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 hypothesis that insulin action involves a membrane proteolytic step was further explored, by using isolated rat adipocytes and liver plasma membranes. (1) The maximal insulin stimulation of 2-deoxyglucose transport and lipogenesis in fat-cells was selectively inhibited (73-88%) by N alpha-p-tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl; active-site inhibitor of trypsin; 30-125 microM), p-nitrophenyl p'-guanidinobenzoate (active-site inhibitor of serine proteinases; 30-125 microM) and p-tosyl-L-arginine methyl ester (arginine ester substrate analogue of proteinases; 1-2 mM), under conditions where neither the basal rate of each metabolic process nor insulin binding nor cellular ATP content were affected. In contrast, N-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester (alanine ester substrate analogue of proteinases; 1-2 mM) was ineffective. (2) Endoproteinase Arg-C (0.25-40 micrograms/ml) exerted dose-dependent insulin-like effects on both 2-deoxyglucose transport and lipogenesis in fat-cells, whereas endoproteinase Lys-C (5-100 micrograms/ml) was ineffective. The maximal activation by endoproteinase Arg-C of both processes (200 and 177% of control values respectively) was shown to occur under conditions where membrane integrity (assessed by measurement of lactate dehydrogenase leakage and passive glucose diffusion) was preserved. This effect was inhibited by Tos-Lys-CH2Cl (125 microM) and was not additive with the maximal insulin effect. (3) Insulin (1-100 ng/ml) produced a dose-dependent increase in the trichloroacetic acid-soluble 125I radioactivity released after a 30 min incubation at 37 degrees C of 125I-labelled liver plasma membranes, but was ineffective on 125I-labelled bovine serum albumin. Insulin effects on both radio-labelled proteins were reproduced by wheat-germ agglutinin (20 micrograms/ml), an insulin mimicker shown to act through the insulin receptor. These data provide further evidence for the hypothesis that insulin bioeffects involve the activation of a membrane serine proteinase with arginine specificity.
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PMID:Further evidence for the involvement of a membrane proteolytic step in insulin action. 388 92

We examined the binding and internalization of unlabeled and 125I-labeled, purified lactate dehydrogenase-elevating virus (LDV) by peritoneal macrophages cultured in vitro. Upon incubation of the cells at 4 degrees C with greater than 100 ID50/cell, the virus was surface-bound on a small subpopulation of macrophages (about 5% of the total cells) as determined by electron microscopy, fluorescent antibody staining, and autoradiography of cells incubated with 125I-labeled LDV. At 37 degrees C, LDV particles were seen in intracellular endocytic vesicles also in about 5% of the cells, and the proportion of cells with virus-containing vesicles correlated with the proportion of cells which became productively infected with LDV as assessed by determining LDV RNA synthesis in individual cells and by fluorescent antibody staining. Pretreatment of the resident peritoneal macrophages with trypsin inhibited the binding of 125I-labeled LDV and the productive infection of the cells with the virus. After removal of the trypsin and incubation in complete medium, permissiveness for LDV reappeared after an 8-12 h lag, whereas Fc and C3 receptors reappeared more rapidly after trypsin treatment. Populations of resident peritoneal macrophages, starch-elicited peritoneal macrophages, splenic macrophages, and bone marrow macrophages contained a similar proportion of cells that could be productively infected with LDV. Little, if any, LDV replication was detected in cultures of lung, liver and peripheral blood macrophages as well as in thioglycollate-elicited and BCG-activated macrophages. We conclude that the permissiveness for LDV of resident peritoneal macrophages correlates with the presence of a trypsin-sensitive receptor present on a subpopulation of these cells. The identity of the receptor has not been definitively established. Treatment of macrophages with neuraminidase or various sugars had no significant effect on LDV replication. Lysis of I-A-positive macrophages with a monoclonal antibody and complement reduced the number of macrophages which could be productively infected by 50%, which suggests that macrophages lacking surface Ia can be productively infected with LDV in vitro.
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PMID:Cell surface receptors for lactate dehydrogenase-elevating virus on subpopulation of macrophages. 389 Apr 5

Skeletal muscle extract contains a previously undocumented 1300- to 1500-Da neurotrophic factor. Incubation of ventral spinal cord neurons in the presence of this factor enhances the rate of de novo acetylcholine synthesis two- to threefold over control cells, after 6 days in culture. This effect on cholinergic activity appears to be selective, since incubation with the factor results in only slight elevations of lactate dehydrogenase activity and DNA content, and no increase in the acetylcholinesterase activity. The 1300- to 1500-Da factor is acid-stable and partially sensitive to proteolysis by proteinase K, Staphylococcus aureus V8 protease, and subtilisin, but insensitive to trypsin. These results indicate that the active moiety is a peptide. The importance of peptides as neurotransmitters or neuromodulators is well accepted, but their role in the regulation of neuronal development is not widely appreciated. The present cholinergic neurotrophic peptide is distinct from previously characterized cholinergic trophic factors and represents the first example of a small, target-derived peptide which influences cholinergic development.
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PMID:Low-molecular-weight peptide stimulates cholinergic development in ventral spinal cord cultures. 390 37

1. Relative rates of enzyme inactivation were measured in liver slices, homogenates and cytosol fractions as well as in the presence of trypsin and at acid pH. The enzymes chosen are all present in the cytosol fraction of rat liver, and have widely different degradation rate constants in vivo. 2. The inactivation rates of lactate dehydrogenase, fructose bisphosphate aldolase, glucose 6-phosphate dehydrogenase, glucokinase, phosphoenolpyruvate carboxykinase (GTP), l-serine dehydratase and thymidine kinase in liver preparations at neutral pH are in a similar order to the rate constants of degradation of these enzymes in the intact animal. 3. The two exceptions of this general correlation were tyrosine aminotransferase, which was stable in vitro but not in vivo, and glyceraldehyde phosphate dehydrogenase, which shows the reverse pattern. 4. These findings generally support the concept that the same factors are responsible for enzyme inactivation in vitro as occur in the intact tissue.
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PMID:The relative stability of liver cytosol enzymes incubated in vitro. 415 34

Electrohydraulic shock was shown to produce oxidation reactions which inactivated certain compounds important in cellular metabolism. Enzymes that were inactivated included lactic dehydrogenase, trypsin, and proteinases of Bacillus subtilis. Free sulfhydryl groups and reduced nicotinamide adenine dinucleotide were oxidized. Adenosine triphosphate was destroyed, but deoxyribonucleic acid was not affected. Intracellular material of Escherichia coli lost its ability to absorb at 260 mmu after electrohydraulic shock. The bactericidal mechanism involved appeared to be due to nonselective oxidation reactions produced by high-voltage discharges in water. These oxidation reactions were probably mediated by free radicals produced in the water.
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PMID:Mechanism of the bactericidal action produced by electrohydraulic shock. 429 20

The activity of the neutral, Mg2+-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, alpha-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg2+-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.
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PMID:Evidence that neutral sphingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane. 609 59

In order to test the possible involvement of surface proteins on some metabolical aspects of chick glial cell differentiation in culture, perturbations were induced on the glial cell surface membrane by limited trypsinization before seeding. The developmental changes of enzymes involved in the energy metabolism of the cell: malate dehydrogenase (MDH), glutamate dehydrogenase (GDH), hexokinase (HK), lactate dehydrogenase (LDH), enolase as well as glutamine synthetase (GS) were determined in trypsin treated cells and controls. The total protein and DNA content per dish was higher in treated cells than in controls, however the protein ratio towards DNA remained unchanged. The levels of GS, GDH, LDH, and enolase activities were significantly enhanced after trypsin treatment of the cells compared to controls. The enhanced value of total LDH activity is essentially the result of the increase of M subunit containing isoenzymes. Considering that a higher level of GS activity characterizes some maturation of the glial cells (as observed during the maturation of the chick brain) it is apparent that modifications of cell surface located factors, by trypsin treatment, induce differentiation phenomena at the functional state of the glial cells in culture. This may indicate that interactions located at the cell surface are involved in the modulation of key enzymes of the energy metabolism pathway.
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PMID:Trypsinization of chick glial cells before seeding: effects on energy metabolism enzymes and glutamine synthetase. 614 Jun 46

A superfusion technique was developed as a model system for the study of stimulus-secretion coupling in collagenase-dispersed rat pancreatic acinar cells. Cells (10(7)) were combined with a slurry of Biogel P-4 beads and the mixture was decanted into a plastic column (1.5 cm X 8.5 cm) and perfused with Krebs-Ringer. Amylase activity was determined in sequentially collected effusate fractions and used to estimate the secretory rate. Carbachol, carbachol plus dibutyryl cyclic AMP, cholecystokinin-pancreozymin, and the ionophore A-23187 all stimulated a rapid increase in the rate of secretion. Cell integrity was unaffected by these stimulants as evidenced microscopically and by the lack of lactate dehydrogenase activity in the effusates. Enzymes secreted in response to secretagogues were collected, concentrated, and isoelectrofocused on polyacrylamide gels. A film detection technique was developed to localize amylase activity. The model system has the following advantages: (1) secreted proteolytic products are removed from the vicinity of cells, thereby preventing direct cellular damage and hydrolysis of peptide agonist; (2) the need to add trypsin inhibitors is eliminated and only a minimal addition of albumin (0.001%) is required, thus allowing the separation and distortion-free analysis of secreted proteins; (3) the perfusion conditions can be changed rapidly without disturbing the cells. The model described is therefore well suited to the study of both molecular and kinetic events involved in the enzyme secretory phenomenon in exocrine pancreas.
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PMID:A model system for the study of stimulus - enzyme secretion coupling in rat pancreatic acinar cells. 616 55

A shift in the incubation temperature of rabbit alveolar macrophages (0 degree C leads to 37 degrees C leads to 0 degree C) resulted in a 40-60% reduction in the ability of cells to bind alphamacroglobulin. 125I-trypsin complexes (alphaM. 125I-T). The reduction in binding activity did not reflect a disruption of cell integrity since the levels of intracellular components (lactate dehydrogenase, beta-N-acetyl-hexosaminidase) or other plasma membrane components (alkaline phosphodiesterase) were unaltered. Analysis of receptor-ligand interaction indicated that the temperature shift effected a decline in receptor number rather than an alteration in ligand-receptor affinity. Studies indicated that a temperature shift resulted in the loss of unoccupied receptors, and that ligand bound to receptors was not lost. However, after ligand internalization, receptors were removed by the temperature shift. The rate of receptor loss was maximal when cells were incubated at temperatures greater than 24 degrees C. Receptor loss was not prevented by treatment of cells with colchicine, cytochalasin B, or N-ethylamaleimide, but was prevented by treatment with the cross-linking agent paraformaldehyde. Data indicate that the reduction in alphaM. 125I-T binding activity resulted from shedding of receptors into the media since media obtained from temperature-shifted cells contained material that competed with cell-bound receptors for alphaM. 125I-T. Additionally, binding of alphaM. 125I-T was diminished on membrane fragments obtained from temperature-shifted cells. Incubation with Triton X-100, of cells whose receptors were occupied with alphaM. 125I-T, led to the extraction of 40% of cell-bound activity. However, no radioactivity was extracted from cells labeled with alphaM. 125I-T after a temperature shift. Measurement of ligand accumulation by control and temperature-shifted cells incubated at 20 degrees C indicated that control cells exhibited a subpopulation of receptors capable of binding ligand but only slowly internalizing it. This subpopulation was not present on temperature-shifted cells. These results indicate that surface receptors for alphamacroglobulin . protease complexes are heterogeneous and that the temperature shift resulted in the selective loss of membrane components.
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PMID:Temperature shifts induce the selective loss of alveolar-macrophage plasma membrane components. 618 Oct 76

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.
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PMID:Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes. 634 85


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