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)

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.
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PMID:Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2. 310 32

Recent studies have suggested a possible role for extracellular ATP in neutrophil function. This report provides evidence for the existence of an ecto-protein kinase activity on the surface of human neutrophils capable of phosphorylating intrinsic cell membrane proteins as well as exogenous proteins. Addition of extracellular [gamma-32P]ATP to neutrophils resulted in rapid incorporation of 32P into cellular proteins that were sensitive to trypsin. The ability of adherent cells to phosphorylate the exogenous substrate casein, while no protein kinase activity was released into the supernate, provided further evidence for a cell surface associated protein kinase activity.
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PMID:Evidence for ecto-protein kinase activity on the surface of human neutrophils. 337 96

A protein kinase activity (S6PK) that phosphorylates ribosomal protein S6 has been detected in cytosolic extracts prepared from an insulin-sensitive mouse fibroblast-melanoma hybrid cell line. The activity of this enzyme is greatly increased in cells that have been stimulated with insulin or serum for 30 min before preparation of the extract. In the parental melanoma cells, which are insensitive to the growth-stimulatory action of insulin, the activity of the enzyme is lower than in the hybrid cells and is not increased in response to insulin. The insulin-sensitive, serum-sensitive S6PK from the hybrid cells is eluted as a single peak from diethylaminoethyl (DEAE)-cellulose between 0.15 and 0.2 M KCl. The apparent mol wt of the enzyme, as determined by gel permeation chromatography, is approximately 105,000. A second S6 kinase activity from the hybrid cells is trypsin dependent and elutes from DEAE-cellulose at a lower salt concentration than S6PK. In contrast to S6PK, the trypsin-dependent S6 kinase activity does not vary in a consistent manner in response to insulin or serum. Fractions obtained from DEAE-cellulose chromatography of extracts of the hybrid cells have also been assayed for ability to phosphorylate the synthetic octapeptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6-1), the structure of which is based on a phosphorylated region of the S6 protein. Two trypsin-dependent peaks of protein kinase activity have been found to phosphorylate this peptide, one eluting at 0.05 M KCl and the other at 0.10-0.15 M KCl. The first peak elutes at the same salt concentration as the trypsin-dependent protein kinase(s) that phosphorylate ribosomal protein S6, while the second elutes slightly, but reproducibly ahead of S6PK. Several properties of the second peak of S6-1 phosphorylating activity suggest that it is not S6PK.
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PMID:Insulin-sensitive, serum-sensitive protein kinase activity that phosphorylates ribosomal protein S6 in cultured fibroblast-melanoma hybrid cells. 352 18

Incubation of rat liver plasma membrane produced histone phosphorylating activity at 75 mM Mg2+ in the soluble fraction. The release of the kinase activity was inhibited by leupeptin and bovine pancreatic trypsin inhibitor, suggesting the involvement of membrane-bound protease. When partially purified protein kinase C from rat liver cytosol was treated with the trypsin-like protease purified from rat liver plasma membrane, histone phosphorylating kinase which was independent of Ca2+ and phospholipids, produced with a molecular weight of about 5 X 10(4). These results suggest that membrane-bound, trypsin-like protease activates protein kinase C in plasma membrane and the activated kinase is released from the membrane to the soluble fraction.
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PMID:Proteolytic activation of protein kinase C by membrane-bound protease in rat liver plasma membrane. 390 21

Two interrelated sites have been detected on the fructose carrier in Rhodopseudomonas sphaeroides: an activity-linked dithiol and a Zn2+-binding site. Binding of Zn2+ brings EIIFru into a new conformation that to some extent mimics the conformation of phosphorylated EIIFru, an essential intermediate in the turnover of the enzyme. Binding of zinc to EIIFru or phosphorylating the enzyme protects it against trypsin inactivation relative to the dephosphorylated zinc-free enzyme. A dithiol is essential for activity. Interchanges between the redox states of the enzyme can be brought about by dithiothreitol and ferricyanide, but not, or very slowly, by molecular oxygen. The dithiol is protected, in the EIIFru-Zn2+ complex, against alkylation by MalNEt, reversible oxidation by Fe(CN)6(3-) and Cu2+, irreversible oxidation by Cu2+. The pK value of the activity linked thiol is 7.8. Protection experiments show that the dithiol is not located in any of the substrate-binding sites. The redox state of the enzyme does not influence the rate of inactivation of EIIFru by trypsin.
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PMID:The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. EIIFru possesses a Zn2+-binding site and a dithiol/disulfide redox centre. 394 72

Antibodies have been produced, in three rabbits, to Na/K-ATPase purified from guinea pig renal outer medulla. Each rabbit produced antibodies to both the alpha (catalytic) and the beta (glycoprotein) subunits of Na/K-ATPase. The titers of the anti-alpha and anti-beta antibodies varied with time and between rabbits. None of the antisera inhibited Na/K-ATPase activity under various preincubation conditions. A method is presented for separating small amounts of anti-alpha subunit from anti-beta subunit antibodies. There was no cross-reactivity of antibodies to one subunit with the other subunit. The alpha subunit of the Na/K-ATPase was cleaved into a 41,000-dalton peptide (that contains the ATP phosphorylating site) and a 58,000-dalton hydrophobic peptide as described by Castro and Farley (Castro, J., Farley, R.A., 1979, J. Biol. Chem. 254: 2221-2228). Anti-alpha antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the alpha subunit of Na/K-ATPase from human, cow, dog, rabbit, rat, mouse, turtle, and toad; and with the beta subunit from human, rat, and mouse. The loci of cross-reactivity were investigated using partially purified canine kidney Na/K-ATPase cleaved with trypsin as described above. The anti-sera from rabbits 1 and 2 cross-reacted with the 41,000-dalton peptide from the dog but very little with the 58,000-dalton peptide. No cross-reactivity was observed with antiserum from rabbit 3 to either fragment. Guinea pig kidney RNA was translated in a rabbit reticulocyte lysate system followed by immunoprecipitation with the antisera. The molecular weight of the cell-free synthesized alpha chain was 96,000 daltons. Its identity was established with purified anti-alpha antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the beta subunit was not detected in this system.
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PMID:Characteristics of antibodies to guinea pig (Na+ + K+)-adenosine triphosphatase and their use in cell-free synthesis studies. 628 56

A bovine heart protein which specifically inhibits calcium-dependent proteases has been purified to near homogeneity. The purified inhibitor had a Stokes radius of 6.8 nm estimated by gel filtration and a molecular weight of 145,000 estimated by sodium dodecyl sulfate-gel electrophoresis. There is evidence that it may be a glycoprotein. The inhibitor could be phosphorylated by bovine heart cyclic AMP-dependent protein kinase, and its inhibitory effect on Peak II (high-calcium-requiring) protease was modestly increased. However, no other phosphorylating or dephosphorylating conditions significantly influenced its activity. The inhibitor was not hydrolyzed by calcium-dependent proteases, but it was very sensitive to proteolytic inactivation by trypsin or proteases present in a lysosomal fraction from rat heart. Thus, proteolysis may represent a mechanism for decreasing the activity of the inhibitor in different physiologic or pathologic conditions.
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PMID:The protein inhibitor of calcium-dependent proteases: purification from bovine heart and possible mechanisms of regulation. 631 92

The possibility of localization of active sites structural components by affinity labelling was investigated. The modification of E. coli MRE-600 phenylalanyl-tRNA synthetase (E.C.6.1.1.20) (alpha 2 beta 2-type) by the phosphorylating analog of ATP-- [14C]adenosine-5'-trimetaphosphate results in the labelling of both heavy (beta) and light (alpha) enzyme subunits. Analysis of the peptide maps of the tryptic enzyme hydrolysate reveals a great number of peptides containing [14C]radioactivity. The decrease of covalent binding at low concentration of the analog did not abolish the plural labelling. The data permit to consider this kind of analogs as unperspective for localization of specific peptides. Modification of phenylalanyl-tRNA synthetase by tRNAPhe containing the photoreactive group (--CH2CONHC6H5N3) at eighth position of molecule (S8U) results in the labelling of only heavy beta-subunits. These data correspond to the previous results which testify to the disposition of tRNA binding sites on beta-subunits of phenylalanyl-tRNA synthetase. After hydrolysis of the modified phenylalanyl-tRNA synthetase by trypsin six peptides covalently bound with tRNAPhe were revealed. This quantity of modified peptides is higher than the number of tRNA binding sites. Hence the method of affinity labelling has definite limitations for localization of peptides of enzyme active sites.
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PMID:[Study of the possibility of identifying the structural elements of the phenylalanyl-tRNA-synthetase active center by affinity labeling]. 639 Jan 78

By quantitative phosphorus determination on the single chains of human fibrinogen it is demonstrated that the covalently bound phosphorus of adult and fetal fibrinogen is exclusively located in the A alpha chain. The A alpha-chain of fetal fibrinogen contains about twice as much phosphorus as the adult A alpha-chain in the well known position of Ser 3 of fibrino-peptide A as well as in a hitherto unknown second position on the A alpha-chain. By consecutive cleavage of the A alpha-chains of fetal and adult fibrinogen with cyanogen bromide, trypsin, and chymotrypsin, separation of the resulting peptide mixtures and analysis for phosphorylated amino acids, this second phosphorylation site could be traced to Ser 345 of the A alpha-chain. There is only one sequence homology between the two now known in vivo phosphorylation sites of human fibrinogen, namely that the second amino acid to the carboxyl side of the phosphorylated Ser is Glu. The sequence specificity of the up to now unidentified protein kinase phosphorylating fibrinogen allows it to be classified as a member of the group of type-2 casein kinases or casein kinases TS.
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PMID:The location of a second in vivo phosphorylation site in the A alpha-chain of human fibrinogen. 671 96

Sulfopropyl (SP) Sephadex chromatography of trypsin-pepsin digests of dinitrophenylated gizzard myosin, pretreated with and without the myosin light-chain kinase calcium-calmodulin phosphorylating system, yielded similar elution patterns and nine peptide fractions were found. From a comparison with trypsin-pepsin digests of the heavy chains of dinitrophenylated myosin, pretreated with and without the phosphorylating system, it was established that peptide I, a major peptide fraction, was part of the 17-kDa dinitrophenylated light chain. Phosphorylation of myosin did not change the dinitrophenyl group content of peptide I but it did result in a significant increase in the dinitrophenylation of other peptides. The peptides contained only S-dinitrophenyl cysteine. Peptide III, previously considered to be part of the light-chain region (Bailin, G. and Lopez, F. (1982) J. Biol. Chem. 257, 264-270), was shown to originate in the heavy chains of myosin based on a comparison of the elution patterns of the digests of modified myosin and its heavy chains. Several neutral and basic peptides (peptides III to IX) originated in the heavy-chain region and they were different from those from the heavy chains of rabbit skeletal myosin. Phosphorylation of the 20-kDa light chain shifted the dinitrophenylation of the sulfhydryl groups from the 17-kDa light chain to the heavy chains of myosin, predominantly. These thiol groups do not resemble the fast-reacting -SH groups of rabbit skeletal myosin. The light chains are involved, in part, in making sites available on myosin that are necessary for actin-myosin interaction.
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PMID:Peptide fragments of the heavy-chain region of phosphorylated and dinitrophenylated gizzard myosin. 688 76


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