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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Metal-binding peptides in proteolytic digest maps have been identified by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS). The plasma and milk metal transport protein chosen to demonstrate this process, histidine-rich glycoprotein (HRG), was purified and then digested with trypsin; the cleavage products were analyzed by LDTOF-MS with dihydroxybenzoic acid as the matrix. The selective interaction of specific peptides with one or more Cu atoms was observed when Cu(II) ions were added to the digest mixture. At least one specific metal-binding peptide was identified by computerized sequence analysis using the molecular mass data and available cDNA sequence. These results demonstrate the first direct observation by mass spectrometry of differential peptide-metal ion interactions in protein digest maps. The ability to evaluate peptide-metal ion interactions, including stoichiometry, with less than 1 pmol of sample improves significantly our ability to identify metal binding domains in metal-binding proteins.
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PMID:Recognition of transition metal ions by peptides. Identification of specific metal-binding peptides in proteolytic digest maps by UV laser desorption time-of-flight mass spectrometry. 173 Mar

We have previously shown that melittin, a bee venom peptide, potently inhibited the catalytic and transport functions of rabbit gastric (H+ + K+)ATPase. A radioactive photoaffinity analog of melittin, ([125I]azidosalicylyl melittin), labeled the (H+ + K+)ATPase. These results suggested that melittin exerted inhibitory effects through direct interaction with the (H+ + K+)ATPase. In this study we attempt to define the melittin-binding domain of the (H+ + K+)ATPase using conformation-dependent proteolytic fragmentation of [125I]azidosalicylyl melittin-labeled hog gastric (H+ + K+)ATPase. In the presence of KCl (E2 form) the 95,000-Da [125I]-azidosalicylyl melittin-labeled (H+ + K+)ATPase was cleaved by trypsin to a 40,000-Da NH2-terminal tryptic fragment and a 56,000-Da COOH-terminal fragment through cleavage at Arg 454 of the (H+ + K+)ATPase. The 40,000-Da fragment was labeled by [125I]-azidosalicylyl melittin. The 56,000-Da fragment was not labeled. When unmodified (H+ + K+)ATPase was trypsinized in the presence of KCl, and the fragments were then reacted with [125I]azidosalicylyl melittin, similar tryptic fragmentation results were obtained. In the absence of KCl (E1 form), the 56,000- and 40,000-Da fragments did not accumulate. Chymotryptic hydrolysis of [125I]azidosalicylyl melittin-labeled (H+ + K+)-ATPase was very slow in the presence of KCl (E2 form). In the absence of KCl (E1 form), chymotryptic hydrolysis was more rapid, with accumulation of a major 42,000-Da fragment which was radiolabeled. The melittin-binding region on the (H+ + K+)ATPase is N-terminal to Arg 454 of the (H+ + K+)ATPase. This region is known to contain the aspartyl phosphate residue (Asp 385), the site of phosphoenzyme formation on the (H+ + K+)ATPase. Melittin is also known to bind to calmodulin and other proteins. Another known calmodulin-binding peptide with a different sequence but similar structure, Trp-3, (Leu-Lys-Trp-Lys-Lys-Leu-Leu-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Leu-Gly) also inhibited the (H+ + K+)ATPase and label incorporation by [125I]azidosalicylyl melittin. These Trp-3 results suggested that the (H+ + K+)ATPase contains a peptide-binding domain which is similar to the peptide-binding domains found on other melittin-binding proteins.
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PMID:[125I]azidosalicylyl melittin binding domains: evidence for a polypeptide receptor on the gastric (H+ + K+)ATPase. 215 80

The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) was purified to homogeneity from 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinol+ ++- labeled bovine heart mitochondrial ubiquinol-cytochrome c reductase. The N-terminal amino acid sequence of the isolated protein is Gly-Arg-Gln-Phe-Gly-His-Leu-Thr-Arg-Val-Arg-His-, which is identical with that of a Mr = 9500 protein in the reductase [Borchart et al. (1986) FEBS Lett. 200, 81-86]. A ubiquinone-binding peptide was prepared from [3H]azidoubiquinol-labeled QPc-9.5 kDa protein by trypsin digestion followed by HPLC separation. The partial N-terminal amino acid sequence of this peptide, Val-Ala-Pro-Pro-Phe-Val-Ala-Phe-Tyr-Leu-, corresponds to amino acid residues 48-57 in the reported Mr = 9500 protein. According to the proposed structural model for the Mr = 9500 protein, the azido-Q-labeled peptide is located in the membrane on the matrix side. These results confirm our previous assessment that the Mr = 13,400 subunit is not the small molecular weight Q-binding protein. Purified antibodies against QPc-9.5 kDa have a high titer with isolated QPc-9.5 kDa protein and complexes that contain it. Although antibodies against QPc-9.5 kDa do not inhibit intact succinate- and ubiquinol-cytochrome c reductases, a decrease of 85% and 20% in restoration of succinate- and ubiquinol-cytochrome c reductases, respectively, is observed when delipidated succinate- or ubiquinol-cytochrome reductases are incubated with antibodies prior to reconstitution with ubiquinone and phospholipid, indicating that epitopes at the catalytic site of QPc-9.5 kDa are buried in the phospholipid environment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The small molecular mass ubiquinone-binding protein (QPc-9.5 kDa) in mitochondrial ubiquinol-cytochrome c reductase: isolation, ubiquinone-binding domain, and immunoinhibition. 216 42

The caudal spinal cord region of teleost fish terminates in a neurosecretory organ, the urophysis. Two peptides have been purified to homogeneity from an extract of the urophysis of a teleost fish, the flounder. The primary structure of one peptide, Ser-Glu-Asp-Pro-Pro-Met-Ser-Ile-Asp-Leu10-Thr-Phe-His-Met-Leu-Arg- Asn-Met-Ile- His20-Met-Ala-Lys-Met-Glu-Gly-Glu-Arg-Glu-Gln30-Ala-Gln-Ile- Asn-Arg-Asn-Leu-Leu - Asp-Glu40-Val, indicates identity with urotensin I. By analogy with other urotensins, the COOH-terminal residue is probably alpha-amidated. A second peptide was present in the extract in a concentration that was approximately equimolar with that of urotensin I. The amino acid composition of this peptide indicated a total of approximately 65 residues. The amino acid sequence of a fragment produced by digestion with trypsin was established as: Ala-Ala-Ala-Ala-Gly5-Asp-Ser-Ala-Ala-Ser10-Asp-Leu-Leu-Gly-Asp1 5-Asn-Ile-Leu- Arg. This sequence shows partial homology to carp prepro-urotensin I(41-59)-peptide as deduced from the nucleotide sequence of a cloned cDNA. It is concluded that the second peptide probably represents the N-terminal flanking peptide of pro-urotensin I which, it has previously been suggested, may function as a urotensin-binding peptide (urophysin) analogous to the neurophysins.
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PMID:Urotensin I and its N-terminal flanking peptide from the flounder, Platichthys flesus. 228 99

A glucan-binding domain of 1,6-alpha-glucan synthase (dextransucrase) (GTF-S) was isolated from a trypsin digest of the Streptococcus sobrinus enzyme. The large 60.5-kilodalton peptide had an affinity for dextran comparable to that of the native enzyme, but had no glucan synthesis activity. The domain was produced in high yield compared with other large cleavage products, which allowed easy purification by size exclusion high-pressure liquid chromatography and affinity chromatography. Two other proteases (mouse submaxillaris protease and lysyl endopeptidase) with specificities similar to trypsin generated a distribution of GTF-S peptides that was also greatly enriched in the glucan-binding peptide. Proteases with markedly different specificities (chymotrypsin and Staphylococcus aureus V8 protease) produced a family of peptides with some evidence of the glucan-binding domain but in far lower yield. The tertiary structure of the domain was critical to its resistance to proteolysis; heat denaturation of GTF-S before trypsin digestion resulted in cleavage of the enzyme to small limit peptides leaving no evidence of the glucan-binding domain. The amino acid composition of the peptide was very similar to that of the native enzyme. The common occurrence of proteases in oral streptococcus cultures and reports of glucosyltransferase degradation during purification and storage raises the possibility that some accounts of glucan-binding receptors are peptides derived from glucosyltransferase. Kinetic implications of a glucan-binding domain are discussed.
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PMID:Isolation of a glucan-binding domain of glucosyltransferase (1,6-alpha-glucan synthase) from Streptococcus sobrinus. 296 13

A cell-binding peptide (Mr = 85,000) which lacks the gelatin- and heparin-binding domains, was purified from trypsin-digested fibronectin. Preincubation of rat hepatocytes in suspension with the peptide, inhibited initial attachment of the cells to immobilized fibronectin while attachment to immobilized laminin and collagen was unaffected. 125I-labeled 85-kDa peptide bound to the cells in suspension at 4 degrees C in a time-dependent, saturable, and partially reversible reaction. Scatchard analysis of the binding data indicated a single class of receptors with a Kd of 1.7 X 10(-8) M. The number of binding-sites was approximately 2.8 X 10(5)/cell. Unlabeled 85-kDa peptide inhibited the binding of 125I-labeled 85-kDa peptide 30-fold more effectively than intact fibronectin. These results provide direct evidence for the presence of a domain in the fibronectin molecule which has or may acquire a high affinity for receptors involved in adhesion of hepatocytes to immobilized fibronectin.
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PMID:Demonstration of high affinity fibronectin receptors on rat hepatocytes in suspension. 315 36

We have found a wide occurrence of alpha,beta-diaminopropionate ammonia-lyase in bacteria and actinomycetes. Considerable amounts of this enzyme were found in Salmonella typhimurium. The enzyme was purified and crystallized from S. typhimurium (IFO 12529). The relative molecular mass of the native enzyme, estimated by the ultracentrifugal equilibrium method, is 89,000 Da, and the enzyme consists of two subunits identical in molecular mass. The enzyme exhibits absorption maxima at 278 and 413 nm and contains 2 mol of pyridoxal 5'-phosphate(pyridoxal-P)/mol of enzyme. The enzyme catalyzes the alpha,beta-elimination reaction of both L- and D-alpha,beta-diaminopropionate, the most suitable substrates, to form pyruvate and ammonia. The L- and D-isomers of serine were also degraded, though slowly. After the internal Schiff base with pyridoxal-P had been reduced with sodium borohydride, followed by trypsin or lysyl endopeptidase digestion of the enzyme, we determined the sequence of about 20 amino acid residues around the lysine residue which binds pyridoxal-P. No homology was found in either the amino acid sequence of the pyridoxal-P binding peptide or the amino-terminal amino acid sequence between the enzyme and other pyridoxal-P-dependent enzymes.
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PMID:Diaminopropionate ammonia-lyase from Salmonella typhimurium. Purification and characterization of the crystalline enzyme, and sequence determination of the pyridoxal 5'-phosphate binding peptide. 327 62

A glycoprotein having a subunit weight of approximately 60,000 was isolated from rabbit liver microsomes. It is a predominant component of the hepatic microsomal membrane and reacts rapidly with diisopropylphosphorofluoridate (DFP), resulting in the loss of enzymatic activity toward artificial substrates such as acyl esters of o-nitrophenols. Automated Edman degradation of this protein together with sequence analysis of peptides provided the NH2-terminal sequence of some 70 residues as follows: His-Pro-Ser- Ala-Pro-Pro-Val-Val-Asp-Thr-Val-Lys-Gly-Lys-Val- Leu-Gly-Lys-Phe-Val-Ser-Leu-Glu-Gly-Phe-Ala-Gln- Pro-Val-Ala-Val-Phe-Leu-Gly-Val-Pro-Phe-Ala-Lys- Pro-Pro-Leu-Gly-Ser-Leu-Arg-Phe-Ala-Pro-Pro-Gln- Pro-Ala-Glu-Ser-Trp-Ser-His-Val-Lys-Asn (CHO)- Thr-Thr-Ser-Tyr-Pro-Pro-Met-Cys-Ser-Ser. A carbohydrate attachment was identified at asparaginyl residue 61. The COOH-terminal peptide of the protein was isolated from two independent enzymatic digests, and its sequence was established as Arg-Glu-Thr-Glu-His-Ile-Glu-Leu. In order to isolate the DFP binding peptide, liver microsomes were labeled with [3H]DFP and the 60-kDa protein containing covalently bound DFP isolated in pure form. Following reduction and carboxymethylation, the DFP-labeled protein was fragmented with trypsin and the digest subjected to gel filtration. Digestion of the labeled peptide preparations with chymotrypsin followed by chromatography of the digest yielded two diisopropylphosphoryl (DIP) peptides. Automated Edman degradation of these peptides provided the following amino acid sequences: Gly-Glu-DIPSer- Ala-Gly-Gly-Gln-Ser-Val-Ser-Ile-Leu-Leu-Leu-Ser- Pro and Thr-Val-Ile-Gly-Asp-DIPHis-Gly-Asp-Glu-Ile-Phe. The active site serine peptide of the 60-kDa protein shows some 70% similarity to the active center region of choline esterases. While the postulated active histidyl residue in choline esterases has not been identified, it is proposed that the DFP binding histidine of the 60-kDa protein corresponds to His-438/440 of choline esterases.
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PMID:Isolation and characterization of a 60-kilodalton glycoprotein esterase from liver microsomal membranes. 366 34

When scallop S1(+LC) (formerly called CaMg S1) is digested by trypsin, the heavy chain degrades while the two light chains remain complexed to each other and a peptide fragment of the heavy chain. The three components of the complex comigrate during electrophoresis under nondissociating conditions and can be purified by chromatography and concentrated by precipitation with ammonium sulphate in the presence of millimolar calcium ions. The truncated regulatory light chain remains associated with the binary complex consisting of the peptide and essential light chain as long as divalent cations are present; in the presence of EDTA it dissociates. This behaviour of the light chains-peptide complex mimics that of the intact molecule. The effect of bound light chains and bound actin on the susceptibility to tryptic digestion was studied using scallop S1(+LC) and S1(-LC) (EDTA S1 according to previous nomenclature). The heavy chains of both types of S1 are labile and have two main sites susceptible to proteolysis. Tryptic digestion on site A produces an N-terminal peptide of around 70 000 and a C-terminal 24 000 fragment from S1(+LC) and a 20 000 C-terminal fragment from S1(-LC); the latter is prone to further proteolysis. Thus S1(-LC), produced in the absence of bound regulatory light chain is shorter on the C-terminal end. Proteolysis on site A abolishes actin-activated ATPase activity; the latter is prevented by digesting acto-S1. The rate of tryptic digestion on site B is somewhat slower than on site A; when either S1 is split at this site an N-terminal 63 000 peptide is produced. The corresponding C-terminal peptide can be obtained from acto-S1 when hydrolysis on site A is prevented; this is estimated as around 31 000 derived from S1(+LC) and 28 000 derived from S1(-LC). The results are compared with similar experiments where vertebrate subfragments were digested by trypsin and the possible localization of the light-chain binding peptide in the intact heavy chain is discussed.
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PMID:Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide. 623 96

Mechanism-based inactivators were used to probe the active site of the broad specificity amino acid racemase from Pseudomonas striata. Kinetic parameters for the inactivation of the racemase with both stereoisomers of beta-fluoroalanine, beta-chloroalanine, and O-acetylserine were determined. By use of 14C-labeled O-acetylserines, the stoichiometry of inactivator binding was found to be one inactivator bound per enzyme subunit. The PLP-dependent enzyme contains one coenzyme per subunit, and after NaB3H4 reduction of the PLP-imine bond, followed by trypsin digestion of the protein, the amino acid sequence of the PLP-binding peptide was determined. Trypsin digestion of the enzyme labeled with either L or D isomer of O-acetylserine and sequencing of the labeled peptide revealed that the inactivators bind to the same lysine residue which binds PLP in native enzyme. The characterization of a PLP adduct released from inactivated enzyme under some conditions is also described. Implications of the formation of this compound with respect to the overall reaction mechanism of inactivation are discussed.
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PMID:Inactivation of the Pseudomonas striata broad specificity amino acid racemase by D and L isomers of beta-substituted alanines: kinetics, stoichiometry, active site peptide, and mechanistic studies. 643 37


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