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)

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.
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PMID:Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation. 620 95

After the rat preputial gland was treated with collagenase and trypsin, five bands of cells were isolated by centrifugation in Ficoll gradients. Homogenates of the heavier cells (Bands IV and V) which contained less lipids, were more active than the homogenates of the lighter cells (Bands I, II and III) in transforming [1,2-3H]-dehydroepiandrosterone ([1,2-3H]-DHA) into [3H]-androstenedione and [3H]-testosterone and the latter into [3H]-dihydrotestosterone (DHT). In the presence of NAD, NADH and NADPH-generating system, [1,2-3H]-DHA was transformed into [3H]-DHT in 50-60% yield by homogenates of cells in Bands IV and V. DHT levels in the preputial gland were measured by radioimmunoassay. The levels in female rats reduced by 77% from 3.14 +/- 0.27 to 0.72 +/- 0.10 pg/mg tissue after adrenalectomy, and by 45% to 1.71 +/- 0.10 pg/mg tissue after ovariectomy. In male rats, the level reduced by 15% from 4.58 +/- 0.55 to 3.88 +/- 0.62 pg/mg tissue after adrenalectomy and by 40% to 2.74 +/- 0.21 pg/mg tissue after orchidectomy. These results demonstrated the transformation of DHA into DHT in the preputial gland of the rat, and that the adrenal is an important source of precursor steroid (DHA) for DHT formation in the preputial gland.
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PMID:Transformation of dehydroepiandrosterone into dihydrotestosterone by isolated cells from rat preputial gland. 622 75

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

The topology of beef heart Complex III has been studied by tryptic and chymotryptic digestion of isolated Complex III, Mg2+-ATP submitochondrial particles, and mitoplasts. Degradation products were detected by the immunoreplication technique using specific antibodies against core protein 1 (50 K) and core protein 2 (47 K). It can be shown that both peptides are digested from the matrix side of the inner membrane. However, no evidence was found that these peptides were digested by trypsin or chymotrypsin from the cytoplasmic side. It is concluded that the beef heart core proteins are membrane-bound peptides containing tryptic and chymotryptic digestion sites only on the matrix surface of the inner membrane. The data also suggest that beef heart core protein 2 contains multiple domains which are inserted into the membrane from the matrix surface. Proteolytic treatment of submitochondrial particles under conditions which digested at least 50% of the core proteins from the matrix surface did not, however, influence NADH oxidation rates or the respiratory control ratios.
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PMID:Studies on beef heart ubiquinol-cytochrome c reductase. Topological studies on the core proteins using proteolytic digestion and immunoreplication. 630 20

A method for purification of detergent-solubilized cytochrome b5 to gel electrophoretic homogeneity from yeast (Saccharomyces cerevisiae) microsomes is described. The purified preparation shows the same absorption spectra as the trypsin-solubilized cytochrome (Y. Yoshida, H. Kumaoka, and R. Sato J. Biochem. 75, 1211-1219 (1974)) in the visible and Soret regions. The detergent-solubilized cytochrome is an amphipathic protein having a monomeric molecular weight of about 18,000 and exists as a hexa- or heptameric aggregate (Mr 122,000) in aqueous media. In the presence of low concentrations of Triton X-100, it interacts effectively with the intact form of NADH-cytochrome b5 reductase purified either from yeast microsomes or from rabbit liver microsomes. Upon trypsin digestion, it is converted to a heme-containing, hydrophilic fragment (Mr 13,000) which retains the spectral characteristics of the original cytochrome, does not form aggregates, and interacts with the reductase only poorly. It is concluded that the preparation purified in this study represents the intact form of yeast cytochrome b5 consisting of a hydrophilic, heme-containing moiety (Mr 13,000) and a hydrophobic, membrane-binding tail (Mr 5000).
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PMID:Purification and characterization of intact cytochrome b5 from yeast microsomes. 633 56

Horse liver alcohol dehydrogenase is inactivated with Michaelis kinetics at pH 7 and 25 degrees C by 3-bromopropionic acid. In the absence of NAD+, the Ki is 2 mM, and the pseudo bimolecular rate constant (k3/Ki) is 0.03 M-1 s-1; in the presence of 1 mM NAD+, Ki is 2.3 mM, and k3/Ki is 0.006 M-1 s-1. 3-Bromopropionic acid is a competitive inhibitor, Ki of 0.4 mM, against ethanol as a substrate. Inactivation was prevented in the ternary complexes with NAD+ X pyrazole and NADH X isobutyramide, was retarded by NAD+, NADH, or bipyridine, and was almost unaffected by imidazole and AMP. Carboxyethylated enzyme did not detectably (as observed spectrophotometrically) bind bipyridine, NAD+, or NADH. Enzyme was inactivated with radioactive 3-bromopropionic acid, aminoethylated, and digested with trypsin and chymotrypsin. Analysis of the labeled peptides showed that Cys-174 was predominantly modified. In the presence of 1 mM NAD+, the reaction was much less specific. The interaction of the carboxyl group of 3-bromopropionic acid with the guanidino group of Arg-369 probably facilitates the selective reaction with Cys-174, which is ligated to the zinc at the active site. Carboxyethylation apparently inactivates by interfering with the proper binding of the pyrophosphate of the coenzyme to the enzyme.
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PMID:Inactivation of horse liver alcohol dehydrogenase by modification of cysteine residue 174 with 3-bromopropionic acid. 636 61

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

Pig heart citrate synthase was subjected to limited proteolytic attack by subtilisin, chymotrypsin, and trypsin in the presence of palmitoyl-CoA. Initial proteolysis by all three proteolytic enzymes resulted in cleavage of the monomeric subunit (Mr 45 000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000 +/- 3000) into a large (Mr 35 000-38 500) and a small (Mr 9000-12 000) fragment. Further proteolysis of the large subunit produced a secondary fragment (Mr 31 000-36 000). The small (Mr 9000-12 000) fragment was stable in the presence of subtilisin but was substantially degraded by both chymotrypsin and trypsin. The actual molecular weight of fragments varied with the choice of the proteolytic enzyme. Limited proteolysis was absolutely dependent on the presence of palmitoyl-CoA and resulted in complete inhibition of the catalytic activity of the enzyme. Citrate, ammonium sulfate, and especially oxaloacetate provided complete protection against proteolysis whereas acetyl-CoA, CoASH, NADH, and ATP were ineffective. Reaction of rabbit anti-citrate synthase with citrate synthase and its proteolytic fragments indicated that the main antigenic region lay primarily in the small fragment. The products of subtilisin cleavage were isolated by gel filtration under denaturing conditions. The large (Mr 35 000-38 500) fragment contained the amino-terminal (approximately)336 amino acids and the small fragment contained the remaining carboxyl-terminal amino acids. The results are discussed in relation to the structure of citrate synthase.
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PMID:Limited proteolysis of pig heart citrate synthase by subtilisin, chymotrypsin, and trypsin. 677 58

NADH-cytochrome b5 reductases purified from human red cell membranes and cytosol were compared with those prepared from human liver microsomes. Minimal molecular weights of the membrane and the cytosol enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were 36,000 and 32,000 daltons, respectively, which are comparable to those of the detergent-solubilized reductase (dfp) and the protease-solubilized one (tfp) of liver microsomes, respectively. All the enzymes contained FAD and had essentially the same turnover numbers and apparent Km values for NADH and protease-solubilized cytochrome b5. The membrane enzyme and liver dfp reduced cytochrome c in the presence of detergent-solubilized cytochrome b5 70-80 times faster than in the presence of trypsin-solubilized cytochrome b5, whereas the cytosol enzyme and liver tfp showed essentially the same low activities with both preparations of cytochrome b5. SDS-PAGE mapping of the limited proteolytic products of the reductases obtained by digestion with staphylococcal protease or a-chymotrypsin showed essentially the same patterns of peptides between the red cell membrane enzyme and liver dfp and between the red cell cytosol enzyme and liver tfp. These results suggest that the NADH-cytochrome b5 reductase of human red cell membranes is identical with that of liver microsomes and that the enzyme of red cell cytosol is a proteolytic product of the membrane enzyme.
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PMID:Human NADH-cytochrome b5 reductases: comparison among those of erythrocyte membrane, erythrocyte cytosol, and liver microsomes. 684 58

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


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