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)

Infection of BHK 21 cells by vesicular stomatitis virus (VSV) results in the intracellular synthesis of the five viral proteins which are easily detectable in polyacrylamide gels after short labeling periods with [35S]methionine. In addition, a 6th prominent radioactive protein band appears intracellularly in VSV-infected BHK cells. This additional polypeptide is also coded by the viral genome, because it is immunoprecipitated by antibodies against viral particles and more specifically by antibodies against purified G-protein. We propose to call this derivative of the G-protein Gsi-protein (short intracellular G-protein). It is associated with intracellular membranes and has an apparent mol. wt. of 58 000. Both G- and Gsi-protein have the same kinetics of appearance in the cell. The ratio of G-:Gsi-protein in BHK 21 cells is approximately 85:15. The mol. wt. difference of approximately 6000 daltons between G- and Gsi-protein is not due to variations in the degree of glycosylation because trypsin digestions of both [3H]mannose-labeled glycoproteins gave rise to identical glycopeptide patterns. Incubation of microsomes with trypsin demonstrates that Gsi-protein is protected in its full length by intracellular membranes. Gsi-protein is lacking an extended carboxy-terminal region of the viral G-protein sequence because it is not modified by palmitic acid and is not immunprecipitated by specific antibodies against a C-terminal peptide of the G-protein. Limited proteolysis by endoproteinase arg C indicates that the structure of Gsi-protein is very similar to the shedded form of the G-protein which has been previously described in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intracellular appearance of a glycoprotein in VSV-infected BHK cells lacking the membrane-anchoring oligopeptide of the viral G-protein. 608 25

Previously a new small subclass of SV40 large T antigen with a high-binding affinity to living target cells was characterized (J. Lange-Mutschler and R. Henning, 1983, Virology 127, 333-344.) In the present study the external binding process, particularly the tight linkage of T antigen to lipid of the target cells, was analyzed. Extraction of SV40-transformed target cells (SV80) first by sonification yielded approx 80% of [35S]methionine-labeled T antigen (mechanical extract). A further 20% was obtained by treatment of cellular debris with hydroxylamine (hydroxylamine extract). As shown by an 125I-protein A radioimmunoassay, hydroxylamine extracts contained significantly higher amounts of cell surface binding T antigen. Correspondingly, after incubating [3H]palmitic acid-prelabeled target cells (HeLa) with unlabeled extracts, predominantly T antigen from hydroxylamine extracts became 3H labeled by the target cells, dependent on metabolic or enzymatic conditions. 3H-labeled T antigen became unlabeled after treatment with hydroxylamine indicating a covalent ester linkage between cell surface-bound T antigen and lipid of the target cells. The cell surface localization of in vitro acylated T antigen was demonstrated by mild trypsin digestion of living target cells. These results strongly support the idea about a novel mechanism by which a minor subclass of T antigen after being bound to the cell surface becomes covalently linked to lipid of the living cell.
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PMID:Cell surface binding simian virus 40 large T antigen becomes anchored and stably linked to lipid of the target cells. 608 50

Fatty acid synthetases isolated from all mammalian tissues synthesize predominately palmitic acid. However, in vivo the mammary gland fatty acid synthetases of some species are responsible for the synthesis of medium chain fatty acids. The objective of this presentation is to outline the mechanism which regulates the product specificity of fatty acid synthetases in general and to illustrate how this control is modified in the mammary gland. Fatty acid synthetases isolated from mammalian tissues are composed of two similar, probably identical, polypeptides, each carrying as many as seven enzyme components. Thioesterase I, the component which functions to terminate growth of acyl chains on the multienzyme, is located at one terminus of each polyfunctional polypeptide and can be detached by limited proteolysis with trypsin. By studying separately the kinetics of chain elongation by the core of the trypsinized complex and of chain termination by the isolated thioesterase I component, it has been possible to establish that the specificities of the elongation and termination reactions account for the synthesis of predominantly the carbon-16 fatty acid by purified fatty acid synthetases. Mammary glands of some species contain an additonal enzyme, thioesterase II, which can modify the product specificity of fatty acid synthetase by hydrolyzing medium chain acyl moieties from thioester linkage to the 4'phosphopantetheine of the multienzyme. At all stages of development of rat mammary gland, the amount of theoesterase II present correlates well with the proportion of medium chain fatty acids synthesized by the gland. This mammary gland-specific thioesterase appears responsible for the ability of this tissue to synthesize medium chain fatty acids characteristic of milk fat.
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PMID:Mechanism of chain length determination in biosynthesis of milk fatty acids. 610

The human transferrin receptor could be fluorographically detected after immunoprecipitation from a leukemic T-cell line labeled with [3H]palmitic acid. The label was found ony in association with the human transferrin receptor and not in association with two other major plasma membrane glycoproteins, demonstrating that the incorporation of radioactivity was not due to metabolism of the palmitate. Treatment of sodium dodecyl sulfate-polyacrylamide gels containing the [3H]palmitate-labeled transferrin receptor with hydroxylamine, prior to fluorography, resulted in release of a substantial fraction of the label from the molecule. In addition, at least part of the label released from immunoprecipitates of the transferrin receptor by treatment with hydroxylamine was identified as palmitohydroxamate, providing further evidence that the labeled fatty acid is covalently bound to the receptor. A proteolytic fragment (Mr = 70,000) derived from the portion of the transferrin receptor exposed on the cell surface can be obtained by trypsin digestion of intact or Nonidet P-40-solubilized cells. When cells were labeled with [3H]palmitic acid, none of the radioactivity could be detected in the tryptic fragment. Thus, the bound palmitate appears to be associated with the region of the molecule that is in close proximity to the plasma membrane.
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PMID:Covalent binding of fatty acid to the transferrin receptor in cultured human cells. 626

A DNA sequence consisting of 24 base pairs was inserted into the structural gene (lpp) coding for the major lipoprotein of the Escherichia coli outer membrane which was carried on a high-copy-number plasmid in which expression was regulated through a lac promoter-operator region. This modification resulted in the insertion of eight amino acid residues, Glu-Glu-Phe-Leu-Glu-Glu-Phe-Leu, between the glutamine residue at position 9 and the leucine residue at position 10 of the wild-type lipoprotein sequence. When production of the mutant lipoprotein was induced by a lac inducer, the cells became swollen, showed unusual morphology, and eventually lysed. When the membrane fraction was analyzed after the induction, the mutant lipoprotein was found to have been normally secreted across the cytoplasmic membrane and assembled in the outer membrane. This lipoprotein was modified with glycerol and palmitic acid and even formed the bound form, which was linked covalently to peptidoglycan. The major difference between the membrane-associated mutant lipoprotein and the wild-type lipoprotein was that the mutant lipoprotein became sensitive to trypsin treatment. These results indicate that the substantial alteration in mutant lipoprotein structure near the amino-terminal end does not interfere with modification of the amino-terminal cysteine residue or cleavage of the signal peptide by the prolipoprotein-specific signal peptidase. However, this mutant lipoprotein assembled in the outer membrane appears to have deleterious effects with respect to envelope structure and cellular morphology and viability.
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PMID:Effects of inserting eight amino acid residues into the major lipoprotein on its assembly in the outer membrane of Escherichia coli. 634 4

The aggregation properties of Streptococcus mitis ATCC 903 cells modified by treatment with heat or different enzymes was investigated. Bacteria that had the ability to aggregate spontaneously lost this capacity by treatment with proteolytic enzymes, beta-galactosidase or heat. Cells subjected to different types of modification were mixed in various proportions and their aggregation properties were recorded. To discriminate between the two kinds of cells in the suspension, one partner in the aggregation reaction was labelled with 14C-palmitic acid. Bacteria treated with beta-galactosidase co-aggregated with spontaneously aggregating cells (not modified) and with cells treated with heat. Heat-treated cells co-aggregated with spontaneously aggregating cells and with cells treated with beta galactosidase. Cells treated with trypsin did not co-aggregate either with spontaneously aggregating cells or cells treated with heat or beta-galactosidase. These findings are consistent with the hypothesis that two surface components are required for specific aggregation of S. mitis cells. We suggest that both components are degraded or released from the bacterial surface by treatment with trypsin (and other proteolytic enzymes) as shown by the inability of these cells to take part in any co-aggregation with spontaneously aggregating cells. Treatment with beta-galactosidase degrades a carbohydrate receptor constituting the terminal part of a glycoprotein. Heat treatment inactivates a protein lectin. The fact that heat-treated bacteria and bacteria treated with beta-galactosidase aggregate when mixed supports the assumption that two components take part in the aggregation reaction.
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PMID:Aggregation of enzymatically modified Streptococcus mitis indicating involvement of lectin-ligand type interaction. 636 25

The present work concerns our studies to search for factor(s) which may influence the hemostatic process in or around metastasis of tumours. We studied the platelet aggregating property of a methyl cholanthrene induced experimental tumour. Platelet aggregating material was found to be different from the known aggregating agents like thrombin, ADP, collagen, thromboxane A2 and trypsin. It depends on a critical level of calcium for its action. PAM is a high molecular weight substance which contains sialic acid. It is trypsin and plasmin insensitive. The activity of this substance is not being destroyed by phospholipase-C. Metabolic study indicates that PAM acts by mitochondrial energy metabolic pathway of the platelets.
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PMID:A new platelet aggregating material (PAM) in an experimentally induced rat fibrosarcoma. 674 May 52

Stearyl-CoA was shown to stimulate the reoxidation rate of cytochrome b5 of gastric microsomes and to decrease the reduction rate of trypsin-purified hog liver cytochrome b5 by the NADH-cytochrome b5 reductase of these microsomes. This latter effect was (1) proportional to microsome concentration and to stearyl-CoA concentration with an apparent Km of 3.3 . 10(-6) M and a Vmax of 71 nmol per min and per mg microsomal protein, (2) insensitive to ATP and inhibited by 1.4 mM KCN, (3) mimicked by palmityl-CoA but not by stearic nor palmitic acid. Direct assays carried out using [14C]stearyl- and [14C]palmityl-CoA as substrates showed a production of 0.12 nmol of oleic and palmitoleic acid, respectively, per min per mg of microsomal protein. In the presence of Tb5 antibodies the reaction was inhibited by 40%. These results support the occurrence of cytochrome b5-dependent fatty acid delta 9 desaturation in gastric microsomes.
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PMID:Cytochrome b5-dependent delta 9 desaturation of fatty acids in gastric microsomes. 684 48

We used porcine pancreatic elastase to isolate type II cells from the lungs of rats; the yield and purity of the type II cells was better than that obtained by methods using trypsin. In 102 experiments we obtained 82 +/- 23 . 10(6) cells/rat, 68 +/- 11% (mean +/- S.D.) of which type II cells. This preparation of cells, when centrifuged over a discontinuous density gradient, yielded 25 +/- 10 . 10(6) cells/rat, 80 +/- 13% of which were type II cells (n = 102). The cells, after density gradient centrifugation, could be futher purified by centrifugal elutriation (94 +/- 3% type II cells, n = 22) or adherence in primary culture (94 +/- 2% type II cells, n = 34). Type II cells isolated with elastase are similar morphologically and biochemically to type II cells isolated from rats with trypsin. The preparations of cells appeared healthy by several different criteria: ultrastructure, exclusion of vital dye, lack of stimulation of oxygen consumption by exogenous sodium succinate, and linear rates of oxidation of [1-14C]palmitic acid and of incorporation of [1-14C]acetate into fatty acids. Type II cells consumed 75 +/- 20 nmol O2/10(6) cell per h, oxidized [1-14C]palmitic acid at a rate of 0.4 nmol/10(6) cells per h, and incorporated [1-14C]acetate into fatty acids at a rate of 7.5 nmol/10(6) cells per h.
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PMID:Metabolic properties and ultrastructure of alveolar type II cells isolated with elastase. 690 14

T protein was extracted with trypsin from an avirulent, M protein-deficient, type 1 group A Streptococcus and purified by ammonium sulfate precipitation and anion-exchange chromatography. The latter procedure removed contaminating lipoteichoic acid (LTA) from the T protein, which consisted of a heterogeneous mixture of polypeptides resistant to digestion by trypsin and ranged in molecular size from 160,000 to 200,000 daltons. Threonine, aspartic acid, glutamic acid, lysine, and valine were the most predominant amino acids. The binding of LTA to an affinity column of T protein was reversible with increasing concentrations of ethanol but not with increasing ionic strength. T protein bound less palmitic acid and LTA than did fatty acid-free bovine albumin and did not stimulate human peripheral lymphocytes. Because the surface and cell wall distribution of the T proteins and LTA appear similar, the possibility exists that T proteins and LTA may interact in situ by weakly hydrophobic bonds. Such ligand-ligand interaction may be indirectly involved in the adherence of group A streptococci to host cell membranes that is known to be mediated by LTA.
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PMID:Lipoteichoic acid-binding and biological properties of T protein of group A streptococcus. 701 85


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