first 28 amino acids of the Alzheimer disease amyloid beta/A4 peptide (3.2 kDa) aggregated to a high molecular weight (15 kDa) on SDS/urea polyacrylamide gels. Proteinase K, V8 protease, trypsin, and endopeptidase Lys-C readily degraded the aggregate. By contrast, when digested by endopeptidase Arg-C, a new polypeptide aggregate of higher molecular weight (16 kDa) was observed on denaturing gels without degraded smaller products. The new aggregate was comprised of three peptides: an intact beta/A4(1-28) and partially degraded peptides beta/A4(1-5) plus beta/A4(6-28). The results were confirmed by treatment of beta/A4 with other arginine-specific proteases: the gamma subunit of nerve growth factor and clostripain. The results indicate that arginine-specific proteases, including a growth factor processing enzyme, can nick aggregated beta/A4(1-28) amyloid and alter the configuration to produce a more complex aggregated form. If similar highly specific proteolytic mechanisms occur in the Alzheimer disease brain, the processing may promote the formation of high molecular weight aggregates that contribute to the development of relatively insoluble senile plaque core protein.
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PMID:Arginine specific endopeptidases modify the aggregation properties of a synthetic peptide derived from Alzheimer beta/A4 amyloid. 151 20

The function of the flexible loop which is disordered in crystal structure analysis of glutathione synthetase from Escherichia coli B has been investigated by limited proteolysis and kinetic measurements for the wild-type and mutant enzymes. Proteolysis of the intact enzyme using arginyl endopeptidase or trypsin brought about a time-dependent decrease in the enzymatic activity and the production of protein fragments. SDS-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only a peptide bond between arginine 233 and glycine 234 in the loop was cleaved. Further, native polyacrylamide gel electrophoresis revealed that the cleaved enzyme retained almost the same quaternary structure as that of the wild-type enzyme. Upon protease treatment, the presence of substrates, ATP and/or gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), protected the loop from cleavage, but the presence of glycine was not capable of protecting it. In addition, replacement of arginine 233 in the loop with lysine by site-directed mutagenesis increased the Michaelis constants for gamma-Glu-Cys and glycine by factors of 28 and 213, respectively. The protection against cleavage on a similar protease incubation of this mutant enzyme was also observed in the presence of ATP and/or gamma-Glu-Cys, but the effect in the presence of both substrates was half as large as that for the wild-type enzyme. These results suggest that the loop covers the active site while ATP and gamma-Glu-Cys bind there and that it protects the unstable gamma-Glu-Cys phosphate intermediate from decomposition by bulk water.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction. 154 May 81

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.
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PMID:Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600. 159 45

A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.
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PMID:Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates. 165 Feb 34

Neurotensin (NT) endopeptidase (EC 3.4.24.16) has been purified about 800-fold from pig brain by four sequential chromatographic steps depending on ion-exchange and hydrophobic interactions. Two types of preparation were studied: one from a Triton X-100-solubilized membrane fraction, and the other from the soluble fraction containing 90% or more of the total activity in the homogenate. NT endopeptidase activity was monitored by high-precision liquid chromatography of the two peptide products, characterized as NT-(1-10) and NT-(1-8), resulting from cleavage of the Pro10-Tyr11 and Arg8-Arg9 bonds respectively. As purification proceeded, from both membranes and cytosol, the yield of the two products achieved a constant ratio of 5:1 and this ratio was reproduced in repeated purifications. However, a distinct peptidase which hydrolysed exclusively at the Arg8-Arg9 bond was partially resolved from NT endopeptidase by chromatography on hydroxyapatite, and this activity was further purified and assigned to endopeptidase-24.15 (EC 3.4.24.15). SDS/PAGE of both preparations of neurotensin endopeptidase revealed a major band of apparent Mr 75000, and treatment of the membrane-associated form with N-Glycanase gave no evidence that the enzyme was a glycoprotein. The membrane-associated and cytosol forms of NT endopeptidase activities, monitored for both NT-(1-10) and NT-(1-8) products, were compared in their responses to 1,10-phenanthroline, EDTA, dithiothreitol (DTT) and some synthetic site-directed inhibitors of endopeptidase-24.15 or peptidyl dipeptidase A. The effects revealed no significant differences between the two preparations, nor did the reagents discriminate between the activities generating the two NT fragments. The partially purified form of endopeptidase-24.15 was also included in this comparison: while some responses were similar, this peptidase was distinguishable in its activation by DTT and its relative resistance to inhibition by EDTA. Both forms of NT endopeptidase were found to hydrolyse other substrates, including Boc-Phe-Ala-Ala-Phe-4-aminobenzoate, bradykinin and substance P (these at faster rates than neurotensin), as well as dynorphin A-(1-8) and luliberin. The bonds hydrolysed in these neuropeptides, as well as in angiotensins I and II and alpha-neoendorphin, were defined. These studies confirm that NT endopeptidase is distinct from endopeptidase-24.15. They further show that the former is a soluble enzyme, not an integral membrane protein, that it is not peptide-specific and that it might be more appropriately named. enzyme, not an integral membrane protein, that it is not peptide-specific and
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PMID:Purification and properties of a neurotensin-degrading endopeptidase from pig brain. 190 21


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