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)

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.
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PMID:The covalent structure of Acanthamoeba actobindin. 237 77

A widely distributed protein methyltransferase catalyzes the transfer of a methyl group from S-adenosyl-methionine to the free carboxyl groups of D-aspartyl and/or L-isoaspartyl derivatives of L-aspartyl and L-asparaginyl residues. This enzyme has been postulated to function in the repair or the catabolism of age-damaged proteins. We present here the complete amino acid sequence of the more basic isozyme I of this enzyme from human erythrocytes. The sequence was determined by Edman degradation and mass spectral analysis of overlapping trypsin, Staphylococcus aureus V8 protease, Pseudomonas fragi endoproteinase Asp-N, cyanogen bromide, and hydroxylamine-generated fragments. The NH2-terminus is modified by acetylation and the protein contains 226 amino acids for a calculated molecular weight of 24,575. This value is in good agreement with the molecular weight determined for the purified protein by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and by gel filtration chromatography under nondenaturing conditions. The identification of 2 different amino acid residues at both positions 22 and 119 may indicate the presence of allelic variants or of two or more closely related structural genes. Finally, comparison of this sequence with those of methyltransferases for RNA, DNA, and small molecules, as well as other S-adenosylmethionine-utilizing enzymes, shows that many of these proteins share elements of three regions of sequence similarity and may be structurally or evolutionarily related.
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PMID:Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases. 268 70

A basic phospholipase A2 (PLA2) was isolated and purified from the venom of Bungarus fasciatus. Four kinds of enzymes, lysyl endopeptidase, endoproteinase Asp-N, endoproteinase Glu-C and trypsin, were employed to elucidate the complete primary structure by means of gas-phase sequencing. The amino-acid sequence reveals 118 amino-acid residues containing seven pairs of half-cystine. It has 78% and 61% structural identities with PLA2 from Bungarus multicinctus and Naja melanoleuca DE-II, respectively.
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PMID:The complete amino-acid sequence of a basic phospholipase A2 in the venom of Bungarus fasciatus. 307 71

1. Two cadmium-binding metallothionein (Mt) isoforms, called Mta and Mtb, were isolated from terrestrial snails (Arianta arbustorum), using various chromatographic techniques, such as gel-permeation chromatography and reversed-phase HPLC. The purified proteins were S-methylated and cleaved by means of different enzymes (trypsin, endoproteinase Glu-C, and endoproteinase Asp-N). Amino acid sequences were determined by automated Edman degradation and collision-induced dissociation (CID) tandem MS. According to their primary structures, both isoforms should be attributed to class-I Mts. 2. The two forms are structurally identical, differing only by one amino acid exchange in position 60 of the peptide chain. Both isoproteins consist of 66 amino acids, 18 of which are cysteine residues. Most of the cysteine residues are arranged in seven Cys-Xaa-Cys motifs. Mta and Mtb possess an N-terminal acetylated-serine residue and contain a short N-terminal motif which shows a high degree of similarity with the N-termini of histones H4 and H2A. 3. A comparison of Mta and Mtb with other invertebrate Mts shows a very high degree of sequence similarity with a cadmium-binding Mt from Helix pomatia, a species that is closely related to Arianta arbustorum. Moreover, Mta and Mtb, as expected, also exhibit structural similarities with Mts from other molluscan species, such as mussels and oysters. It is suggested that Mta and Mtb represent two allelic isoforms, reflecting the genetic polymorphism of Mt in Arianta arbustorum.
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PMID:Mass spectrometry and amino acid sequencing of two cadmium-binding metallothionein isoforms from the terrestrial gastropod Arianta arbustorum. 748 56

We determined the complete amino acid sequence of a zinc metalloendoprotease from Streptomyces caespitosus (ScNP). Peptide fragments obtained by digestion of Rcm-ScNP with trypsin, ScNP and endoproteinase Asp-N were purified by reverse-phase HPLC and their amino acids were analyzed using an automatic sequencer. ScNP consisted of a single polypeptide chain of 132 amino acid residues with one disulfide bond between residues 99 and 112 (M(r) 14376). Thus, the number of amino acid residues determined for this enzyme is much lower than the number of residues previously reported for metalloendoproteases. The amino acid sequence indicated that although ScNP has the zinc-binding motif. His-Glu-Xaa-Xaa-His, which is found at the active site of most zinc metalloendoproteases, it does not share overall significant similarity to the sequences of other zinc metalloendoproteases. Moreover, an analysis of the X-ray structure of ScNP at 0.2-nm resolution (Kirisu et al., unpublished results) revealed that Asp93, together with two histidine residues in the zinc-binding motif (His83 and His87) and a water molecule, is a zinc ligand. We propose that ScNP, which bears the HEXXHXXGXXD motif, represents a novel subfamily of zinc-containing metalloendoproteases.
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PMID:Complete amino acid sequence of a zinc metalloendoprotease from Streptomyces caespitosus. 758 17

The structural and functional domains of Escherichia coli carbamoyl phosphate synthetase (CPS) have been identified by limited proteolysis. Incubation of CPS with several proteases, including trypsin, chymotrypsin, subtilisin and endoproteinase Asp-N, under native conditions, causes a time-dependent loss of enzymatic activity and the generation of a common fragmentation pattern. Amino-terminal sequencing studies demonstrated that the initial cleavage event by trypsin occurred at the carboxy-terminal end of the large subunit. The ultimate fragments produced in most of the proteolysis studies, 35- and 45-kDa peptides, were derived from areas corresponding to the putative ATP binding regions. Substrate protection studies showed that the addition of ligands did not affect the final fragmentation pattern of the protein. However, ornithine and UMP were found to significantly reduce the rate of inactivation by inhibition of proteolytic cleavage. MgATP and IMP provided modest protection whereas bicarbonate and glutamine showed no overall effect on proteolysis. Limited proteolysis by endoproteinase Asp-N resulted in the production of a fragment (or multiple fragments) which contained enzymatic activity but had lost all regulation by the allosteric ligands, UMP and ornithine. The small subunit has been shown to be protected from proteolysis by the large subunit. Proteolysis of the isolated small subunit resulted in the generation of a stable 31-kDa species which contained 10% of the original glutaminase activity. These studies demonstrate that a portion of the C-terminal end of the large subunit can be excised without entirely destroying the ability of CPS to catalyze the formation of carbamoyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mapping the structural domains of E. coli carbamoyl phosphate synthetase using limited proteolysis. 764 1

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.
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PMID:Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label. 768 65

The complete amino acid sequence of two forms of crustacean hyperglycemic hormone (CHH) from the X-organ sinus gland complex of crayfish (Procambarus clarkii) has been determined. There are two variants of P. clarkii CHH (Prc-CHH), I and II, which can be separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Each variant was oxidized by performic acid and then cleaved with lysyl endopeptidase. Intact hormone was also digested with trypsin and endoproteinase Asp-N, successively. The resulting fragments were separated by RP-HPLC and subjected to sequence analyses by a gas-phase sequencer and tandem mass spectrometry. Both variants contain 72 amino acid residues with three disulfide linkages, at positions 7-43, 23-39, and 26-52, and differ from each other by the D/L epimerization of phenylalanine at position 3; Prc-CHH-II contains D-amino acid. Injections of Prc-CHH-I and Prc-CHH-II at a dose of 12.5 pmol resulted in significant increase of hemolymph glucose levels in the crayfish. The hormones are also active in repressing ecdysteroid synthesis at concentrations of 250 mM (Prc-CHH-I) and 25 nM (Prc-CHH-II) in Y-organ culture. These results may indicate that the stereoinversion in the CHH molecule leads to an important alternation in hormonal functions during crustacean development.
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PMID:Characterization of crustacean hyperglycemic hormone from the crayfish (Procambarus clarkii): multiplicity of molecular forms by stereoinversion and diverse functions. 782 76

The complete amino acid sequence of three acyl-binding/lipid-transfer proteins, AB/LTP I, AB/LTP II and AB/LTP III from germinated rape seeds were determined. AB/LTP I and AB/LTP II consist of 93 residues and the M(r) was determined as 9408 by mass spectrometry and calculated as 9406.8 from the sequence. AB/LTP III consists of 92 residues and the M(r) was determined as 9424 by mass spectrometry and calculated as 9422.8 from the sequence. The primary structures were determined by automated Edman degradations of the intact proteins and peptides obtained from digestion with trypsin and endoproteinase Asp-N and cyanogen bromide cleavage. Use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides.
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PMID:Amino acid sequences of three acyl-binding/lipid-transfer proteins from rape seedlings. 782 22

Proteus vulgaris RO104 strain produces a chromosomally encoded beta-lactamase that confers resistance to various beta-lactam antibiotics including methoxyimino third-generation cephalosporins. The beta-lactamase hydrolyzes first- and second-generation cephalosporins efficiently and cefotaxime to a lesser extent. Catalytic activity is inhibited by low concentrations of clavulanic acid and sulbactam. By its broad-spectrum substrate profile, beta-lactamase of Proteus vulgaris RO104 belongs to the group 2e defined by Bush. The protein purified to homogeneity by a four-step procedure was characterized by a pI of 8.31 and a specific activity of 1200 U/mg. The beta-lactamase was digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino-acid sequence determinations of the resulting peptides allowed the alignment of the 271 amino-acid residues of the protein which did not contain any cysteine residue. From amino-acid sequence comparisons, Proteus vulgaris RO104 beta-lactamase was found to share about 68% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca D488 and E23004. Therefore, the cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.
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PMID:Chromosomally encoded cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A. 804 7


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