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)

The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of 32P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the [beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The sequence is Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The phosphothreonine residue, which appeared as either an anomalous proline or an unidentifiable phenylthiohydantoin derivative during sequencing, was verified by two-dimensional phosphoamino acid analysis of the phosphopeptides and by resequencing the tryptic peptide after dephosphorylation with exogenous alkaline phosphatase. This sequence, starting at position 4, is completely homologous to the previously published sequence of the tryptic dodecapeptide harboring the catalytically essential (phospho)histidyl residue in the active-site domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19, 5805-5809). These comparative results indicate that the regulatory phosphothreonine causing complete inactivation of maize leaf dikinase is separated from the critical active-site (phospho)histidine by just one intervening residue in the primary sequence.
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PMID:Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase. 283 85

Three to six mg of the millimolar Ca2+-requiring proteinase (m-calpain) were obtained from 1 kg bovine cardiac muscle (fresh wt) and some enzymatic properties of this proteinase were determined. Activity of bovine cardiac m-calpain decreases as ionic strength increases from 75 to 1000 mM. Maximal activation of m-calpain by Ca2+, La3+, Ba2+, and Mn2+ occurs at 2 to 3 mM concentrations of each of these divalent cations, but La3+ activation is only 20 to 25% and Ba2+ and Mn2+ activation only 6 to 10% as great as Ca2+ activation. Maximum Sr2+ activation occurs at 20 mM Sr2+ and is 90 to 95% of maximum Ca2+ activation. Mg2+, Zn2+, Cr2+, and Cd2+ do not activate m-calpain when added alone; Mg2+ does not affect, but Zn2+ inhibits, Ca2+-stimulated activity. The nonionic detergents, Triton X-100 and Brij 35, activate m-calpain 1.6- to 2.0-fold but do not change its Ca2+ requirement. Sodium dodecyl sulfate and urea inhibit m-calpain completely at 0.045% and 2.0 M, respectively. Because they bind Ca2+ needed for activation, ATP, ADP, and ITP inhibit m-calpain. The trypsin inhibitors, phenylmethylsulfonyl fluoride, ovomucoid trypsin inhibitor, ovoinhibitor, aprotinin, alpha 1-antiproteinase inhibitor, soybean trypsin inhibitor, and lima bean trypsin inhibitor do not affect m-calpain activity; m-calpain does not release measureable quantities of acid-soluble peptides from a rabbit skeletal sarcoplasmic protein fraction but does degrade rabbit skeletal myofibrils and casein.
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PMID:Some properties of the millimolar Ca2+-dependent proteinase from bovine cardiac muscle. 285 32

Photoaffinity labeling experiments with diphtheria toxin fragment A have implicated glutamic acid 148 as a constituent of the NAD binding site. To evaluate the role of this residue in ADP-ribosylation of elongation factor 2, we replaced it with aspartic acid by in vitro mutagenesis of a toxin gene fragment cloned in Escherichia coli. Fragment A containing aspartic acid at position 148 had less than 0.6% the ADP-ribosylation activity of wild-type fragment A. The mutation produced no change in sensitivity of fragment A to trypsin and little, if any, reduction in affinity of fragment A for NAD. These results indicate that glutamic acid 148 is essential for the ADP-ribosylation of elongation factor 2 and are consistent with other data suggesting that this residue may be at or near the catalytic center of the toxin.
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PMID:Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity. 286 66

Salivary gland homogenates of adult female Lutzomyia longipalpis inhibited platelet aggregation induced by ADP and collagen. Apyrase (ATP diphosphohydrolase) activity was prominent, requiring Ca2+ but not Mg2+ and a pH optimum of 8.0. Human as well as rabbit hosts developed a well delimited erythema, evident 2-3 min after initial probing and lasting for as long as 2 days. Erythema, not accompanied by itching or swelling, developed in previously exposed hosts as well as in those not previously exposed to this insect. When injected intradermally into the shaved back of a rabbit, salivary gland homogenates induced marked erythema, even with 1/250 of a homogenized salivary gland. This erythema-inducing factor was insoluble in 90% ethanol and was destroyed by incubation with trypsin. These apyrase and erythema-inducing factors, together with short mouthpart stylets, appear to adapt Lutzomyia sandflies to feed on blood released from superficial skin capillaries.
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PMID:Blood-finding strategy of a capillary-feeding sandfly, Lutzomyia longipalpis. 287 Aug 60

When beef heart mitochondrial F1-ATPase is photoirradiated in the presence of 2-azido[alpha-32P]adenosine diphosphate, the beta subunit of the enzyme is preferentially photolabeled [Dalbon, P., Boulay, F., & Vignais, P. V. (1985) FEBS Lett. 180, 212-218]. The site of photolabeling of the beta subunit has been explored. After cyanogen bromide cleavage of the photolabeled beta subunit, only the peptide fragment extending from Gln-293 to Met-358 was found to be labeled. This peptide was isolated and digested by trypsin or Staphylococcus aureus V8 protease. Digestion by trypsin yielded four peptides, one of which spanned residues Ala-338-Arg-356 and contained all the bound radioactivity. When trypsin was replaced by V8 protease, a single peptide spanning residues Leu-342-Met-358 was labeled. Edman degradation of the two labeled peptides showed that radioactivity was localized on the following four amino acids: Leu-342, Ile-344, Tyr-345, and Pro-346.
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PMID:Identification of amino acid residues photolabeled with 2-azido[alpha-32P]adenosine diphosphate in the beta subunit of beef heart mitochondrial F1-ATPase. 287 32

Under appropriate conditions tight, noncovalent binding of 2-azido-adenine nucleotides to either catalytic or noncatalytic binding sites on the E. coli F1-ATPase occurs. After removal of unbound ligands, UV-irradiation results primarily in the covalent incorporation of nucleotide moieties into the beta-subunit in both catalytic and noncatalytic site labeling experiments. Minor labeling of the alpha-subunit was also observed. After trypsin digestion and purification of the labeled peptides, microsequencing studies identified two adjacent beta-subunit tryptic peptides labeled by 2-azido-ADP or -ATP. These beta-subunit peptides were labeled on tyrosine-331 (catalytic sites) and tyrosine-354 (noncatalytic sites) in homology with the labeling patterns of the mitochondrial and chloroplast enzymes.
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PMID:Catalytic and noncatalytic nucleotide binding sites of the Escherichia coli F1 ATPase. Amino acid sequences of beta-subunit tryptic peptides labeled with 2-azido-ATP. 288 23

Photolabeling of nucleotide binding sites in nucleotide-depleted mitochondrial F1 has been explored with 2-azido [alpha-32P]adenosine diphosphate (2-N3[alpha-32P] ADP). Control experiments carried out in the absence of photoirradiation in a Mg2+-supplemented medium indicated the presence of one high affinity binding site and five lower affinity binding sites per F1. Similar titration curves were obtained with [3H]ADP and the photoprobe 3'-arylazido-[3H]butyryl ADP [( 3H]NAP4-ADP). Photolabeling of nucleotide-depleted F1 with 2-N3[alpha-32P]ADP resulted in ATPase inactivation, half inactivation corresponding to 0.6-0.7 mol of photoprobe covalently bound per mol F1. Only the beta subunit was photolabeled, even under conditions of high loading with 2-N3[alpha-32P]ADP. The identification of the sequences labeled with the photoprobe was achieved by chemical cleavage with cyanogen bromide and enzymatic cleavage by trypsin. Under conditions of low loading with 2-N3[alpha-32P]ADP, resulting in photolabeling of only one vacant site in F1, covalently bound radioactivity was located in a peptide fragment of the beta subunit spanning Pro-320-Met-358 identical to the fragment photolabeled in native F1 (Garin, J., Boulay, F., Issartel, J.-P., Lunardi, J., and Vignais, P. V. (1986) Biochemistry 25, 4431-4437). With a heavier load of photoprobe, leading to nearly 4 mol of photoprobe covalently bound per mol F1, an additional region of the beta subunit was specifically labeled, corresponding to a sequence extending from Gly-72 to Arg-83. The isolated beta subunit also displayed two binding sites for 2-N3-[alpha-32P]ADP. When F1 was first photolabeled with a low concentration of NAP4-ADP, leading to the covalent binding of 1.5 mol of NAP4-ADP/mol F1, with the bound NAP4-ADP distributed equally between the alpha and beta subunits, a subsequent photoirradiation in the presence of 2-N3[alpha-32P]ADP resulted in covalent binding of the 2-N3[alpha-32P]ADP to both alpha and beta subunits. It is concluded that each beta subunit in mitochondrial F1 contains two nucleotide binding regions, one of which belongs to the beta subunit per se, and the other to a subsite shared with a subsite located on a juxtaposed alpha subunit. Depending on the experimental conditions, the subsite located on the alpha subunit is either accessible or masked. Unmasking of the subsite in the three alpha subunits of mitochondrial F1 appears to proceed by a concerted mechanism.
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PMID:Mapping of nucleotide-depleted mitochondrial F1-ATPase with 2-azido-[alpha-32P]adenosine diphosphate. Evidence for two nucleotide binding sites in the beta subunit. 288 35

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.
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PMID:Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin. 289 47

Bovine liver glutamate dehydrogenase reacts covalently with 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of 1 mol reagent/mol enzyme subunit and loss of one of the two ADP sites of native enzyme [S. P. Batra and R. F. Colman, J. Biol. Chem. 261, 15565-15571 (1986)]. Incorporation of reagent is prevented specifically by ADP. The modified enzyme has now been digested with trypsin. The nucleotidyl peptide has been purified by chromatography on phenylboronate-agarose, followed by reverse-phase HPLC. On the basis of amino acid composition following acid hydrolysis, and gas-phase sequencing, the modified tryptic peptide was established as Ala-Gln-His-Ser-Gln-His-Arg, corresponding to amino acids 80-86 of the known glutamate dehydrogenase primary structure. The evidence presented indicates that the target amino acid attacked by 2-BDB-TAMP is histidine-82 and that this residue is located within the high-affinity ADP-activating site of glutamate dehydrogenase. In the course of this work, it was found that the positions of Gln84 and His85 had been reported as reversed in the revised sequence of bovine liver glutamate dehydrogenase [J. H. Julliard and E. L. Smith, J. Biol. Chem. 254, 3427-3438 (1979)]. Three additional corrections are here reported in the amino acid sequence of the native enzyme on the basis of gas-phase sequencing of other peptides purified by HPLC: Asp168 (not Asn); His221-Gly222 (not Gly-His); and Glu355 (not Gln).
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PMID:Identification of histidyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate in an ADP regulatory site of glutamate dehydrogenase. 293 Jan 90

The antifibrinolytic activity was found in the medium of platelet suspension in the process of platelet aggregation induced by thrombin, ADP and 5-hydroxytryptamine. The antifibrinolytic activity was closely associated with inhibitors in platelets, which specifically inhibited plasmin activity and not inhibited other proteases such as urokinase, thrombin and trypsin. One casein unit of plasmin activity was inhibited by the inhibitors released from approximately 10(8) platelets during the aggregation with thrombin. By the activity staining analysis, it was found that there are two kinds of plasmin inhibitors with molecular weights of 25,000 and 17,000. The physiological function of these inhibitors was discussed in relation to the formation of thrombus.
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PMID:Novel plasmin inhibitors released from bovine platelets during aggregation. 293 57


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