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)

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) has been shown to react with bovine liver glutamate dehydrogenase in the region of the GTP-dependent NADH inhibitory site with incorporation of about 1 mol of reagent/mol of subunit [Ozturk, D. H., Safer, D., & Colman, R. F. (1990) Biochemistry 29, 7112-7118]. The modified enzyme was shown to contain only 5 free sulfhydryl groups upon 5,5'-dithiobis (2-nitrobenzoate) titration as compared with 6 in the unmodified enzyme. In the unmodified enzyme digested with trypsin, 6 cysteinyl peptides were detected by high-performance liquid chromatography upon treatment with iodo [3H]acetic acid. In contrast, only 5 (carboxymethyl)cysteinyl peptides were detected in 8-BDB-TA-5'-TP-modified enzyme. When carboxymethylated modified and unmodified enzymes were digested with thermolysin, 6 peptide sequences containing (carboxymethyl)cysteine were obtained in the unmodified enzyme, but only 5 were observed in the modified enzyme. The (carboxymethyl)cysteine which was absent in the modified enzyme was determined to be Cys-319, leading to the conclusion that 8-BDB-TA-5'-TP reacts with Cys-319, thereby preventing it from subsequent reaction with radioactive iodoacetate. It was previously reported that 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TA-5'-DP) modifies Cys-319 in this enzyme [Batra, S. P., & Colman, R. F. (1986) Biochemistry 25, 3508-3515].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of cysteine-319 as the target amino acid of 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate in bovine liver glutamate dehydrogenase. 185 24

Native rat liver methylmalonate semialdehyde dehydrogenase was proteolyzed by lysylendopeptidase C, chymotrypsin, and trypsin to generate different cleavage fragments of molecular masses: 50, 8, 55, 44, 39, 53, 45, and 40 kDa. A proteolytic cleavage map of MMSDH was constructed based on sequencing data and a comparison of appearance and degradation rates of the different protein fragments as shown by SDS-PAGE. NAD+ was highly effective as a protector against proteolysis in both the N-terminal and the C-terminal parts of the intact enzyme. NADH did not efficiently protect the intact enzyme; however, it stabilized proteolytic fragment L50 from further degradation. This suggests that the NAD(+)-binding domain is not destroyed by cleavage of the N-terminal part of MMSDH. CoA had no effect on the proteolytic cleavage patterns of MMSDH. However, CoA esters reduced the protective effect of NAD+ with an order of effectiveness of acetyl-CoA greater than propionyl-CoA greater than butyryl-CoA. p-Nitrophenyl acetate, substrate for esterase activity by the enzyme, partially prevented the protective effect of NAD+ against proteolysis. These results suggest that S-acylation of the enzyme prevents a stabilizing conformational change induced in MMSDH by NAD+ binding.
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PMID:The effect of ligand binding on the proteolytic pattern of methylmalonate semialdehyde dehydrogenase. 189 92

Legionella pneumophila has been shown to survive and multiply in a variety of intracellular environments, including protozoa and human mononuclear phagocytes. However, the mechanism by which this organism acquires iron in the intracellular environment has not been studied. Since L. pneumophila does not produce siderophores, alternative methods of iron acquisition were investigated. Virulent strains of L. pneumophila were able to grow in media containing as little as 3 microM iron, whereas avirulent cells required a minimum of 13 microM iron for growth. Neither virulent nor avirulent cells were able to utilize 55Fe bound to transferrin. When incubated in the presence of 55Fe in the form of ferric chloride, both virulent and avirulent cells accumulated equal amounts of iron. The uptake of iron was energy dependent as indicated by inhibition of 55Fe uptake at 4 degrees C and preincubation of the cells with KCN. Treatment of virulent cells with pronase or trypsin had no effect on iron uptake. In contrast, pronase or trypsin treatment of avirulent cells resulted in increased uptake of iron. Iron reductase activity in both virulent and avirulent cells was demonstrated, with the highest specific activity associated with the periplasmic fraction. Maximum reductase activity of virulent cells occurred with NADH as the reductant. In contrast, avirulent cells showed a twofold increase in enzyme activity when NADPH was used as the reductant. These results suggest that an iron reductase is important in iron acquisition by L. pneumophila.
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PMID:Acquisition of iron by Legionella pneumophila: role of iron reductase. 190 41

35S-labeled Drosophila melanogaster apocytochrome c was made by in vitro transcription/translation of the gene and purified to the monomeric, fully reduced form. It was found that in the presence of a wheat germ extract factor there was a high-affinity phase of the uptake into mouse liver mitochondria at 10-300 pM apocytochrome c, and a lower-affinity phase through 4000 pM. Without the factor, the high-affinity phase was absent. The stimulatory effect of the factor could not be elicited with various reductants, such as NADH, FMN, and ferrous protoheme IX. Conversely, when mitochondria loaded with apocytochrome c were resuspended in fresh medium, the protein readily reequilibrated. Successive washings depleted greater than 95% of the associated apoprotein but removed no holoprotein. Proteases (proteinase K or trypsin) added to a suspension of mitochondria loaded with apoprotein digested an amount of apoprotein similar to that which would have been dissociated during the same time, as measured by successive washings in the absence of protease. Mitochondria loaded with apoprotein and similarly treated with protease continued exporting the apoprotein, even after the protease was inhibited and removed, suggesting that most of the apoprotein associated with the organelle was in a protease-resistant compartment. Apocytochrome c mutants in which serines or alanines replaced cysteines 14 and 17, which bind the prosthetic group, behaved like the cysteine-containing protein, indicating that the covalent attachment of the heme is unrelated to the translocation of the apoprotein.
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PMID:Reversible import of apocytochrome c into mitochondria. 216 15

The effects of culture variables on the specific content and activity of various enzymes of the drug metabolizing system were assessed in colon tumor cell line LS174T. The NADH reduced cytochrome b5 (cyt b5)4 spectrum of these cells was similar to rat liver cyt b5. When released from the membrane by trypsin and concentrated, the cyt b5 was found to cross react with rabbit antibody to rat liver cyt b5 and human liver cyt b5. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 mumol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b5 and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone showed a consistent, but not always significant increase in the NADPH and NADH cyt c reduction and benzanthracene an increase in the NADH cyt c reducing activity and cyt b5 content. Griseofulvin lowered the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5 mM) caused a significant decrease in the specific activity of all enzymes, as judged by a student's t test, with a p less than 0.001.
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PMID:Human colon tumor cell line LS174T drug metabolizing system. 234 45

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.
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PMID:Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites. 236 Nov 37

The effect of Ca2+ or Mg2+ on cytochrome b5 reduction by porcine liver microsomes was examined using trypsin-solubilized cytochrome b5 as a substrate. The reduction of exogenous cytochrome b5 by microsomes was low at 1.2 microM cytochrome b5 (3.9 or 2.7 nmol/min/mg protein, respectively, with NADH or NADPH). The addition of CaCl2 greatly enhanced either NADH-dependent or NADPH-dependent cytochrome b5 reduction. At 2 mM CaCl2, the reduction rate was increased to 23- or 18-fold of control, respectively with NADH or NADPH. The concentration for half-maximal effect (EC50) was 0.5 or 0.6 mM in the NADH or NADPH systems, respectively. MgCl2 also stimulated cytochrome b5 reduction with a EC50 value of 1.0 mM in the NADH system or 0.6 mM in the NADPH system. The comparison with the result with KCl indicated that the activation by CaCl2 or MgCl2 is caused mainly by their divalent cation moiety. The Km value for cytochrome b5 was decreased and the Vmax was increased by calcium with either the NADH- or the NADPH-dependent system. NADH-ferricyanide reductase activity was not affected by calcium, but NADPH-ferricyanide reductase activity was stimulated as well as NADPH-cytochrome c reductase activity. In the presence of Triton X-100, divalent cations were inhibitory in NADH-dependent cytochrome b5 reduction, and in contrast, stimulative in NADPH-dependent reaction. These findings suggest that the activation of cytochrome b5 reduction by divalent cations in the NADH system is mainly due to an increasing accessibility of the substrate, and in the NADPH system, in addition to this, a direct effect of divalent cations on NADPH-cytochrome P450 reductase is also involved.
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PMID:Effect of divalent cations on NADH-dependent and NADPH-dependent cytochrome b5 reduction by hepatic microsomes. 236 23

The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.
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PMID:The pathway of electron transfer in NADH:Q oxidoreductase. 249 59

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.
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PMID:Topological investigations. Study of the trypsin sensitivity of the N-acetylglucosaminyl and mannosyl-transferase activities located in the outer mitochondrial membrane. 252 39

Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.
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PMID:Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina. 253 41


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