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)

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.
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PMID:Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle. 642 Dec 35

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

A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.
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PMID:Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography. 652 98

The proteins of submitochondrial particles solubilized with 0.1% Triton X-100 were separated by polyacrylamide gel electrophoresis. Hydrolysis of several proteinase substrates was registered directly in the gel after completion of electrophoresis. According to the data obtained the inner mitochondrial membrane contains one or two enzymes which catalyze hydrolysis of cytochrome c as well as one or two enzymes splitting synthetic substrate of trypsin-like proteinases, e. g. N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPA) and N-alpha-benzoyl-L-arginine-beta-naphthylamide (BANA). Submitochondrial particles were shown to catalyze hydrolysis of 3H-labelled cytochrome c. This activity is suppressed by the same inhibitors as the hydrolysis of mitochondrial translation products, i. e. phenyl-methylsulfonylfluoride, p-chloromercuribenzosulfonate, leupeptin and antipain. Presumably these two processes are catalyzed by the same enzyme localized in the inner mitochondrial membrane. Physiological functions of BAPA- and BANA-hydrolyzing enzyme(s) are still unclear.
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PMID:[Mitochondrial proteinases of the yeast Saccharomyces cerevisiae: electrophoretic detection, substrate specificity and sensitivity to inhibitors]. 675 14

Methods are described for incorporation of purified forms of rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450LM2, P-450LM3 and P-450LM4 (LM, liver microsomes) into phospholipid vesicles. It was found that each cytochrome could individually be incorporated into preformed phospholipid vesicles in the absence of cholate. However, NADPH-cytochrome P-450 reductase prevented incorporation of P-450 by this method, a phenomenon possibly inherent in the formation of complexes between P-450 and the reductase in solution. Using the cholate-gel filtration technique it was possible to prepare monolamellar phosphatidylcholine vesicles containing any of the cytochromes and P-450 reductase in good yields. It was found that P-450LM3-containing vesicles had a mean diameter of 47 nm, whereas vesicles formed under the same conditions but containing P-450LM4 were much smaller (mean diameter 33 nm). Vesicles formed with P-450LM2 were homogeneous in density (1.04 g/cm3) according to isopycnic centrifugation in Ficoll but not in size (44-72 nm). These findings, taken together with results obtained from treatment of the cytochromes in soluble form and in reconstituted vesicles with the non-penetrating reagent, p-diazobenzene sulphonate, indicate a unidirectional, relatively peripheral orientation of P-450LM4 with the major part localized on the outside of the vesicles. Experiments with trypsin and cytochrome c-reduction demonstrated a unidirectional orientation of P-450 reductase towards the outside of the vesicles.
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PMID:Incorporation of purified components of the rabbit liver microsomal hydroxylase system into phospholipid vesicles. 677 67

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

The complete amino-acid sequence of the copper-zinc superoxide dismutase of the Photobacterium leiognathi was determined. The fragmentation strategy employed included cyanogen bromide cleavage at its methionine residues and the only tryptophan residue. The S-carboxymethylated chain was further cleaved by means of trypsin, in order to obtain overlapping fragments. For sequence determination automated solid or liquid-phase techniques of Edman degradation were used. C-Terminal amino acids of the entire chain were determined after treatment with carboxypeptidase A. Comparison of the primary structure of this bacterial Cu-Zn superoxide dismutase with the established amino-acid sequences of the other eukaryotic Cu-Zn superoxide dismutases revealed clear homologies. Correspondingly, the Cu-Zn-binding amino-acid residues of the active centre were localized: His45, His47, His70, His79, His125 and Asp91. The two cysteine residues in position 52 and 147 were homologous to the cysteine residues, modelling the essential intrachain disulfide bridge of the corresponding bovine enzyme. As only 25-30% of aligned sequence positions were found to be identical, the enzyme of P. leiognathi shows only a remote phylogenetic relationship towards eukaryotic Cu-Zn superoxide dismutases. When compared to the established phylogenetic tree of the cytochrome c family, this indicates a separate evolution of Cu-Zn superoxide dismutase in Photobacterium. Therefore, a natural gene transfer from the eukaryotic host (ponyfish) to the prokaryotic photobacterium, which Martin and Fridovich postulated 1981 (J. Biol. Chem. 256, 6080-6089) on the basis of amino-acid compositions, can be excluded.
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PMID:The primary structure of Cu-Zn superoxide dismutase from Photobacterium leiognathi: evidence for a separate evolution of Cu-Zn superoxide dismutase in bacteria. 688 93

Tissue extract of paranasal mucous membrane from patients with chronic sinusitis was found to show a large lysis area on plasminogen-rich fibrin plates, but not on plasminogen-free fibrin plates. This indicates the existence of tissue plasminogen activator in the tissue extract. Further studies by the gel filtration technique showed that two plasminogen activators of different molecular weights were present in the tissue extract. The existence of tissue plasminogen activator with a low molecular weight has not previously been reported. This activator is labile at neutral pH at 37 degrees C, but stable on fibrin under the above conditions. The molecular weight of this compound is lower than that of cytochrome c. It may be a compound which is proteolytically modified by proteases, ie, trypsin-like enzymes, existing in the paranasal mucous membrane tissue of patients with chronic sinusitis.
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PMID:Characteristics of tissue plasminogen activator from paranasal mucous membrane in chronic sinusitis. 704 70

The rate of mitochondrial phosphorylation, evaluation by coefficient of respiratory control and the ratio ADP/O, was decreased as a result of lowering in the rate of phosphorylation and DNP-stimulated oxidation, occurred in rat liver tissue under conditions of long-term starvation (5 days) as compared with control animals. In starvation rat liver mitochondria were most distinctly impaired by high temperature, phospholipase A2 and trypsin. Latent destructions, formed in mitochondrial membranes, were responsible for high lability of the organelles in starvation. In long-term starvation incorporation of exogenous cytochrome c into mitochondrial membrane was impaired also due to deterioration in structural relationship between phospholipids and protein of the membrane.
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PMID:[Oxidative phosphorylation and the activity of the polyenzyme systems of rat liver mitochondrial membranes in starvation]. 711 54

The first application of deuterium nuclear magnetic resonance spectroscopy (2H NMR) to fully deuterated pyridine (d5-pyridine)-iron(III) porphyrin complexes is described. (1) d5-Pyridine gives very broad 2H NMR signals in the presence of hemin or horseradish peroxidase to which pyridine is hardly (or not) bound, probably due to relatively long electronic relaxation times of the high-spin ferric ions. d5-Pyridine in the presence of horse-heart metmyoglobin gives resolved and less broadened 2H NMR signals, probably due to the relatively short electronic relaxation times of the ferric ion and/or to slow chemical exchange of the ligand. (2) The three resonances of free d5-pyridine coalesce into a single or a double resonance in the presence of a heme octapeptide prepared by trypsin digestion of Candida krusei cytochrome c. Nuclear spin-lattice and spin-spin relaxation times of the d5-pyridine-heme octapeptide complex are markedly shorter than those of free d5-pyridine. These findings are interpreted by the chemical exchange mechanism from the temperature dependences of the relaxation times. Thus, on certain assumptions the residence time of pyridine in the bound state and the exchange rate are estimated as approximately 10(-3) s and approximately 400 s-1, respectively, at 25 degrees C. Since 1H NMR signals of axial ligands of paramagnetic hemoproteins are hard to observe, the usefulness of deuterium magnetic resonance for investigating ligand exchange in the paramagnetic hemoproteins is emphasized.
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PMID:Deuterium nuclear magnetic resonance spectroscopy of deuterated pyridine-iron(III) porphyrin complexes. Locations and relaxation times of bound deuterated pyridine resonances. 711 56


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