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)

Eight proteins of diverse lengths, functions, and origin, are examined for compositional non-randomness amino acid by amino acid. The proteins investigated are human fibrinopeptide A, guinea pig Insulin, rattlesnake cytochrome c, MS2 phage coat protein, rabbit triosephosphate isomerase, bovine pancreatic deoxyribonuclease A, bovine glutamate dehydrogenase, and Bacillus thermoproteolyticus thermolysin. As a result of this study the experimentally testable hypothesis is put forth that for a large class of proteins the ratio of that fraction of the molecule which exhibits compositional non-randomness to that fraction which does not is on the average, stable about a mean value (estimated as 0.32 plus or minus 0.17) and (nearly) independent of protein length. Stochastic and selective evolutionary forces are viewed as interacting rather than independent phenomena. With respect to amino acid composition, this coupling ameliorates the current controversy over Darwinian vs. non-Darwinian evolution, selectionist vs. neutralist, in favor of neither: Within the context of the quantitative data, the evolution of real proteins is seen as a compromise between the two viewpoints, both important. The compositional fluctuations of the electrically charged amino acids glutamic and aspartic acid, lysine and arginine, are examined in depth for over eighty protein families, both prokaryotic and eukaryotic. For both taxa, each of the acidic amino acids is present in amounts roughly twice that predicted from the genetic code. The presence of an excess of glutamic acid is independent of the presence of an excess of aspartic acid and vice versa.
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PMID:Deviations from compositional randomness in eukaryotic and prokaryotic proteins: the hypothesis of selective-stochastic stability and a principle of charge conservation. 17 58

Flavocytochrome b2 (L-lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains. Its first 100 residues constitute the heme-binding core, which is homologous to cytochrome b5 [B. Guiard, O. Groudinsky & F. Lederer (1974) Proc. Natl Acad. Sci. USA 71, 2539-2543]. We report here the amino acid sequence of the heme-binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2. The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C. As isolated, the domain consists of 84 residues without any sulfur amino acids. It shows 49 identities with the heme-binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5. Using the recently published three-dimensional structure of S. cerevisiae flavocytochrome b2 [Z-x. Xia, N. Shamala, P. H. Bethge, L. W. Lim, H. D. Bellamy, N. H. Xuong, F. Lederer and F. S. Mathews (1987) Proc. Natl Acad. Sci. USA 84, 2629-2633], it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates. The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex [F. R. Salemme (1976) J. Mol. Biol. 102, 563-568].
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PMID:Amino-acid sequence of the cytochrome-b5-like heme-binding domain from Hansenula anomala flavocytochrome b2. 331 13

The location of membrane-associated proteins of vesicular stomatitis virus was investigated by using two monofunctional and three bifunctional probes that differ in the degree to which they partition into membranes and in their specific group reactivity. Two hydrophobic aryl azide probes, [(125)I]5-iodonaphthyl-1-azide and [(3)H]pyrenesulfonylazide, readily partitioned into virion membrane and, when activated to nitrenes by UV irradiation, formed stable covalent adducts to membrane constituents. Both of these monofunctional probes labeled the glyco-protein G and matrix M proteins, but [(125)I]5-iodonaphthyl-1-azide also labeled the nucleocapsid N protein and an unidentified low-molecular-weight component. Protein labeling of intact virions was unaffected by the presence of cytochrome c or glutathione, but disruption of membrane by sodium dodecyl sulfate greatly enhanced the labeling of all viral proteins except G. Labeling of G protein was essentially restricted to the membrane-embedded, thermolysin-resistant tail fragment. Three bifunctional reagents, tartryl diazide, dimethylsuberimidate, and 4,4'-dithiobisphenylazide, were tested for their capacity to cross-link proteins to membrane phospholipids of virions grown in the presence of [(3)H]palmitate. Only G and M proteins of intact virions were labeled with (3)H-phospholipid by these cross-linkers; the reactions were not affected by cytochrome c but were abolished by disruption of virus with sodium dodecyl sulfate. Dimethylsuberimidate, which reacts with free amino groups, cross-linked (3)H-phospholipid to both G and M protein. In contrast, the hydrophilic tartryl diazide cross-linked phospholipid primarily to the M protein, whereas the hydrophobic 4,4'-dithiobisphenylazide cross-linked phospholipid primarily to the intrinsic G protein. These data support the hypothesis that the G protein traverses the virion membrane and that the M protein is membrane associated but does not penetrate very deeply, if at all.
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PMID:Localization of membrane-associated proteins in vesicular stomatitis virus by use of hydrophobic membrane probes and cross-linking reagents. 625 16

The amino acid sequence of the soluble monohaem cytochrome c-556 from Agrobacterium tumefaciens, strain B2a, has been determined. The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin. Sequencing of the various peptides was achieved by a combination of manual dansyl-Edman degradation and automatic liquid-phase sequence analysis. The main characteristic of this cytochrome is that the haem-binding sequence Cys-Xaa-Yaa-Cys-His occurs in the C-terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C-terminal lysine-122. As such, the protein belongs to cytochrome c sequence class II (sensu Ambler). The cytochrome c-556 is the first example known of a class II cytochrome of the low-spin type isolated from an obligate aerobic organism.
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PMID:The complete amino-acid sequence of the low-spin class II cytochrome c-556 from Agrobacterium tumefaciens strain B2a. 629 89

Horse heart cytochrome c is cleaved by thermolysin in 50% aqueous TFE (v/v) at neutral pH (25 degrees C, 24 h) at the Gly56-Ile57 peptide bond of the 104-residue chain of the protein. Additional, but anyway minor, fragmentation at the Gly45-Phe46 and Met80-Ile81 peptide bonds is also observed. On the other hand, in buffer only and in the absence of TFE, cytochrome c is digested by thermolysin to numerous small peptides. Considering the broad substrate specificity of the TFE-resistant thermolysin, clearly the conformational state of the protein substrate dictates the observed selective proteolysis. It is proposed that the highly helical secondary structure acquired by cytochrome c when dissolved in aqueous TFE hampers binding and adaptation of the protein substrate at the active site of the protease and that peptide bond fission occurs at flexible chain segments characterized by a low alpha-helix propensity.
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PMID:Limited proteolysis of cytochrome c in trifluoroethanol. 772 10

Here we present a comparison between protein fragments produced by limited proteolysis and those identified by computational cutting based on the building block folding model. The principles upon which the two methods are based are different. Limited proteolysis of natively folded proteins occurs at flexible sites and never at the level of chain segments of regular secondary structure such as alpha-helices. Therefore, the targets for limited proteolysis are locally unfolded regions. In contrast, the computational cutting algorithm considers the compactness of the fragments, their nonpolar buried surface area, and their isolatedness, that is, the surface area which was buried prior to the cutting and becomes exposed subsequently. Despite the different criteria, there is an overall correspondence between sites or regions of limited proteolysis with those identified by computational cutting. The computational cutting method has been applied to several model proteins for which detailed limited proteolysis data are available, namely apomyoglobin, cytochrome c, ribonuclease A, alpha-lactalbumin, and thermolysin. As expected, more cuts are obtained computationally than experimentally and the agreement is better when a number of proteolytic enzymes are used. For example, cytochrome c is cleaved by thermolysin at 56-57, 45-46, and at 80-81, and by proteinase K at 48-49 and 50-51. Incubation of the noncovalent and native-like complex of cytochrome c fragments 1-56 and 57-104 with proteinase K yielded the gapped protein species 1-48/57-104 and finally 1-40/57-104. Computational cutting of cytochrome c reproduced the major experimental observations, with cuts at 47, 64-65 or 65-66 and 80-81 and an unstable 32-47 region not assigned to any building block. The next step, not addressed in this work, is to probe the ability of the generated fragments to fold independently. Since both the computational algorithm and limited proteolysis attempt to dissect the protein folding problem, the general agreement between the two procedures is gratifying. This consistency allows us to propose the use of limited proteolysis to produce protein fragments that can adopt an independent folding and, therefore, to study folding intermediates. The results of the present study appear to validate the building block folding model and are in line with the proposal that protein folding is a hierarchical process, where parts constituting local minima of energy fold first, with their subsequent association and mutual stabilization to finally yield the global fold.
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PMID:Comparison of protein fragments identified by limited proteolysis and by computational cutting of proteins. 1207 Mar 28

The general mitochondrial processing peptidase that removes the N-terminal targeting signals from proteins imported into mitochondria forms part of a respiratory protein complex in potato (Solanum tuberosum L.). We have termed this complex the "cytochrome c reductase/processing peptidase complex" and show that it acts on a variety of precursor proteins from different intramitochondrial locations. In potato, biochemical methods fail to separate the ubiquinol cytochrome c oxidoreductase function from the function of the processing protease. On the other hand, inhibition of electron flow with antimycin A or myxothiazol does not affect processing activity. The integration into an oligomeric protein complex causes the unique properties of the processing enzyme. It is fully active at high pH and in the presence of high salt. It does not need externally added metal ions, but it is inhibited by EDTA and 1,10-phenanthroline. Other protease inhibitors have no effect on the processing activity. Taken together, the molecular genetic and physiological results indicate that the mitochondrial processing protease does not belong to the thermolysin superfamily of metalloproteinases but may be a member of a new class of metalloendoproteases.
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PMID:The Cytochrome c Reductase Integrated Processing Peptidase from Potato Mitochondria Belongs to a New Class of Metalloendoproteases. 1223 67