Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The complete primary structures of the two main forms, PRP-IV and PRP-V, of a proline-rich polypeptide bound in vivo to rat prostatic binding protein has been determined. Their sequences were established using manual Edman degradation of the native polypeptide and of purified fragments derived from trypsin and thermolysin digestions. Both polypeptides contain 38 amino acid residues (Mr = 4397 and 4339); cysteine, methionine, and serine are missing. In spite of the high proline content (21%), no polyproline stretches were detected. PRP-IV and PRP-V show an extensive structural homology and differ only by three substitutions. These amino acid replacements are located in the NH2-terminal part of the molecule at positions 6 (His leads to Pro), 10 (Pro leads to His), and 11 (Asp leads to Gly). Moreover, each component displays a microheterogeneity at several positions in the sequence which indicates that multiple structural variants exist for PRP-IV and PRP-V. These data not only suggest the existence in rat ventral prostate of a multigene family coding for the proline-rich polypeptides but also the occurrence of a pronounced genetic polymorphism for these components. In addition, a remarkable sequence homology is observed between the PRP components and the region of the B chain in the precursor of mouse renin.
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PMID:Proline-rich polypeptides bound to rat prostatic binding protein. The primary structure of the two main components, proline-rich polypeptides IV and V. 668 33

Incubation of purified prostaglandin endoperoxide synthetase from sheep vesicular glands with aspirin results in a covalent binding of the acetyl group of acetylsalicylic acid to the protein. During this acetylation, the cyclooxygenase activity is lost, but not the peroxidase activity. The reaction is completed when almost one acetyl group is bound per polypeptide chain (Mr = 68 000). After proteolysis of [3H]acetyl-protein with pronase, radioactive N-acetylserine was obtained. Originally, however, the hydroxyl group of an internal serine residue in the chain is acetylated. The formation of N-acetylserine can be explained by a rapid O leads to N acetyl shift as soon as the NH2 group of serine is liberated. A radioactive dipeptide was isolated from a thermolysin digest of the [3H]acetyl-enzyme containing phenylalanine and serine, phenylalanine being its N-terminal amino acid. Automatic Edman degradation of native and acetylated enzyme showed that only one polypeptide sequence was present: Ala-Asp-Pro-Gly-Ala-Pro-Ala-Pro-Val-Asn-Pro-X-X-Tyr-. The N-terminal sequence has an apolar character.
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PMID:Acetylation of prostaglandin endoperoxide synthetase with acetylsalicylic acid. 677 69

Glycogen synthase is a substrate for five distinct protein kinases in skeletal muscle which phosphorylate seven different serine residues on the enzyme. Cyclic-AMP-dependent protein kinase phosphorylates sites 1a, 1b and 2, phosphorylase kinase, site 2, glycogen synthase kinase 3, sites 3a, 3b and 3c, glycogen synthase kinase 4, site 2 and glycogen synthase kinase 5 site 5. Site 2 is seven residues from the N-terminus of glycogen synthase and is located in a cyanogen bromide peptide termed CB1 (apparent Mr = 9000). The other six phosphorylation sites are located in a cyanogen bromide peptide termed CB2 (apparent Mr = 24 000) at the C-terminal end of the molecule. The sequence of the N-terminal 123 residues of peptide CB2, has been completed. Sites 3a, 3b, 3c, 5, 1a and 1b are located at residues 30, 34, 38, 46, 87 and 100 from the N-terminus of CB2 respectively. Site 1a is the next serine residue after site 5. The region surrounding sites 3a, 3b and 3c is very rich in proline residues while that surrounding sites 1a and 1b contains many serine and threonine residues. The 23 residues following site 5 contain 15 aspartic acid and glutamic acid residues, while the region immediately N-terminal to site 1a is very basic. The whole region is remarkably hydrophilic and is the region at which the native enzyme is attacked by proteinases. The sites at which glycogen synthase is cleaved by trypsin, chymotrypsin and thermolysin have been identified. The finding that trypsin cleaves the enzyme C-terminal to site 3c while chymotrypsin cleaves N-terminal to site 3a has formed the basis of a simple procedure for determining the state of phosphorylation of the seven serine residues in vivo [Parker, P.J., Embi, N., Caudwell, F.B., and Cohen, P. (1982) Eur. J. Biochem. 124, 47-55].
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PMID:Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Organisation of the seven sites in the polypeptide chain. 680 97

The 22076-Mr Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak beta-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+/serine-containing beta-lactamases.
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PMID:The complete amino acid sequence of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of streptomyces albus G. 682 89

The methylamine dehydrogenase from Pseudomonas AM1 is a tetramer composed of two subunits, light(L)- and heavy-subunits. The amino acid sequence of the L-subunit was determined by sequence analyses of trypsin, chymotrypsin, staphylococcal protease, and thermolysin peptides of Cm-protein. The subunit consisted of a single polypeptide chain of 129 amino acid residues, with alanine and serine at the amino(N)- and carboxyl(C)-terminus, respectively. Yellow-colored peptides containing a prosthetic group were composed of two polypeptide chains and the prosthetic group was covalently bound to two residues at positions 55 and 106, which could not be identified yet. The molecular weight of the subunit was 13,500 excluding the binding residues and the prosthetic group. Various structural features are discussed.
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PMID:Amino acid sequence studies of the light subunit of methylamine dehydrogenase from Pseudomonas AM1: existence of two residues binding the prosthetic group. 684 24

The structure of cytoplasmic malate dehydrogenase has been partially refined by crystallographic least squares methods. Using x-ray phases based on the refined coordinates, analysis of the resultant electron density maps has led to a new model of cytoplasmic malate dehydrogenase and a tentative "x-ray sequence." The two crystallographically independent subunits comprising the dimeric enzyme are nearly identical in structure and are related to each other by roughly 2-fold rotational symmetry. The best fit of the molecular structure of cytoplasmic malate dehydrogenase to that of lactate dehydrogenase has been obtained by least squares methods. The active sites of these two enzymes contain similarly oriented His-Asp pairs linked by a hydrogen bond which may function as a proton relay system during catalysis. This pair could also provide an explanation for the relatively stronger binding by cytoplasmic malate dehydrogenase and lactate dehydrogenase of NADH versus NAD. Similar His-Asp pairs have been observed in the serine proteases, thermolysin, and phospholipase A2, and the His-Asp pair may play a similar functional role in all of these enzymes.
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PMID:The presence of a histidine-aspartic acid pair in the active site of 2-hydroxyacid dehydrogenases. X-ray refinement of cytoplasmic malate dehydrogenase. 684 15

The complete primary structure of elongation factor Tu from Escherichia coli has been elucidated. The protein, which is a mixture of two gene products, consists of a single polypeptide chain of 393 residues. After tryptic digestion of S-carboxymethylated protein, 50 tryptic peptides were isolated covering the complete protein chain. Their alignment was established with overlapping peptides obtained by chemical cleavage with cyanogen bromide and subsequent enzymic subdigestion with Staphylococcus aureus protease, chymotrypsin, elastase and thermolysin. Peptides were sequenced by manual dansyl-Edman and direct Edman degradation procedures. The N-terminal amino acid of EF-Tu is serine and is N-acetylated. The lysine residue at positon 56, in the polypeptide chain is partly methylated. The C-terminal residue is a mixture of serine and glycine, and this was the only heterogeneity found in the EF-Tu preparation used in this study.
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PMID:The complete amino-acid sequence of elongation factor Tu from Escherichia coli. 699 43

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.
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PMID:L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. 704 72

The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.
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PMID:The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II. 743 94

Aspergillus fumigatus (Afu) and A. flavus (Afl), two causative agents of invasive aspergillosis, produce highly homologous serine proteinases. In addition, the former produces a 42-kDa metalloproteinase (MEP), whereas the latter produces a 23-kDa MEP. The cDNA and the gene encoding the 42-kDa MEP were cloned and sequenced. Here, we report the cloning of the cDNA and the gene encoding the 23-kDa MEP from Afl and a homologous gene from the Afu. Using degenerate primers based on the amino acid (aa) sequence of A. oryzae (Ao) MEP and thermolysin-like proteinases, a 282-bp fragment of the 23-kDa MEP-encoding gene of Afl was cloned by PCR. A 6.5-kb KpnI fragment of Afl genomic DNA containing the complete gene was cloned. The open reading frame (ORF) in this gene encodes a protein of 381 aa. Since the mature enzyme from this and other aspergilli would have a theoretical molecular mass of about 20 kDa, this MEP-encoding gene is designated mep20. A Western blot of the protein in the culture filtrate of Afl with polyclonal antibodies prepared against the MEP showed a single band at 23 kDa. The N-terminal sequence of the extracellular MEP20, TKVAS, was found at aa 194-198 within the ORF. Thus, the primary translation product has a putative 19-aa signal and a pro region of 174 aa. A homologous gene cloned from a genomic DNA library of Afu showed an ORF encoding 365 aa. Comparison of the nucleotide (nt) sequences of the cDNAs cloned by RT-PCR with their respective genes showed that there are no introns in the ORF of mep20 in Afl, but there is a 59-bp intron in the gene from Afu. The MEP20 of Afl and Afu have 68% identity and show weak immunological cross reactivity. MEP20 from both these fungi share about 60% sequence identity with the penicillolysin of Penicillium citrinum and the neutral protease II of Ao. MEP20 of Afl and Afu show only the conserved sequence, HEFTHA, but not the two other conserved sequences seen in thermolysins and similar MEP.
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PMID:Cloning and characterization of the cDNAs and genes (mep20) encoding homologous metalloproteinases from Aspergillus flavus and A. fumigatus. 748


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