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)

Phagocytic vesicles from rabbit lung macrophages produced superoxide in the presence of NADH or NADPH. At 37 degrees C, these vesicles generated 51+/-7.8 nmol O(2) (-)/min per mg protein in the presence of 0.5 mM NADPH. The apparent K(m) for NADPH and NADH (66 and 266 muM, respectively), the pH optimum for the reaction (6.9), and the cyanide insensitivity were similar to properties of plasma membrane-rich fractions of stimulated polymorphonuclear leukocytes studied by others. The activity of the phagocytic vesicles was trypsin sensitive. The specific superoxide-generating activity of macrophage phagocytic vesicles isolated from cells incubated up to 90 min with phagocytic particles remained constant. Calcium in micromolar concentrations inhibited the NADPH-dependent O(2) (-)-generating activity of phagocytic vesicles. In a physiological ionic medium (100 mM KCl, 2.5 mM MgCl(2), 30 mM imidazole-HCl, pH 6.9), a maximal inhibition of O(2) (-) generation by phagocytic vesicles of 80% was observed at 40 muM free Ca(2+). The half maximum inhibitory effect was at 0.7 muM Ca(2+). Variations of the calcium concentration resulted in rapid and reversible alterations in O(2) (-)-forming activity. Preincubation of phagocytic vesicles in the presence of EGTA rendered their O(2) (-) generation rate in the presence of NADPH insensitive to alterations in the free calcium concentration. This desensitization by low EGTA concentrations (</=100 muM) was reversible by the addition of excess calcium, but desensitization by high EGTA concentrations (>1 mM) was not reversible by the addition of calcium either in the presence or absence of purified rabbit lung macrophage or bovine brain calmodulins. Furthermore, trifluoperazine, a drug that inhibits calmodulin-stimulated reactions, did not alter the activity or the calcium sensitivity of the superoxide-generating system of sensitive phagocytic vesicles. Peripheral plasma membrane vesicles (podosomes) prepared by gentle sonication of macrophages possessed on O(2) (-)-generating system with similar properties to those of phagocytic vesicles. We conclude that the activated O(2) (-)-generating system of rabbit lung macrophages has its initial localization in the plasmalemma and undergoes subsequent internalization into phagocytic vesicles, where it can function for prolonged periods of time. Calcium at concentrations likely to exist in macrophage cytoplasm exerts a regulatory effect on the activated system.
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PMID:Effect of calcium on superoxide production by phagocytic vesicles from rabbit alveolar macrophages. 625 9

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH-cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH-cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH-cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH-cytochrome P-450 reductase and cytosolically orientated membrane factor(s).
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PMID:Asymmetric distribution of cytochrome P-450 and NADPH--cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver. 625 76

Cytochrome c synthetase in yeast mitochondria catalyzes the formation of a yeast cytochrome c-like species from the apoprotein and hemin (Basile, G., DiBello, C., and Taniuchi, H. (1980) J. Biol. Chem. 255, 7181-7191). To test the specificity of this enzyme, 125I-labeled horse apocytochrome c was incubated with the yeast mitochondrial fraction in the presence of hemin, NADPH, and an ethanol extract of the postmitochondrial fraction. A radioactive 125I-labeled cytochrome c-like species was formed in yields of up to 26%. This 125I-labeled species is indistinguishable from horse cytochrome c by ion exchange chromatography (under the conditions which allow separation of horse and yeast cytochrome c), resistance in its reduced form to digestion by trypsin, resistance against autoxidation, reduction by cytochrome b2, and generation of the apoprotein after treatment with silver sulfate and dithiothreitol. With unlabeled horse apoprotein and [59Fe]hemin, the yield of a [59Fe-labeled horse cytochrome c-like species was up to 7% with respect to the apoprotein incubated. The yield of the 59Fe-labeled species was not altered by the addition of unlabeled FeCl3. Conversely, synthesis of the 59Fe-labeled species was not detectable after incubation of yeast mitochondria with unlabeled horse apoprotein, unlabeled hemin, and 59FeCl3. The formation of both 125I- and 59Fe-labeled cytochrome c-like species was sensitive to heat. Thus, we conclude that cytochrome c synthetase catalyzes direct bonding of heme (or hemin) to the apoprotein. Since the amino acid sequences of horse and yeast cytochromes c differ considerably, cytochrome c synthetase may recognize only a limited region(s) of the apoprotein.
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PMID:Formation of a cytochrome c-like species from horse apoprotein and hemin catalyzed by yeast mitochondrial cytochrome c synthetase. 626 48

Electrophoretic isolation of a membrane-bound NADPH oxidase of guinea-pig polymorphonuclear leukocytes was attempted with the O2- -generating membranes of cells unstimulated or stimulated with C3b-zymosan or sodium dodecyl sulfate, and also with the phagosomes isolated from the phorbol myristate acetate-coated latex particle-phagocytosing cells. When these vesicles were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of Triton X-100 and then assayed for NADPH-Nitroblue tetrazolium reducing activity, the activity was detected by the appearance of a single, blue band of the reduced dye on the gel, independent of the source of vesicles. In addition, the enzyme was able to generate O2- and its activity was significantly augmented with the homologous liver microsomal cytochrome b5. Its activity was heat-labile and inactivated by N-ethylmaleimide and p-chloromercuribenzene sulfonate. The enzyme, with an apparent molecular weight of 150 000, in the phagosomes was easily susceptible to limited proteolysis by trypsin and formed an active fragment with a molecular weight of 70 000, accompanying the loss of O2- -generating activity of the vesicles.
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PMID:Electrophoretic isolation of a membrane-bound NADPH oxidase from guinea-pig polymorphonuclear leukocytes. 630 74

Sequential treatment of the chicken liver fatty acid synthetase by trypsin and subtilisin cleaved the Mr 267,000 subunit to 6-8 polypeptides, ranging in molecular weights from 15,000 to 94,000. Fractionation of the digest by ammonium sulfate and chromatography on a Procion Red HE3B affinity column permitted the isolation of a polypeptide (Mr = 94,000) containing the beta-ketoacyl reductase activity but no other partial activities normally associated with the synthetase. The specific activity of the beta-ketoacyl reductase increased 2 to 3 times in this fraction, an increase that is within the expected range based on relative molecular weight. The kinetic parameters of this fraction towards NADPH and N-acetyl-S-acetoacetyl cysteamine were essentially the same as the beta-ketoacyl reductase component of the intact synthetase. However, the purified fragment did not catalyze the reduction of acetoacetyl-S-CoA derivative, a substrate that is readily reduced by the intact synthetase. Fluorescence measurements with etheno-NADP+ indicate the binding of about 1 mol of NADP+/94,000 daltons, a value which is in agreement with the results obtained from fluorescence measurements with NADPH and the binding of a radiolabeled photoaffinity analog of NADP+. Phenylglyoxal inhibits the beta-ketoacyl reductase activity of either the intact synthetase or the isolated fragment, suggesting the involvement of an essential arginine at or near the active site. Another fragment (Mr 36,000) containing beta-ketoacyl reductase activity was isolated from the synthetase after kallikrein/subtilisin double digestion. Previous mapping studies had shown that this fragment lies adjacent to the COOH-terminal thioesterase domain and overlaps the tryptic Mr 94,000 peptide by approximately 21 daltons. This fragment, but not the Mr 94,000 fragment, was found to contain the phosphopantetheine prosthetic group, indicating that the acyl carrier protein moiety is located in the 15,000-dalton segment that separates the beta-ketoacyl reductase from the thioesterase domain.
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PMID:The architecture of the animal fatty acid synthetase. III. Isolation and characterization of beta-ketoacyl reductase. 636 Oct 31

Scanning transmission electron microscopy of individual unfixed molecules of methylenetetrahydrofolate reductase has been used to determine the molecular mass distribution of the protein. Methylenetetrahydrofolate reductase, which has a subunit molecular mass of 77 kilodaltons, was found to exist predominantly as a dimer with an apparent molecular mass of 136 +/- 29 kilodaltons. The mass distribution of the enzyme molecules was unchanged in the presence of the allosteric inhibitor S-adenosylmethionine. Examination of negatively stained protein molecules suggested that each subunit of the dimer consists of two globular domains of approximately equal size. Limited proteolysis of the enzyme by trypsin gave results which were entirely consistent with the presence of two domains per subunit. In the presence of 1% trypsin, the enzyme was cleaved into two fragments. The masses of these fragments were 39 and 36 kilodaltons as assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Tryptic cleavage did not lead to loss of NADPH-menadione or NADPH-methylenetetrahydrofolate oxidoreductase activity, and the flavin prosthetic group remained bound to the protein. However, the cleaved protein was completely desensitized with respect to inhibition by S-adenosylmethionine. These results suggest that each subunit of methylenetetrahydrofolate reductase contains two domains and that allosteric inhibition requires specific interactions between these domains. The region between these two domains appears to be very sensitive to proteolysis, while the domains themselves are relatively resistant to further degradation.
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PMID:Methylenetetrahydrofolate reductase. Evidence for spatially distinct subunit domains obtained by scanning transmission electron microscopy and limited proteolysis. 638 10

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisole demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminishes the cytochrome b5 reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.
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PMID:The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes. 641 23

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.
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PMID:Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide. 643 80

The degradation of cytochrome P-450 as a result of proteolytic action of trypsin is a biphasic process. Lipid peroxidation (LPO) increases the rate of the fast phase of cytochrome P-450 degradation and its accessibility to protease. The efficiency of this process depends on the mode of LPO induction and decreases in the following order: NADPH----NADH----ascorbate-dependent LPO. The induction of the monooxygenase system increases the efficiency of proteolysis. LPO and proteolysis seem to be mutually enhancing processes which provide for a high efficiency of cytochrome P-450 degradation. LPO can be regarded as a triggering mechanism which makes various forms of cytochrome P-450 accessible to endogenous proteases.
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PMID:Mechanisms of disassembly of a mixed function oxygenase system in liver endoplasmic reticulum: II. The interrelation between lipid peroxidation and proteolytic degradation of cytochrome P-450. 648 26

It has been shown that endogenous lipid peroxidation (LPO) is an effective mechanism participating in the destruction of endoplasmic reticulum membranes (cytochrome P450) in liver. Antioxidants are able to control the rate of degradation of cytochrome P450 in vivo. Stock of the constitutive cytochrome P450 as compared with induced P450 is more resistive to LPO in vivo and in vitro. Spontaneous as well as induced by Fe2+--ADP+ +NADPH system destruction of cytochrome P450 due to accumulation of LPO products malonic dialdehyde (MDA) occurs during incubation of isolated rats hepatocytes. The LPO inhibitors (4-methyl-2,6- ditretbutilphenol , pyrogallol) stabilize cytochrome P450 preventing accumulation MDA hepatocytes. Degradation of cytochrome P450 in microsomes during trypsin proteolysis has been found to be enhanced by PLO induction. Efficiency of proteolysis depends on the way of induction and decreases in such an order: NADPH-- HNDH --ascorbate-dependent LPO. LPO may be considered as a trigger mechanism that makes some forms of cytochrome P450 available for endogenous proteases.
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PMID:[Mechanisms of mixed-function oxidase system breakdown in the hepatic endoplasmic reticulum. The role of membrane phospholipid peroxidation]. 671 28


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