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)

A protein kinase activity has been detected in two strains of murine Oncornaviruses, MSV/MLV and EFV. This activity phosphorylates not only endogenous viral proteins but also exogenous substrates (histones and phosvitin). The stimulation of enzyme activity by detergents along with the increase of specific activity in viruses treated with trypsin during purification suggest that the enzyme is located in the viral particle.
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PMID:[Evidence of protein kinase activity in 2 murine oncornaviruses]. 7 Dec 20

Endogenous protein kinase activity was detected in the outer plasma membrane of 373 and SV40 transformed 3T3 cells. When intact cells were incubated with [gamma-32P]ATP, there was a transfer of [32P]phosphate into an acid-insoluble product. The reaction was: (a) linear as a function of time (up to 30 min), (b) proportional to the number of cells present and (c) dependent on temperature and Mg2+ concentration. The acid-insoluble product was susceptible to pronase but not RNase or DNase. More specifically, phosphomonoester bonds to serine and threonine were identified. There was less than 3% hydrolysis of the [gamma-32P]ATP during the reaction; moreover, free [32P]phosphate failed to substitute for the ATP. The reaction product was located on the cell surface, as evidenced by the fact that it could be removed by mild trypsin treatment of intact 3T3 cells. Further evidence for the surface location of the kinase was shown by its activity in phosphorlating exogenous substrate, histone, and phosvitin. The level of phosphorylation increased by 2- to 4-fold prior to the start of S phase when quiescent 3T3 cells were stimulated to reinitiate growth by the addition of serum. The SV40 3T3 cells had from 5- to 10-fold more activity per cell than the quiescent 3T3 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioautography show at least 25 phosphorylated proteins; the surface label pattern of 3T3 cells differs from that of SV40-transformed 3T3 cells.
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PMID:Endgoenous protein kinase in outer plasma membrane of cultured 3T3 cells. Nature of the membrane-bound substrate and effect of cell density, serum addition, and oncogenic transformation. 18 98

A heat-and acid-stable protein inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does no occur when phosphorylated histone or phosphorylated protamine are used as substrates.
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PMID:Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeleta muscle. 19 98

Protein kinase associated with rat liver microsomes was only partly extracted by treatment with 1.5 M KCl. The enzyme was solubilised by Triton X-100 or sodium deoxycholate at the same or slightly higher detergent concentrations than microsomal marker components. The enzyme activity increased 2-3 fold upon solubilisation. Three peaks with protein kinase activity (fractions MI, MII and MIII) were resolved on DEAE-cellulose chromatography. Fraction MIII but not fractions MI or MII was activated by adenosine 3':5'-monophosphate (cyclic AMP). All fractions catalysed the phosphorylation of protamine and histones but not that of casein or phosvitin. Fractions MI and MIII had a similar substrate specificity and phosphorylated histones at a relatively much higher rate than did fraction MII. The isoelectric points were 8.1 for fraction MI, 5.5 for fraction MII and 4.9 for fraction MIII. On incubation of fraction MIII with cyclic AMP it was split into two catalytically active components with pI 8.1 and 7.35. The component with pI 8.1 was predominant and corresponded to fraction MI. Five protein kinase peaks were resolved from rat liver cytosol by DEAE-cellulose chromatography. Three of them (fractions CIa, CIIb and CIII) had the same properties as each of the microsomal kinase fractions. A forth fraction (CIIa) was cyclic-AMP-dependent and had the same substrate specificity as fractions MI and MIII. Its pI was 5.1, and it was split into two components by cyclic AMP (pI 8.1 and 7.35). In binding studies fraction CIIb bound more efficiently to microsomes than fraction CIII, while fractions CIa, CIIa and the microsomal protein kinase fractions did not bind appreciably. When microsomes were treated with trypsin exposed protein kinase was inactivated and the latency of the remaining enzyme increased substantially. Most of fraction MII was inactivated by trypsin while fraction MIII was resistant. The possible orientation of protein kinase fractions MII and MIII in the microsomal membrane is discussed.
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PMID:Protein kinases of rat liver endoplasmic reticulum. Solubilisation, partial characterisation and comparison with protein kinases of rat liver cytosol. 20 48

A protein phosphokinase (EC 2.7.1.1.37) was isolated from baker's yeast (Saccharomyces cerevisiae) after a 17,000-fold purification; the purified enzyme is homogeneous according to the criteria of gel electrophoresis and ultracentrifuge analysis. The enzyme has a high isoelectric point of ca. 9 and appears to exist as a monomer with a molecular weight of 42,000 plus or minus 1500. It is neither stimulated by cyclic 3',5'-AMP, -GMP, -CMP or -ump nor inhibited by the regulatory subunit of rabbit muscle protein kinase (Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971), J. Biol. Chem. 246, 1986). In the presence of divalent metal ions, preferably Mg-2+ or Mn-2+, the enzyme readily transfers the terminal phosphate group of ATP to phosvitin, alphaS1B- and beta a-casein and an NH2-terminal tryptic peptide derived from beta a-casein, but not to protamine, lysine, or arginine-rich histones or to yeast enzymes such as phosphorylase, phosphofructokinase, or pyruvate carboxylase; serine and polyserine were also inactive as phosphate acceptors. Km values of 0.17 mM for beta a-casein and 0.2 mMfor ATP were determined at 10 mM Mg-2+. The urified yeast protein kinase also catalyzes the reverse reaction, namely, the transfer of phosphate from fully phosphorylated beta a-casein or its NH2-terminal peptide to ADP resulting in the formation of ATP. AMP, GDP, UDP, and CDP did not serve as phosphate acceptors in this reaction. As observed by Rabinowitz and Lipmann (Rabinowitz, M., and Lipmann, F. (1960), J. Biol. Chem. 235, 1043) both reactions have different pHoptima with values of 7.5 for the forward reaction (phosphorylation of the proteins) and ca 5.2 for the formation of ATP; both are differently affected by salts. Phosphorylation of beta a-casein with [gamma-32-P]ATP followed by digestion of the labeled protein with trypsin indicated that all the radioactivity was exclusively introduced in an NH2-terminal peptide possessing the unique sequence: Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu...(Ribadeau-Dumas, B., Brignon, G., Grosclaude, F., and Mercier, J.-C. (1971), eur J. Biochem. 20, 264). By subjecting beta a-casein and its NH2-terminal peptide to the combined action of almond acid phosphatease and purified yeast protein kinase, it was determined that the phosphorylation and dephosphorylation reactions proceed randomly, i.e., all seryl phosphate residues are equally susceptible and that the rate of phosphorylation decreases drastically as the number of bound phosphate groups in the substrate diminishes.
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PMID:Purification and properties of a yeast protein kinase. 23 75

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].
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PMID:Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein. 84 74

A form of glycogen synthase kinase designated GSK-M3 was purified 4000-fold from rat skeletal muscle by phosphocellulose, Affi-Gel blue, Sephacryl S-300 and carboxymethyl-Sephadex column chromatography. Separation of GSK-M from the catalytic subunit of the cAMP-dependent protein kinase was facilitated by converting the catalytic subunit to the holoenzyme form by addition of the regulatory subunit prior to the gel filtration step. GSK-M had an apparent Mr 62,000 (based on gel filtration), an apparent Km of 11 microM for ATP, and an apparent Km of 4 microM for rat skeletal muscle glycogen synthase. The kinase had very little activity with 0.2 mM GTP as the phosphate donor. Kinase activity was not affected by the addition of cyclic nucleotides, EGTA, heparin, glucose 6-P, glycogen, or the heat-stable inhibitor of cAMP-dependent protein kinase. Phosphorylation of glycogen synthase from rat skeletal muscle by GSK-M reduced the activity ratio (activity in the absence of Glc-6-P/activity in the presence of Glc-6-P X 100) from 90 to 25% when approximately 1.2 mol of phosphate was incorporated per mole of glycogen synthase subunit. Phosphopeptide maps of glycogen synthase obtained after digestion with CNBr or trypsin showed that this kinase phosphorylated glycogen synthase in serine residues found in the peptides containing the sites known as site 2, which is located in the N-terminal CNBr peptide, and site 3, which is located in the C-terminal CNBr peptide of glycogen synthase. In addition to phosphorylating glycogen synthase, GSK-M phosphorylated inhibitor 2 and activated ATP-Mg-dependent protein phosphatase. Activation of the protein phosphatase by GSK-M was dependent on ATP and was virtually absent when ATP was replaced with GTP. GSK-M had minimal activity toward phosphorylase b, casein, phosvitin, and mixed histones. These data indicate that GSK-M, a major form of glycogen synthase kinase from rat skeletal muscle, differs from the known glycogen synthase kinases isolated from rabbit skeletal muscle.
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PMID:Characterization of GSK-M, a glycogen synthase kinase from rat skeletal muscle. 282 16

In oviparous vertebrates estrogens induce hepatic synthesis of vitellogenin (VG), a blood protein sequestered in vitellogenic oocytes and from which lipovitellin (LV) and phosvitin are derived. Our objective was to identify VG in the channel catfish, Ictalurus punctatus. An intraperitoneal injection of estradiol-17 beta into adult male fish induced a dose-dependent accumulation of a 150 kDa protein (EP) in the plasma. EP was detectable in Coomassie blue-stained polyacrylamide gels within 24 hr after injection of 2 mg hormone/100 g body weight. During the next 4 days, EP increased from 5 to about 25% of the total plasma protein. Electrophoretic mobility, peptide mapping, and immunological crossreactivity showed EP to be indistinguishable from a plasma protein in adult females with vitellogenic ovaries. Two major yolk polypeptides, YP1 (120 kDa) and YP2 (29.6 kDa), were precipitated by (NH4)2SO4 from a yolk protein extract. YP1 but not YP2 reacted with an anti-EP polyclonal antiserum in Western blots. Peptide mapping after proteolysis with trypsin showed YPs 1 and 2 to be unique and revealed structural homologies between YP1 and EP. Liver but not pancreatic explants from an estradiol-treated male synthesized and secreted a [35S]methionine-labeled, 150 kDa protein beginning about 2 hr after initial exposure to the label. We tentatively conclude that EP and YP1 represent VG and LV, respectively. YP2 remains unidentified.
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PMID:Vitellogenin induction by estradiol in channel catfish, Ictalurus punctatus. 292 Aug 97

Glycogen synthase I from human polymorphonuclear leukocytes was phosphorylated with cAMP dependent protein kinase, synthase kinase or phosvitin kinase prepared from these cells. Limited tryptic hydrolysis released four phosphopeptides (t-A, t-B, t-C, t-D). Subsequent alpha-chymotryptic hydrolysis of the trypsin resistant core released three phosphopeptides (c-A, c-B, c-C). The kinetic changes of glycogen synthase were compared with the phosphorylation of the peptides. Equivalent kinetic changes (Kc equals 0.2-0.3 mM Glc-6-P) were obtained when 1 Pi/subunit was introduced by cAMP dependent protein kinase, 0.5 Pi/subunit by synthase kinase and 0.8 Pi/subunit by both kinases. Initially, cAMP dependent protein kinase phosphorylated peptides c-A and t-C in parallel and somewhat later also t-B, whereas synthase kinase initially phosphorylated only c-A. The ultimate effect of the two kinases on c-A was additive. It was concluded that the initial kinetic changes were dependent on phosphorylation of c-A, which contained two sites, one for each kinase. The same kinetic changes were induced by phosphorylation on each of the sites. In the subsequent phosphorylation the kinases, separately or together, phosphorylated peptide c-C indicating one non-specific phosphorylatable site in this peptide. The cAMP dependent protein kinase alone phosphorylated t-C maximally, whereas both kinases were required for an equal phosphorylation of t-A and t-B. It is suggested that the cAMP dependent protein kinase phosphorylated t-A and t-C, whereas the data did not allow a similar suggestion for t-B. The kinetic changes occurring during the later stages of phosphorylation were an increase in Kc for Glc. 6-P to 4-5 mM at 1.85 Pi/subunit and to 20 mM at 3.3 Pi/subunit, but the changes could not be assigned to phosphorylation of any specific peptide. Phosphorylation of the peptides t-D and c-B were insignificant, but c-B may be phosphorylated under other experimental conditions (25). The phosvitin kinase phosphorylated glycogen synthase extremely slowly to an extent of 0.8 Pi/subunit, mainly in peptide c-C. Glycogen synthase would appear without physiological importance as substrate for this kinase. Phosphorylase kinase from rabbit skeletal muscle incorporated 0.7 Pi/subunit, mainly in peptide c-A causing a decrease in RI to 0.3, which upon further incubation remained constant. The rate of decrease in RI in 0.5 was unaffected by several synthase modifiers, including Glc-6-P, but was inhibited by ADP and Pi. The rate of phosphorylation by cAMP dependent protein kinase and synthase kinase was diversely affected in different buffers, however, without affecting the ultimate phosphorylation pattern.
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PMID:Phosphorylation of glycogen synthase I from human polymorphonuclear leukocytes. 626 29

A rabbit liver protein kinase (PC0.7), able to phosphorylate glycogen synthase and phosvitin, has been extensively purified. The enzyme had apparent Mr = 170,000-190,000 as judged by gel filtration and was associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000). Two other polypeptides, Mr = 38,000 and Mr = 35,000, were also detected. Treatment with trypsin led to an enzyme composed only of polypeptides of Mr = 35,000 and Mr = 25,000. The beta-polypeptide underwent autophosphorylation when incubated with Mg2+ and ATP or GTP. The protein kinase was effective in utilizing both ATP and GTP as the phosphoryl donor (apparent Km values 5-11 microM and 9-19 microM, respectively). The enzyme phosphorylated phosvitin, casein, and glycogen synthase but not histone or phosphorylase and was inhibited by heparin. Phosphorylation of glycogen synthase proceeded to approximately 0.5 phosphate/subunit with little inactivation of the glycogen synthase. The phosphorylation occurred predominantly in a 21,000-dalton CNBr fragment of glycogen synthase that had been previously shown to reside toward the COOH terminus of the molecule. The liver PC0.7 appeared very similar to an analogous enzyme isolated from rabbit muscle (DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962). The present work, therefore, provides a point of contact between the Ca2+ and cyclic nucleotide-independent glycogen synthase kinases of rabbit liver and muscle.
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PMID:Rabbit liver glycogen synthase kinases. Characterization of a protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin. 627 17


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