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)

MTX peptides in which the amino acid was linked to the alpha-carboxyl group have been prepared and examined for cytotoxicity before and after treatment with proteolytic enzymes. The alanine, aspartic acid and arginine derivatives (MTX-ala, MTX-asp and MTX-arg) were synthesized by a regio-specific route, following the general procedures of Rosowsky and Montgomery. Each compound was obtained in good yield, and purity was established by TLC, HPLC, absorbance spectra and elemental analyses. The MTX peptides were not hydrolyzed by a variety of proteolytic enzymes (e.g., trypsin, plasmin, urokinase, aminopeptidase). Pancreatic carboxypeptidase A, however, hydrolyzed MTX-ala readily, MTX-asp slowly and MTX-arg not at all. The MTX-ala and, to a lesser extent, MTX-arg were substrates for pancreatic carboxypeptidase B. MTX-arg was also hydrolyzed by the endogenous carboxypeptidase N in human serum. The cytotoxicity of these MTX peptides toward L1210 cells was measured in a microculture assay system using a tetrazolium dye. MTX-ala was weakly cytotoxic (ID50 = 2.0 x 10(-6)M) compared to MTX (ID50 = 2.4 x 10(-8)M). When MTX-ala was tested in the presence of carboxypeptidase A, the ID50 value improved to 8.5 x 10(-8)M. MTX-arg gave an ID50 of 5.0 x 10(-8)M, which was not unexpected in view of its susceptibility to hydrolysis by the carboxypeptidase activity present in the fetal calf serum of the culture medium. Inclusion of carboxypeptidase B lowered the ID50 value to 2.5 x 10(-8)M. Possible clinical uses of MTX peptides are discussed.
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PMID:Chemotherapeutic potential of methotrexate peptides. 307 29

Pseudomonas fluorescens strains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular. Two peptidases were partly purified from strain 32A. One specifically hydrolysed N-alpha-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase. The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase. The endopeptidase had a mol. wt of 33,000 and a pH optimum of 8.0. The enzyme was stimulated by Ca2+ and Mg2+ and inhibited by Co2+, Mn2+, Hg2+, Zn2+ and leupeptin. Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity. The aminopeptidase had a mol. wt of 44,000 and a pH optimum of 8.0. It was inhibited by all the metal ions mentioned above and by PMSF. Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A. It was concluded that the cell-associated peptidases from Pseudomonas strains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.
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PMID:Peptidases from two strains of Pseudomonas fluorescens: partial purification, properties and action on milk. 311 Feb 31

N-Benzyloxycarbonyl-Gly-Pro-diazomethyl ketone (Z-Gly-Pro-CHN2) was synthesized and tested as inhibitor of the post proline cleaving enzyme from bovine brain. The compound was found to inactivate the enzyme completely and irreversibly at low concentrations (0.3 microM) without affecting other proteolytic enzymes such as post proline dipeptidyl aminopeptidase, pyroglutamate aminopeptidase or trypsin. Substrates of post proline cleaving enzymes such as luliberin (LH-RH; pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and Benzyloxycarbonyl-Gly-Pro-Ala protected the enzyme from the reaction with Z-Gly-Pro-CHN2. Thus, Z-Gly-Pro-CHN2 seems to be an active site directed, specific inhibitor of post proline cleaving enzyme. When administered intraperitoneally to rats, this inhibitor (8 mg/kg) completely inactivated the post proline cleaving enzyme in all tissues studied including brain. Therefore, Z-Gly-Pro-CHN2 should be a valuable tool for studies on the physiological function of this enzyme within the metabolism of neuropeptides.
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PMID:Specific inhibition of post proline cleaving enzyme by benzyloxycarbonyl-Gly-Pro-diazomethyl ketone. 351 11

The activity of a glycopeptide prepared from rat serum by treatment with trypsin and ultrafiltration was investigated in several in vivo proliferation systems. In baby rat hepatocytes synchronized by a subcutaneous injection of casein solution it caused a G1-S block, stopping cells at the end of the G1 phase and sending them back to the G0 phase. The glycopeptide also caused a G1-S block in young adult rats during the first semi-synchronized wave of proliferation that followed partial hepatectomy. Inhibition of hepatocyte proliferation by the glycopeptide was suppressed by blood proteins from normal rats but not from acute phase rats. Alpha 1-acid glycoprotein, an acute phase protein, increased this inhibition and reversed the antagonistic effect of normal blood proteins. In normal baby rats a G1-S block of non-synchronously proliferating hepatocytes was produced in two situations in which the antagonistic effect of normal blood proteins was eliminated: after treatment of the glycopeptide with leucine-aminopeptidase, and after mixing it with alpha 1-acid glycoprotein. The glycopeptide did not inhibit cell proliferation in kidney, submaxillary gland, or tongue epithelium. It seems to be the active component of a system that inhibits the proliferation of hepatocytes, probably by reducing their sensitivity to various mitogenic stimuli.
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PMID:Inhibition of hepatocyte proliferation in vivo by a glycopeptide from rat serum. 369 Jun 25

We followed the time course of the activities of various hydrolytic enzymes in murine brain for 30 months to investigate their roles in the aging process. Although most of the 15 enzymatic activities tested tended to rise with the increase in age, by analyzing them by multivariate analysis their movements could be divided into three types, each having a particular mode of nonlinear regression: exponential, logarithmic, or parabolic. Several enzymatic activities, including those of angiotensin-converting enzyme, esterase, trypsin-like enzyme, post-proline-cleaving enzyme, and Gly-Pro-aminopeptidase, showed rhythmic oscillations with about one cycle per 3 months throughout the period of 30 months. The behavior of these enzymes probably represents some particular aspect of metabolism relatively independent from the aging process. These findings may provide a fundamental clue in association with the process of maturation and aging in the brain.
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PMID:Three modes of age-related changes of hydrolytic enzymes in murine brain. 382 79

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.
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PMID:A unique trypsin-like protease associated with plasma membranes of rat liver. 394 38

1. S-Aminoethylcysteinyl derivatives of the A and B chains of cod insulin were prepared from the individual S-sulpho chains. 2. Studies on small peptides derived from the S-aminoethylated peptide chains by treatment with trypsin allowed the amino acid sequences in the region of the cysteinyl residues of the A and B peptide chains to be defined. 3. The six amide groups in cod insulin were located by complete digestion of small peptides from the A and B chains with aminopeptidase followed by amino acid analyses. 4. The results, together with previous studies on the oxidized A and B chains, define the sequences of the 51 amino acids that constitute cod insulin.
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PMID:The sequence of amino acids in insulin isolated from islet tissue of the cod (Gadus callarias). 488 74

Purification of pronase by ion-exchange chromatography gave four proteolytically active fractions. Fraction A(2) contained an endopeptidase that attacks poly l-valine. Fraction B contained an endopeptidase, an aminopeptidase and carboxypeptidases. The activities against hippuryl-l-arginine and hippuryl-l-phenylalanine could be inhibited to a considerable extent by di-isopropyl phosphorofluoridate and by EDTA. Fraction C contained an endopeptidase resembling bovine trypsin. The pure enzyme was completely inactivated by di-isopropyl phosphorofluoridate and pancreatic trypsin inhibitor and to about 90% by other naturally occurring trypsin inhibitors. Fraction D contained an apparently homogeneous endopeptidase, inhibited by diisopropyl phosphorofluoridate, that adsorbed to and hydrolysed elastin. The activity of all these fractions was tested qualitatively against a wide range of small peptides and synthetic substrates.
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PMID:The specificity of proteinases from Streptomyces griseus (pronase). 498 92

Microvillus aminopeptidase (EC 3.4.11.2) is an enzyme with a molecular weight around 300 000. Normal preparations contain three different subunits (subunit A, Mr 162 000; subunit B, Mr 123 000; subunit C, Mr 61 000). The relationship between the three subunits was studied by immunoelectrophoresis using specific antibodies against individual denatured subunits and by densitometric scanning of polyacrylamide gels after separation of the three subunits. The results suggest that microvillus aminopeptidase initially appears in the membrane as a symmetric molecule built up to two identical A subunits. These subunits are then split into equimolar amounts of subunit B and subunit C by trypsin. Subunit B cannot generate subunit C but may be further degraded. The reaction sequence described is one which occurs in vivo. Treatment of purified aminopeptidase with trypsin increases the specific activity twofold. This phenomenon does not seem to be correlated to the generation of subunit B and subunit C or to the transformation of amphiphilic form into hydrophilic form.
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PMID:Changes of the quaternary structure of microvillus aminopeptidase in the membrane. 612 Aug 37

Glucose transporter proteins (zone 4.5) which had been photoaffinity labeled with [3H]cytochalasin B in human erythrocyte ghosts were subjected to enzymatic dissection in order to study the transmembrane disposition of the protein in situ. Proteolytic enzymes as well as glycosidases were used to treat unsealed and resealed ghosts in order to explore the various membrane domains of the transporter in a topographically defined manner. Limited digestion of sealed ghosts with trypsin had no effect on the apparent Mr of the transporter (55,000). Similar treatment in unsealed ghosts, however, resulted in the generation of a major fragment of 21.5 kDa, along with several minor fragments. Thermolysin also had no effect on sealed ghosts but caused a complete loss of radiolabel from the zone 4.5 region with no lower-molecular-weight fragments being retained on the gel. Chymotrypsin treatment resulted in the generation of a single peak, Mr = 18,400, in both sealed and unsealed ghosts indicating its action occurs at the outer surface. Digestion with carboxypeptidase and aminopeptidase indicate the C-terminal end of the transporter is located exterior to the membrane with the N terminus located at the cytoplasmic surface. Treatment with endoglycosidase resulted in a shift of mobility of the transporter to a lower Mr of 49,000. The results obtained indicate that the carbohydrate is located near the C-terminal end and that the cytochalasin B-binding site is located near the cytoplasmic N-terminal end.
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PMID:Orientation of the glucose transporter in the human erythrocyte membrane. Investigation by in situ proteolytic dissection. 615 39


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