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)

In perchloric acid extracts of normal lung and colonic tumors, 3 NCA molecules were identified by monoclonal antibodies that cross-reacted with CEA, which itself gave 2 bands in SDS-PAGE. The proteins had molecular weights of 50, 75 and 97 kd, while the 2 CEA molecules banded at 180 and 160 kd in SDS-PAGE. No MAb recognized only one molecule, with the exception of MAb 3/13 which precipitated solely the upper CEA band. Analysis of the biochemical relationship of the cross-reactive antigens showed that none of them contained any internal methionine. Furthermore, after digestion by thermolysin, the peptide maps of the immunoprecipitated molecules showed very close similarities, if not identity. When the cross-reactive and the CEA were compared, the only differences found were in the upper CEA band, which apparently lacked one hydrophobic peptide, while the 97 kd cross-reacting protein showed one extra peptide. We conclude from our results that CEA and the cross-reacting molecules are composed of nearly identical, small (i.e. less than 50 kd) polypeptide chains.
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PMID:Two CEA and three NCA species, although distinguishable by monoclonal antibodies, have nearly identical peptide patterns. 383 68

Hen egg white ovomacroglobulin purified by Miller and Feeney without reference to its activity was shown to have a protease inhibitory activity towards trypsin, papain, and thermolysin. It has four subunits of equal molecular weight (175,000 by SDS-PAGE) and each two of which are disulfide bonded. Upon incubation with trypsin it yields a fragment of Mr = 80,000 plus smaller ones. The subunit composition, amino acid composition and a newly found protease inhibitory activity place ovomacroglobulin as a closely related protein to human serum alpha 2-macroglobulin.
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PMID:Hen egg white ovomacroglobulin has a protease inhibitory activity. 618 73

A model showing the topological distribution, functions, and serological specificities of eight distinct, monoclonal antibody-defined epitopes on the tick-borne encephalitis (TBE) virus glycoprotein has been presented in a previous publication (F. X. Heinz, R. Berger, W. Tuma, and Ch. Kunz (1983). Virology 126, 525-537.) In the present report the influence of conformational change, chemical modification, and fragmentation on the antigenic reactivity of each epitope has been analyzed by the use of blocking enzyme immunoassays and "Western blotting." One of the two major antigenic domains (A), composed of three different epitopes, completely lost its antigenicity upon incubation at pH 5.0 or by treatment with guanidine-HCl/urea, SDS, reduction and carboxymethylation, as well as by proteolytic (trypsin, alpha-chymotrypsin, thermolysin) and chemical (CNBr) fragmentation. The second major antigenic domain (B), however, defined by four distinct monoclonal antibodies, three of which are hemagglutination (HA)-inhibiting, neutralizing, and protective, was shown to be resistant to low pH, guanidine-HCl/urea treatment, and proteolytic cleavage of the native protein. Also, polyclonal immune sera from mice and rabbits contained antibody populations reactive with antigenic determinants which are resistant and others which are sensitive to conformational change and fragmentation. Glycoprotein fragments with molecular weights of about 9000, generated by proteolysis of the native protein, were immunoreactive with neutralizing and protective monoclonal antibodies (defining domain B) as well as with a polyclonal mouse immune serum. Thus, these fragments appear to contain antigenic determinants which are immunodominant on the native protein and play an important role in the induction of a protective immune response against TBE virus. In addition, these results show that antibody binding to antigenic domains which are topologically and structurally completely unrelated may result in neutralization and/or HA inhibition. As the presence of two receptor-binding sites is unlikely, different effector mechanisms may account for the effects of these antibodies. The antigenic reactivity of domain A is sensitive to the same treatments which also inactivate HA activity of TBE virus, whereas domain B is resistant. These treatments include a change of domain A induced by incubation at slightly acidic pH which also results in inactivation of virus infectivity. Antibodies to domain A therefore presumably block viral activities by direct binding at or near the putative receptor-binding site whereas antibodies to domain B may cause loss of biological activities by inducing a conformational change of the receptor-binding site.
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PMID:Location of immunodominant antigenic determinants on fragments of the tick-borne encephalitis virus glycoprotein: evidence for two different mechanisms by which antibodies mediate neutralization and hemagglutination inhibition. 619 9

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.
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PMID:The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro. 645 76

Purified platelet thrombospondin binds to immobilized fibrinogen if both Ca++ and Mg++ are present. Digestion of the purified molecule with thermolysin results in a limited number of discrete proteolytic fragments. When such digests are subjected to affinity chromatography on immobilized fibrinogen, only the fragments with Mr of 120,000 and 140,000 are specifically bound and subsequently eluted by the addition of EDTA to the column buffer. Examination by SDS-PAGE under both reducing and nonreducing conditions reveals that the fibrinogen-binding domain is derived from the region of the thrombospondin molecule containing the interchain disulfide bonds. The requirement for Ca++ and Mg++ for optimal binding to fibrinogen is also manifest by the Mr 120,000/140,000 thermolytic fragments.
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PMID:Isolation of the fibrinogen-binding region of platelet thrombospondin. 671 66

A major allergen has been identified in an aqueous extract of the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae). Following chromatography on Sephadex G-100 allergenic activity, as assessed by skin ('prick') testing, eluted as two closely related peaks (pools I and II) at about 50% bed volume. When these pools were applied separately to columns of CM-cellulose, activity in each eluted with 0 . 05 M NaCl. Isoelectric focusing of the unfractionated allergen gave a single peak of activity at pI 4 . 3. By SDS--PAGE, biological activity in the whole 'green nimitti' extract and the material eluting from both pools I and II of the Sephadex G-100 column migrated to the same positions and were associated with a molecular size of 15,000--20,000 daltons. Skin test reactivity of the unfractionated material and the Sephadex G-100 pool I and II eluates were all destroyed following incubation with trypsin, chymotrypsin, thermolysin and neuraminidase. These experiments indicate that a major allergen derived from the 'green nimitti' midge, a cause of widespread and severe immediate-type allergy in the Sudan, is an acidic glycoprotein of 15,000--20,000 molecular weight.
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PMID:Widespread IgE-mediated hypersensitivity in the Sudan to the 'green nimitti' midge, Cladotanytarsus lewisi (Diptera: Chironomidae) II. Identification of a major allergen. 743 59

The structure of the ca. 250 amino acid hormone binding domain of the human estrogen receptor (hER-LBD), expressed in E. coli and purified as a complex with estradiol, has been probed by selective proteolysis, with analysis of the protein fragments both by classical methods (SDS-PAGE and Edman N-terminal sequencing) and by mass spectrometry (HPLC-coupled electrospray ionization mass spectrometry (LC/ESI-MS)). Rapid cleavage by several proteases (trypsin, chymotrypsin, thermolysin, and Asp-N endoproteinase) is observed within a localized region (residues 297-303) at the N-terminus. In contrast, proteolytic scission at the C-terminus is less localized and more progressive; initial cuts by trypsin, chymotrypsin, thermolysin, V8, and Asp-N proteinases are observed to occur in the region 553-571, followed by further cleavage with thermolysin (548) and trypsin (548, 531, and 529). Thus, N304 and K529 define the protease-resistant N- and C-termini of a core structure for this domain that appears to contain the elements sufficient for ligand binding. The remaining segment of this domain (530-553), which is known to embody elements essential for ligand-modulated transcription activation (AF-2), is likely a surface-exposed region that, through these studies, is shown to be accessible to proteases. Only a single region within the 26 kDa ligand-binding core (N304-K529) has been identified as being readily accessible to proteases; rapid proteolysis using the proteases trypsin, chymotrypsin, and thermolysin, is localized to residues 465-468, with cleavage occurring at residues K467, L466, and both T465 and S468, respectively. The flexibility implied by the cuts in this internal 465-468 region suggest that the hER-LBD may actually consist of two subdomains. These proteolysis studies provide a substantially refined view of the conformational nature of the human estrogen receptor ligand binding domain.
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PMID:Analysis of the structural core of the human estrogen receptor ligand binding domain by selective proteolysis/mass spectrometric analysis. 754 10

The ligand-filled 32-kDa fragment of the porcine estradiol receptor extending from His267 to the C-terminal Ile595 was purified to homogeneity by adsorption to mAb 13H2. The native protein was exposed at 4 degrees C to a panel of proteases: thermolysin, subtilisin, pronase, elastase, ficin, bromelain, endopeptidase Lys-C, both in the dimer and the monomer state, and chymotrypsin at pH 8.2 only. The digests were analysed by SDS/PAGE/Western blotting for Coomassie staining and immunostaining. Peptides were sequenced from blots. The majority of cleavage sites in upper domain E (8 out of 11) amassed in the Leu296-Leu310 stretch. Cleavage at Leu319 was seen with subtilisin and at Tyr328 with chymotrypsin. Susceptability to enzymic proteolysis was also pronounced in Thr465-Glu470 at the center of domain E. Three peptides, 13 kDa with thermolysin, beginning at Leu337, 6 kDa and, in low yield, 5 kDa with endopeptidase Lys-C beginning at Asp473 resp. Cys417 were only obtained from the monomer substrate. The various digests featured either 27-23-kDa peptides or mixtures of 17-13-kDa and 12-7-kDa peptides separable by SDS/PAGE. All peptides with N-termini between Leu297 and Ser329 reacted with mAb 13H2. The digests showed high peaks of bound estradiol in the dimer position of 32-kDa fragment controls on density gradient centrifugation at pH 7.4. However, the property of proton-driven dissociation was only preserved in the pronase, elastase and chymotrypsin digests with peptides extending beyond the His547-ArgLeuHis550 motif. The preservation of the estradiol-binding niche in the tightly complexed peptides of domain E was also demonstrated by refilling after steroid removal. The sites exposed to proteolytic enzymes and the epitope for 13H2 attachment are in good agreement with surface probability plots.
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PMID:Surface mapping of the ligand-filled C-terminal half of the porcine estradiol receptor by restricted proteolysis. 763 63

A cDNA coding for the E isoform of alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) was isolated by oligonucleotide hybridization following immunochemical screening of the rabbit liver cDNA library. The deduced amino acid sequence of the E isoform showed 96.4% identity in 413 residues of the F and S-1 isoforms of rabbit alpha-1-antiproteinase. The N-terminal half of the amino acid residues of the three isoforms was almost identical, but the putative reactive-site loop structure (P8-P'8) was significantly different in the various forms, the P1 site of the E form being glutamic acid. Interaction of the recombinant E form with the various proteinases was investigated by SDS/PAGE, followed by immunoblot analysis. The recombinant protein and trypsin formed a 62 kDa equimolar complex, which gradually became graded to the 37 kDa fragment through several intermediates. The E form also formed a complex of a similar size with elastase and became degraded to the 31 kDa fragment. Several proteinases which cleaved the E form without forming a detectable complex on SDS/PAGE are chymotrypsin, protease V8, pancreas kallikrein, thermolysin, papain and ficin. Other proteinases, with a stringent substrate specificity, such as thrombin, factor Xa, plasmin, plasma kallikrein and cathepsin G, did not attack the E form. Unlike the F and S-1 forms of rabbit plasma alpha-1-antiproteinase, the recombinant E form did not inhibit the amidolytic and proteolytic activities of trypsin. Neither elastase nor protease V8 was inhibited by the E form. Thus the change in the amino acid residues in the reactive-site loop, probably in the P1 site, is responsible for the loss of inhibitory activity of rabbit alpha-1-antiproteinase E. The novel character of the E form could provide a new insight into the interaction of serpin and proteinases.
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PMID:Rabbit alpha-1-antiproteinase E: a novel recombinant serpin which does not inhibit proteinases. 773 71

A periplasmic insulin-cleaving proteinase (ICP), purified to its electrophoretic homogeneity in the SDS-PAGE from the Gram-negative bacterium Acinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory effects exerted by EDTA and o-phenanthroline. Furthermore, dialysis against EDTA and o-phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+. Similar to protease III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B-chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters, p-nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-specificity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of protease III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sites in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe25-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the protease III and four sites with the IDE.
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PMID:A periplasmic insulin-cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes. 773 84


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