Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Subtilosin A, a new antibiotic produced by Bacillus subtilis 168, was extracted from culture medium with n-butanol and purified to homogeneity by a combination of gel filtration and thin-layer chromatography. The yield was 5.5 mg from a liter of culture. It had bacteriocidal activity against some gram-positive bacteria. Amino acid analysis and mass spectrometry showed that it was a peptide with a molecular weight of 3398.9, consisting of 32 usual amino acid and some non-amino acid residues. Its amino- and carboxyl-termini were blocked. By analysis of the fragments obtained by partial acid hydrolysis, as well as by chymotryptic and thermolysin digestions of reduced and S-carboxymethylated samples and Achromobacter protease I digestion of performic acid-oxidized samples, the amino acid sequence was determined to be as follows: X-Gly-Leu-Gly-Leu-Trp-Gly-Asn-Lys-Gly-Cys-Ala-Thr-Cys-Ser-(sequence; see text) Ile-Gly-Ala-Ala-Cys-Leu-Val-Asp-Gly-Pro-Ile-Pro-Asp-Glx-Ile-Ala-Gly-Ala. The analyses of cross-linking structures revealed that there were linkages between the amino- and carboxyl-termini and between the Cys-19 and the Glx-28 residues through an unknown residue with a residue weight of 163. Consequently, subtilosin A was deduced to be a cyclic peptide antibiotic with a novel cross-linking structure. The production of subtilosin A begins at the end of vegetative growth and finishes before spore formation. Studies on the correlation between the production of subtilosin A and spore formation with decoyinine in the original strain and in asporogenous mutants of B. subtilis 168 suggested that there was no close correlation between the two phenomena. The production of subtilosin A was repressed by inhibitors of protein and RNA synthesis in contrast to that of many other antibiotic peptides, suggesting that it is synthesized by the mechanism of usual protein synthesis.
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PMID:Subtilosin A, a new antibiotic peptide produced by Bacillus subtilis 168: isolation, structural analysis, and biogenesis. 393 39

An acid DNase (DNase II) from porcine spleen was purified by sequential chromatography over carboxymethyl-cellulose, blue dextran-Sepharose, hydroxylapatite, and sulfoxyethyl-cellulose. The purified enzyme shows two polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 35,000 (alpha chain) and 10,000 (beta chain). The sum of the two molecular weights is that of the native enzyme (45,000). Thus, the DNase II molecule is an alpha,beta dimer. The two polypeptides are not joined by disulfide bonds, but can be cross-linked chemically with dimethyl suberimidate. They are dissociable in 8 M urea, after which they can be isolated by gel filtration on Sephadex G-100, eluting with 1 M acetic acid. Once dissociated, the two polypeptides cannot be reassociated to regenerate DNase II activity. The sum of the amino acid compositions of the two polypeptides is that of the native enzyme, and both contain carbohydrate. The beta chain is devoid of histidine, half-cystine, valine, and methionine. The NH2-terminal amino acid of the alpha chain is leucine, while that of the beta chain cannot be identified by either dansylation or Edman degradation. Alkylation of an essential histidine residue of DNase II occurs on incubation of the enzyme with [2-14C] ICH2COOH (Oshima, R. G., and Price, P. A. (1973) J. Biol. Chem. 248, 7522-7526). Radioactivity is found only in the alpha chain. After hydrolysis of the alpha chain with trypsin, chymotrypsin, and thermolysin, radioactive peptides were isolated by gel filtration on Sephadex G-25 and reversed-phase high performance liquid chromatography. Sequence analyses of the radioactive peptides show alkylation of 1 of 9 histidines in the entire amino acid sequence of DNase II. The sequence around this histidine, determined by manual microsequencing and by the release of amino acids with carboxypeptidases A and B, is Ala-Thr-Glu-Asp-His-Ser-Lys-Trp.
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PMID:The subunit structure and active site sequence of porcine spleen deoxyribonuclease. 403 Jul 66

The synthetic tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine (Ac-DVDA) is a model of the calcium binding site of proteins such as carp parvalbumin, thermolysin and calmodulin. 1H n.m.r. spectra of the tetrapeptide are presented and assigned for D2O and DMSO solutions to determine the conformational mobility. The resonance of the two aspartyl side chains could be completely analysed and the vicinal coupling (C alpha H-C beta H and NH-C alpha H) indicated that the free peptide has considerable conformational mobility. The Ca(II) complex generates a different 1H n.m.r. spectrum for the aspartyl resonances at neutral pH. The solution conformation of Pr(III) complex of Ac-DVDA has been investigated using induced chemical shifts. The observed trends in the magnitude of the shift ratios and the rotamer population suggest that the metal ion binds predominantly to both carboxylates of two aspartyl residues in a bidentate fashion. We discuss the consistency of the differentiated spectra for aspartyl residues in the complex with the stepwise binding of Ca2+ to the carrier.
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PMID:Calcium and praseodymium complexes in solution. 1H n.m.r. conformational study of the model tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine. 405 28

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.
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PMID:Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria. 406 97

An endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle under mild conditions, comprised high- and low-molecular-weight components. The latter (LMW-inhibitor; Mr=50,000) was purified to homogeneity by means of chromatography on DEAE-cellulose and phenyl-Sepharose CL-4B and chromatofocusing. The purified inhibitor is a protein composed of two polypeptide chains with molecular weights of 26,000 and 24,000 daltons. It contains large amounts of glutamic acid, alanine, and serine, and small amounts of aromatic amino acids. It was specific for CANPs having low (m-type) and high (mu-type) Ca2+-sensitivity, had no effect on any other protease examined (trypsin, alpha-chymotrypsin, bromelain, ficin, papain, thermolysin, etc.), and inhibited rabbit mCANP more effectively than rabbit muCANP or chicken mCANP. It was demonstrated that the inhibition is due to the formation of a stoichiometric complex between two molecules of rabbit mCANP and one inhibitor molecule.
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PMID:Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle: purification of 50,000-dalton inhibitor. 609 76

Pig muscle phosphoglucose isomerase modified with pyridoxal 5'-phosphate under conditions that cause at least 90% inactivation of its catalytic activity was found to incorporate about 1.5 eq of pyridoxal 5'-phosphate per subunit. After digestion with thermolysin, two pyridoxal 5'-phosphate-containing peptides were isolated and their amino acid sequences were determined to be Leu-Gly-pyridoxyl-Lys-Gln and Ile-Ala-Ser-pyridoxyl-Lys-Thr.
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PMID:Isolation and sequence determination of two pyridoxal 5'-phosphate-labeled thermolysin peptides from pig muscle phosphoglucose isomerase. 621 48

The N-terminal tryptic peptide of Crithidia oncopelti cytochrome c557 X-Pro-Me3Lys-Ala-Arg in which X represents an unknown N-terminal blocking group was characterized by electrophoresis at pH 2 and by 1H and 13C nuclear magnetic resonance. 1H-NMR spectra of the tryptic peptide suggested that the blocking group X was N,N-dimethylproline although the electrophoretic mobility of the peptide suggested a larger molecular weight. The peptides X-Pro-Me3Lys and X-Pro were generated by treatment of the tryptic peptide with thermolysin and carboxypeptidase and the free blocking group X was prepared by acid hydrolysis. Comparison of the 1H-NMR spectra of these peptides with spectra of synthetic N,N-dimethylproline and N,N-dimethylprolylproline demonstrated that the blocking group was indeed N,N-dimethylproline. The 13C-NMR spectrum of the tryptic peptide was consistent with this conclusion although unambiguous assignments to all resonances could not be obtained because of the small amount of material available. The origin of the dimethylproline blocking group is discussed.
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PMID:Identification of N,N-dimethylproline as the N-terminal blocking group of Crithidia oncopelti cytochrome c557. 625 58

Difference spectra generated during thermolysin digestion of camel beta-endorphin at pH 8.2 or at pH 6.5 indicate rapid blue shifting of the near-UV absorption bands of the NH2-terminal tyrosine. A similar spectral change is not observed for the NH2-terminal tyrosine in [Met]enkephalin when it is digested under similar conditions. These results suggest that enzymatic digestion destroys or alters some structural interaction between the NH2-terminal tyrosyl residue of the endorphin and a residue(s) within the COOH-terminal segment of the molecule. Peptide mapping of the digest as a function of time suggests that cleavage of the bond linking the alanine-21 and isoleucine-22 residues produces most of the observed effect. These data provide evidence for the existence of a tertiary structure for beta-endorphin in aqueous solutions.
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PMID:beta-Endorphin: demonstration of a tertiary structure in aqueous solution. 627 66

The inhibition mechanism of ovostatin was studied using rabbit synovial collagenase and thermolysin. When enzymes were complexed with ovostatin, only the proteolytic activity towards high molecular weight substrates was inhibited. Activity towards low molecular weight substrates was partially modified: the catalytic activity of collagenase bound to ovostatin was inhibited by only 40% towards 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and that of thermolysin bound to ovostatin was activated about 2.6-fold towards benzyloxycarbonyl-Gly-Leu-NH2 and benzyloxycarbonyl-Gly-Phe-NH2. Collagenase-ovostatin complexes failed to react with anti-(collagenase) antibody. Saturation of ovostatin with thermolysin prevented the subsequent binding of collagenase. Ovostatin-proteinase complexes ran faster than free ovostatin on 5% polyacrylamide gel electrophoresis. Complexing ovostatin with either collagenase or thermolysin resulted in the cleavage of the quarter-subunit of ovostatin (Mr = 165,000) into two fragments with Mr = 88,000 and 78,000. On the other hand, when the inhibitory capacity of ovostatin was tested with trypsin, chymotrypsin, and papain, only partial inhibition of their proteolytic activities was observed towards azocasein. Stronger inhibition was noted when Azocoll was a substrate, however. Analyses of ovostatin-enzyme complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the quarter-subunit of ovostatin was cleaved into several fragments by those enzymes. These results led us to propose that ovostatin inhibits metalloproteinases in preference to proteinases of other classes in a manner similar to alpha 2-macroglobulin; hydrolysis of a peptide bond by a proteinase in the susceptible region of the ovostatin polypeptide chain triggers a conformational change in the ovostatin molecule and the enzyme becomes bound to ovostatin in such a way that the proteinase is sterically hindered from access to large protein substrates and yet is accessible to small synthetic substrates. A kinetic study of collagenase binding to ovostatin gave the value of k2/Ki = 6.3 X 10(5) M-1 min-1. The results indicate that ovostatin is equally as good a substrate for collagenase as type I collagens.
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PMID:Ovostatin: a novel proteinase inhibitor from chicken egg white. II. Mechanism of inhibition studied with collagenase and thermolysin. 630 43

A thermolysin-like metalloendopeptidase, optimally active at a neutral pH, was identified in human serum. The enzyme cleaves the synthetic substrate glutaryl-Ala-Ala-Phe-2-naphthylamide at the Ala-Phe bond. Activity was determined by measuring the rate of formation of Phe-2-naphthylamide in a coupled enzyme assay in the presence of excess aminopeptidase M. 2-Naphthylamine released during the reaction was determined by a diazotization procedure. Enzyme activity is not affected by inhibitors of serine, thiol, or carboxyl proteases, but is sensitive to inhibition by metal chelators such as EDTA and o-phenanthroline. Dialysis against EDTA leads to loss of activity, which can be fully restored by zinc and cobalt ions. The serum enzyme closely resembles a membrane-bound metalloendopeptidase (EC 3.4.24.11) abundant in lung, spleen, and kidney in that both enzymes are inhibited by the same active-site-directed inhibitors. In addition, an antiserum obtained against the metalloendopeptidase from rabbit kidney shows strong cross-reactivity with the serum enzyme. Metalloendopeptidase activity was measured in 150 controls and in 95 patients with sarcoidosis; the two groups had significantly different enzyme activities (p less than 0.001). The mean enzyme activity in the sarcoidosis group was more than threefold higher than that of the control group. The mean enzyme activity for patients with active disease was more than double that of patients with inactive disease and more than four times that of controls (p less than 0.001). This is noteworthy because angiotensin converting enzyme, a zinc-dipeptidyl carboxypeptidase with a mechanism of action similar to that of the metalloendopeptidase, has also been reported to be increased in the serum of patients with active sarcoidosis. Enzyme activity in patients with active tuberculosis, primary pulmonary neoplasms, and idiopathic interstitial pulmonary fibrosis did not differ significantly from that of controls.
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PMID:Identification of a thermolysin-like metalloendopeptidase in serum: activity in normal subjects and in patients with sarcoidosis. 636 93


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