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)

We described earlier the facilitated purifications of the trypsin and aminopeptidase components present in Pronase (Vosbeck, K. D., Chow, K. -F., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 6029-6034). A partially resolved protein mixture left over after one of the steps in that procedure was passed through a Sephadex G-75 column. By this means, a component with carboxypeptidase activity was separated from associated serine endopeptidases. Further purification of this exopeptidase to apparent homogeneity was acheived by refiltration through the same Sephadex column and by CM-cellulose chromatography. A single protein band was observed after acrylamide gel electrophoresis; analysis by sedimentation equilibrium using the meniscus depletion method gave a molecular weight of 30,300. This enzyme demonstrates activity against Nalpha-benzyloxycarbonylglycyl-L-leucine and hippuryl-D,L-phenyllactate; no activity was found against Nalpha-acetyl-L-tyrosine ethyl ester, Nalpha-benzoyl-D,L-arginine-p-nitroanilide, or L-leuckne-p-nitroanilide. The maximum activity lies between pH values of 7 and 8; the enzyme is stable between pH values of 6 and 10. At room temperature 1,10-phenanthroline inactivates the enzyme completely whereas EDTA has no effect. Of the many cations tested, only Co2+, Ni2+, or Zn2+ restores activity to the 1,10-phenanthroline-treated enzyme; Co2+ provided 3 times the native activity. The metal in the native protein was found to be zinc. These findings are similar to those recorded with bovine pancreatic carboxypeptidase A, and suggest the possibility that the present enzyme may ge genetically related to the mammalian protein, as in previously noted examples of homology of three Pronase endopeptidases to pancreatic serine enzymes.
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PMID:Proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from a commercial preparation (Pronase). Purification and characterization of the carboxypeptidase. 0 Mar 99

Demetallized concanavalin A is degraded rapidly at pH 7.0 and 8.2 by alpha-chymotrypsin, thermolysin or trypsin, yielding peptide fragments devoid of ability to bind to Sephadex G-75. Addition of Ni2+ and of Ca2+ confers on concanavalin A high resistance towards proteolytic attack so that even after long periods of exposure to the enzymes, almost all of the saccharide-binding capacity is preserved. Ni2+ alone protects strongly at pH 7.0 but not at pH 8.2. Apparently, both the transition metal ion and Ca2+ play an important role in stabilizing the native conformation of the protein molecule. Digestion of demetallized concanavalin A with alpha-chymotrypsin or thermolysin readily yields small peptide fragments (Mr less than 10 000), while trypsin yields as the major product(s) larger peptide(s) (Mr approximately 20 000) of appreciable resistance to further fragmentation.
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PMID:Effect of divalent metal ions on the digestibility of concanavalin A by endopeptidases. 1 27

To obtain gingival cell cultures, human gingival tissue was minced and/or subjected to trypsin treatment with or without prior separation into epithelial and connective tissue portions. The tissues were then incubated in Eagle/Earle's MEM with 10% fetal calf serum in a humidified atmosphere of 5% CO2 in air. Fibroblast-like cell cultures were regularly obtained, and one culture showed epithelial-like cell islets that could be transferred and kept in continuous culture. These epithelial-like cells exhibited bone resorption stimulating activity as seen in gingival tissue and retained their growth pattern after prolonged storage. They were able to grow at serum concentrations down to 2.5% and with equal doubling time (about 17h) in rich or minimum essential media. Exposure to nickel gave toxic effects on the growth at concentrations down to 2.5 microgram/ml of nickel. In spite of the affinity of nickel to certain serum components, the serum concentration did not appear to be of specific importance to protect or aggravate the toxicity. It is felt that these cells may be of value for research on cytotoxicity of dental materials.
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PMID:Epithelial-like cells in culture derived from human gingiva: response to nickel. 27 21

Tryptic action occurs when a layer of bovine serum albumin adsorbed on a nickel-plated slide and protected by a Formvar membrane 120 A thick is treated with dilute trypsin solution. But experimental evidence indicates that the trypsin molecules to not cross the membrane. Thus the proposal that trypsin can exert its enzymatic action without intimate contact with the substrate, first set forth in 1948 but later abandoned in favor of a "forced diffusion" hypothesis, now appears the correct interpretation. Analogously, antibodies can be specifically immobilized on one side of a membrane separating them from adsorbed antigens located on the other side.
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PMID:Tryptic action across a membrane. 54 33

The membrane properties of isolated neurons from Helix aspersa were examined by using a new suction pipette method. The method combines internal perfusion with voltage clamp of nerve cell bodies separated from their axons. Pretreatment with enzymes such as trypsin that alter membrane function is not required. A platinized platinum wire which ruptures the soma membrane allows low resistance access directly to the cell's interior improving the time resolution under voltage clamp by two orders of magnitude. The shunt resistance of the suction pipette was 10-50 times the neuronal membrane resistance, and the series resistance of the system, which was largely due to the tip diameter, was about 10(5) omega. However, the peak clamp currents were only about 20 nA for a 60-mV voltage step so that measurements of membrane voltage were accurate to within at least 3%. Spatial control of voltage was achieved only after somal separation, and nerve cell bodies isolated in this way do not generate all-or-none action potentials. Measurements of membrane potential, membrane resistance, and membrane time constant are equivalent to those obtained using intracellular micropipettes, the customary method. With the axon attached, comparable all-or-none action potentials were also measured by either method. Complete exchange of Cs+ for K+ was accomplished by internal perfusion and allowed K+ currents to be blocked. Na+ currents could then be blocked by TTX or suppressed by Tris-substituted snail Ringer solution. Ca2+ currents could be blocked using Ni2+ and other divalent cations as well as organic Ca2+ blockers. The most favorable intracellular anion was aspartate-, and the sequence of favorability was inverted from that found in squid axon.
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PMID:Properties of internally perfused, voltage-clamped, isolated nerve cell bodies. 66 Jan 59

Two heifers were implanted with 300 mg of the radiolabeled anabolic steroid, trenbolone acetate (TBA). After a 60 day slaughter and a 60 day removal followed by 76 day slaughter, total 3H-content in various tissues was 0.5--25 ng/g equivalents of TBA. Radioimmunoassay of the tissues showed that only 1--5% of the total residue present was TBA, its main metabolite trenbolone (TBOH), and TBOH glucuronide, plus up to 5% of other organic-soluble material. Of the radioactivity remaining about half was directly water-soluble, and the insoluble residue could be made water-soluble by treatment with the proteolytic enzymes pepsin and trypsin. These 2 portions were purified with Sephadex G-25 to give a low and high molecular weight fraction. Raney nickel reduction of sulfur bonds in either fraction released up to 50% of radioactivity into the organic phase. TLC showed that the latter contained 2 components which had characteristics similar to TBOH and its metabolites, and thus were at least partly drug-related metabolites. In vitro experiments with bovine liver also showed a small but definite protein binding. It is proposed that in dealing with these covalently bound residues, priority be given to the reactive drug intermediate and the type of binding to macromolecules rather than to the presence of the bound residue itself.
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PMID:Trenbolone acetate: experiences with bound residues in cattle tissues. 72 40

The isolation and characterization of a microsomal arylaminopeptidase from rat kidney is reported. By treatment of a microsomal arylaminopeptidase-phosphatase-complex with trypsin and subsequent gel filtration of the solubilized proteins on Sepharose 6B a electrophoretic homogeneous arylaminopeptidase was obtained (yield, 3%; enrichment, 900 times). The following properties of the purified enzyme were determined: 1. Molecular weight: 182000 (gel filtration on Sepharose 6B) to 192000 (SDS-polyacrylamide gel electrophoresis). 2. Subunit structure: In the presence of 6 M guanidine - HC1 + 1% BETA-mercaptoethanol the enzyme dissociates into subunits (MW 46700, ESTIMATED BY SDS gel electrophoresis method). 3. Isoelectric point: 4,71 (agarose gel electrophoresis method). 4. UV characteristics: E 280nm/E260NM=1.3. 5. Substrate specifity: optimal substrates L-alanyl derivatives (anilide, beta-naphthyl amide, p-nitroanilide, 4-(phenylazo)-phenylamide and hydrazide). Among these compounds the anilide derivative was hydrolyzed most rapidly. Furthermore, di- and tripeptides, especially L-methionyl-L-leucine, were also split. No hydrolysis was observed with hemoglobin (pH 4.5 and 7.5) and amino acid- or peptide-ester substrates. 6. Optimal pH: 7.5 +/- 0,1; optimal temperature: 45 to 50 degrees C. 7. The enzyme has no transamidation activity with L-alanyl amide both as aminoacyl donator and -acceptor. 8. Influence of effectors: Heavy metal ions (Ni2+, Cd2+, Cu2+, Zn2+), chelating agents (EDTA, o-phenanthroline) and puromycin inhibit the enzyme significantly. SH-group reagents are without any influence. 9. L-alanyl-L-alanyl-4 (phenylazo)-phenylamide, a dipeptide aryl aminopeptidase substrate, is hydrolyzed by the purified enzyme preparation according to a consecutive or step by step mechanism.
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PMID:[Isolation and characterization of a microsomal arylaminopeptidase from rat kidney]. 97 46

The effect of trace metals on plasma alpha1-antitrypsin was studied in vitro by adding known concentrations of trace metals, either alone or in combination, to plasma. Cadmium was the only trace metal that reduced the concentration of alpha1-antitrypsin and depressed the trypsin inhibitory capacity. No such effects were found with divalent lead, mercury, nickel, iron, and zinc ions. The present study appears to offer a plausible explanation for the emphysema that occurs in industrial workers exposed to cadmium.
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PMID:Influence of cadmium and other trace metals on human alpha1-antitrypsin: an in vitro study. 108 68

This study examines the molecular basis for paralysis of ciliary motility by Ni2+. At concentrations above 0.1 mM, Ni2+ slowed and subsequently stopped swimming of living, axenically grown Paramecium tetraurelia. However, some cilia still beat in the presence of 0.1 mM Ni2+. When permeabilized and reactivated with 4 mM ATP at pCa greater than 7, cells resumed ciliary beat and swam forward at approximately 170 +/- 28 microns s-1; swimming speed increased in the presence of 10 microM cyclic AMP. Addition of Ni2+ (pNi less than 5) caused rapid arrest of all ciliary beat in a single position. This was fully reversible when EGTA was added to raise the pNi. Axonemes were then isolated and sliding was observed in the presence of trypsin and ATP. When pNi was lowered to about 5, sliding was reduced dramatically. This too was reversible with EGTA. Dynein was then extracted from the axonemes and used for in vitro translocation assays. At concentrations of Ni2+ where microtubule-sliding and axonemal beat were greatly inhibited or absent, microtubule translocation in vitro by 22 S dynein was only slightly affected. However, translocation by 14 S dynein was stopped completely. When pNi was raised by repeated washing with solutions containing EGTA, microtubule translocation by 14 S dynein resumed. We conclude that Ni2+ induces a reversible paralysis by a direct effect on 14 S dynein while 22 S dynein is not a primary target.
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PMID:Analysis of Ni(2+)-induced arrest of Paramecium axonemes. 183 92

A recombinant trypsin was designed whose catalytic activity can be regulated by varying the concentration of Cu2+ in solution. Substitution of Arg-96 with a His in rat trypsin (trypsin R96H) places a new imidazole group on the surface of the enzyme near the essential active-site His-57. The unique spatial orientation of these His side chains results in the formation of a stable, metal-binding site that chelates divalent first-row transition-metal ions. Occupancy of this site by a metal ion prevents the imidazole group of His-57 from participating as a general base in catalysis. As a consequence, the primary effect of the transition metal ion is to inhibit the esterase and amidase activities of trypsin R96H. The apparent Ki for this inhibition is in the micromolar range for copper, nickel, and zinc, the tightest binding being to Cu2+ at 21 microM. Trypsin R96H activity can be fully restored by removing the bound Cu2+ ion with EDTA. Multiple cycles of inhibition by Cu2+ ions and reactivation by EDTA demonstrate that reversible regulatory control has been introduced into the enzyme. These results describe a novel mode of inhibition of serine protease activity that may also prove applicable to other proteins.
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PMID:Regulation of serine protease activity by an engineered metal switch. 212 68


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