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)

Two tryptic phosphopeptides containing the sites on the alpha and beta subunits of phosphorylase kinase which are phosphorylated by protein kinase, dependent on adenosine 3':5'-monophosphate (cyclic AMP), have been isolated and their amino acid sequences have been determined. 32P-labelled phosphorylase kinase, containing 1.9 mol phosphate per mol enzyme, was digested with an equimolar quantity of trypsin for 2.5 min at pH 7.0, 20 degrees C. This treatment released nearly all the 32P radioactivity associated with the beta subunit as trichloroacetic-acid-soluble material. Only a small proportion of the 32P radioactivity associated with the alpha subunit was solubilised, the remainder being removed in the trichloroacetic acid pellet. The beta-subunit tryptic phosphopeptide was completely resolved from traces of the alpha-subunit phosphopeptide by gel filtration on Sephadex G-25. Further purification by peptide mapping separated the phosphopeptide into four components, each derived from the same nine-amino-acid segment of the betachain, which was found to possess the sequence: Gln-Ser-Gly-Ser(P)-Val-Ile-Tyr-Pro-Leu-Lys. The four components were produced by the partial cyclisation of the N-terminal glutaminyl residue, and by the presence of two alleles for the beta subunit in the rabbit population, which led to a valine-isoleucine ambiguity. The alpha-subunit phosphopeptide was liberated from the trichloroacetic acid pellet by redigestion with trypsin. It was the largest component in the digest which remained soluble in 5% trichloroacetic acid, and obtained in a highly purified form by a single filtration on Sephadex G-50. The peptide comprised 39 amino acids of which nine were serine and three were threonine residues. Only one residue, the serine at position three from the amino terminus, was phosphorylated. The amino-terminal sequence of the peptide was shown to be: Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly. The sequences confirm the stoichiometry of the reaction and the absolute specificity of cyclic-AMP-dependent protein kinase for just two of the 200 serine residues in the enzyme. These results and an inspection of the rate of phosphorylation of a number of skeletal muscle proteins, including each enzyme of the glycolytic pathway, lead to the conclusion that cyclic-AMP-dependent protein kinase is an extremely specific enzyme. The molecular basis of this specificity is discussed.
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PMID:The hormonal control of activity of skeletal muscle phosphorylase kinase. Amino-acid sequences at the two sites of action of adenosine-3':5'-monophosphate-dependent protein kinase. 16 50

Sequences of amino acids at the N-termini of virus proteins VP1, VP2, and VP3 were determined for foot-and-mouth disease virus types A12 strain 119, O1Brugge and C3Resende. In the polyacrylamide gel electrophoresis system used to purify the proteins, VP3 migrated faster than VP1 or VP2; and in the virion, VP3 could be cleaved by trypsin into VP3a and VP3b. The N-terminal amino acids for each of the virus types were glycine in VP1, aspartic acid in VP2, and threonine in VP3. No divergences in sequence across the virus types were indicated until at least the fourth position in VP1, and the third in VP3. For virus types A12, O1 and C3, the sequences were, respectively: for VP1 (Gly-ile-phe,pro,val---), (Gly,ile,phe---) and Gly-ile-phe,ala---); for VP2 (Asp,X,met---), (Asp---) and Asp-leu---); and for VP3 (Thr-thr-ala-thr---), (Thr-thr-ser---) and (Thr-thr---). Unresolved mixtures of VP3a and VP3b, from either A12 or O1 viruses, appeared to have the N-terminal amino acids threonine, which is presumed to be the same threonine as in uncleaved VP3 and serine, which is generated by the tryptic cleavage.
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PMID:N-terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, O, and C. 17 52

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

1. It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End-group analysis after trypsin treatment yields aspartic acid as the new N-terminal amino acid while the C-terminal threonine remains unchanged. 2. NADH, especially in the presence of 2-oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated. 3. The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed. 4. The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N-terminal part of the polypeptide chain is not located in the contact domains between the polypeptide chains. The association of the hexamer to large associated particles is reduced but not abolished. 5. It is shown by treatment of the enzyme with iodo[2(-14)C]acetic acid as well as with Ellman's reagent that the six - SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rat of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme. 6. Cross-linking of the enzyme molecule with bifunctional reagents and subsequent dodecylsulfate-polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three. 7. The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.
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PMID:Studies of glutamate dehydrogenase: analysis of functional areas and functional groups. 24 Jun 78

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.
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PMID:Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology. 26 15

Str. griseus protease hydrolyzes essentially insoluble collagen of bone tissue, with 34.5% of protein solubilized and 6.0% of peptide bonds splitted. 60.0 M of N-terminal amino acids is formed per 10(5) g of protein, out of them 16.8 in the fraction of free amino acids, 32.3 M in the fraction of soluble DNP-peptides and 10.9 M in that of insoluble DNP-peptides. Under the effect of trypsin the amount of collagen changing to the soluble form is thrice as low and the splitted peptide bonds are ten times as low as in case of the Str. griseus protease action. The peptide bonds incorporating the N-end of serine, threonine, glycine are more available for protease. It is supposed that under used conditions Str. griseus protease hydrolyzes not only telepeptides but also the main molecule of collagen.
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PMID:[Study of bone tissue insoluble collagen hydrolysis by Streptomyces griseus protease using the method of N-terminal analysis]. 40 89

The heterophile antigen (Paul-Bunnell antigen, PBA) of infectious mononucleosis was isolated by extraction of an aqueous suspension of bovine erythrocyte stromata with chloroform-methanol (2:1). The upper aqueous layer contained gangliosides, PBA, and a high-molecular-weight glycoprotein. PBA and gangliosides were separated from the high-molecular-weight glycoprotein by extraction of lyophilized upper layer with chloroform-methanol solvents. Separation of PBA from gangliosides was carried out by chromatography on DEAE-cellulose with chloroform-methanol solvents. PBA appeared to be a minor glycoprotein component of the erythrocyte membrane and had both hydrophobic and hydrophilic properties. It was soluble in either organic or aqueous solvents. On SDS-polyacrylamide gel electrophoresis, it migrated as a single component that stained for protein with Coomassie blue, for carbohydrate with periodic acid-Schiff reagent, and for lipid with oil red 0; it had an apparent molecular weight of 26,000. It was composed of 62% protein with major amino acids; glutamic acid, proline, glycine, isoleucine, leucine, and threonine (158, 116, 98, 90, 85, and 82 residues per 1,000 residues, respectively). Carbohydrate content was 9.2% with major sugar constituents: sialic acid, galactosamine, and galactose. Serologic activity of PBA was destroyed by pronase but not by trypsin.
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PMID:Isolation and partial characterization of the heterophile antigen of infectious mononucleosis from bovine erythrocytes. 40 89

The largest fragment produced by complete cyanogen bromide digestion of the alpha chain of human fibrinogen contains 236 residues and has a calculated molecular weight of 23,949. The complete amino acid sequence of the fragment was determined by the isolation of peptides generated by plasmin, trypsin (including digestion of citraconylated material), staphylococcal protease, and chymotrypsin. In addition, some key subfragmentation was achieved by selective chemical cleavage at tryptophan residues. The fragment has an unusual amino acid composition, more than half of its residues being glycine, serine, threonine, and proline. There are very few nonpolar residues, although 7 of the alpha-chain's 10 tryptophans occur in this fragment. The fragment contains 2 cysteine residues located 30 residues apart which are connected by an intrachain disulfide bond in the native molecule. The tryptophans occur with a definite periodicity that highlights a series of 13-residue homology repeats. The fragment also contains the two principal alpha-chain cross-linking sites.
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PMID:Amino acid sequence studies on the alpha chain of human fibrinogen. Complete sequence of the largest cyanogen bromide fragment. 51 44

Casein epsilon-aminolysyl residues were converted to the methyl (and dimethyl), isopropyl or cyclopentyl derivatives in high yield with formaldehyde, acetone or cyclopentanone, respectively, in the presence of sodium borohydride. When incorporated into diets at 10% as the sole protein source, the chemically modified caseins failed to support growth of young rats. Methyl casein did, however, support limited growth after about 5 days. Plasma threonine levels increased and lysine levels decreased markedly in rats fed the alkyl caseins. The respective alkyllsine derivatives were present in plasma and urine. In another experiment, nearly normal or normal growth was obtained by feeding lysine-supplemented methyl or isopropyl casein, respectively. A preparation of partially methylated casein, containing approximately equal amounts of monomethyl- and dimethyllysines, supported normal rat growth. These results demonstrate that lysine deficiency was produced by feeding highly alkylated caseins. Digestibility of the chemically modified caseins in vivo was not affected, although in vitro studies with trypsin and alpha-chymotrypsin showed lowered digestibility. Since no apparent toxicity was observed limited methylation of food proteins may be useful for protection of lysyl residues against deteriorative reactions during processing and storage.
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PMID:Effect of reductive alkylation of the epsilon-amino group of lysyl redsidues of casein on its nutritive value in rats. 56 44

Intraduodenal amino acids are known to stimulate the release of gastric inhibitory polypeptide and cholecystokinin. In order to separate and quantitate gastric inhibitory polypeptide secretion selectively, 12 normal subjects received an intraduodenal perfusion of a mixed amino acid solution (158 mM) containing either methionine, phenylalanine, tryptophan, and valine (perfusate 1), or an amino acid solution containing arginine, histidine, isoleucine, leucine, lysine, and threonine (perfusate 2). Serum concentrations of gastric inhibitory polypeptide and insulin were significantly greater in the group receiving perfusate 2 (P less than 0.001). In contrast, after administration of amino acid perfusate 1, there was only a slight increase in serum gastric inhibitory polypeptide concentration and insulin secretion increased only slightly. Mean trypsin and bilirubin outputs in the group receiving perfusate 1 were nearly 3 times greater than the outputs of the group receiving the other amino acid mixture. This study expands the importance of intraduodenal amino acid mixtures in stimulating secretion of gastric inhibitory polypeptide and insulin and quantitatively separates gastric inhibitory polypeptide release from release of hormones that stimulate pancreatic enzyme secretion, such as cholecystokinin.
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PMID:Selective release of gastric inhibitory polypeptide by intraduodenal amino acid perfusion in man. 64 19


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