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)

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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PMID:The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase. 155 Mar 61

Human phagocyte cytochrome b is the terminal component of the microbicidal superoxide generating system. Although the primary structure of this protein has been determined, little is known about the placement of the heme prosthetic groups in this heterodimeric integral membrane protein. Analysis of the cytochrome using lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 0 degree C followed by tetramethylbenzidine heme staining demonstrated the presence of heme in both the 91- and 22-kDa subunits identified by Western blot analysis using peptide specific antisera. Exposure of cytochrome b (purified or in isolated neutrophil plasma membranes) to Staphylococcal protease V8 or trypsin did not affect absorbance spectra. However, such treatment resulted in degradation of both subunits to smaller fragments, including characteristic immunoreactive 20-kDa fragments of both the large and small subunits of the cytochrome that retained one or both of the hemes. The spectral stability to proteolysis and size of the proteolytic heme-containing fragments generated explains previous reports which suggested that the heme resided in the small subunit. Our current results indicate that human neutrophil cytochrome b is a bi-heme or possibly tri-heme molecule with at least one heme residing in the large subunit and one shared between both subunits and that the heme-containing regions of the cytochrome probably lie within the membrane lipid bilayer. Such a multi-heme structure would be consistent with an electron transfer function for this cytochrome by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create superoxide.
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PMID:Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits. 155 74

A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE. The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous-CO and ferrous-O2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain, and that the two domains are folded almost independently of each other. An NADPH-cytochrome-P-450 reductase system composed of NADPH and detergent-solubilized NADPH-cytochrome-P-450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.
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PMID:Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization. 165 Dec 44

The Ah receptor binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related aryl hydrocarbons and mediates their biochemical and toxic effects by modifying gene expression. In order to interact with DNA, the TCDD.receptor complex must undergo a poorly understood transformation to a form which is distinguishable by its increased affinity for DNA-Sepharose and for its specific enhancer element upstream from the cytochrome P450IA1 gene. We have found that this transformation process is inhibited in vitro by treatment of rat hepatic cytosol with activated charcoal prior to addition of [3H]TCDD. The transformed form of the receptor can be generated in the charcoal-inhibited cytosol by incubation with hepatic cytosol from either DBA/2J mice (in which [3H]TCDD-specific binding is undetectable under these conditions) or rat (in which Ah receptor was prebound with unlabeled ligand). Transformation is observed whether this addition occurs before or after [3H]TCDD is bound to the charcoal-treated receptor. Thus, transformation is (i) mediated by some additional cytosolic component(s) and (ii) separable from ligand binding. The untransformed [3H]TCDD.receptor complex, isolated by DNA-Sepharose chromatography, can also be transformed if DBA mouse hepatic cytosol is added. This partially purified untransformed receptor preparation and gel retardation analysis were used to further characterize the transforming activity in DBA cytosol. We observed that the "Ah receptor transforming factor" is heat-labile, trypsin-sensitive, removed or inactivated by charcoal, of greater than approximately 50 kDa, and elutes from Superose at a Rs of approximately 6 nm. In conjunction with our previous studies documenting the increased molecular weight of the transformed compared to the untransformed Ah receptor, and identifying the heteromeric structure of the transformed receptor, we postulate that the ligand-binding subunit (the untransformed receptor) must associate tightly with another cytosolic protein, which is also present in the receptor-defective DBA mouse, in order to transform to the transcriptionally active receptor.
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PMID:Inhibition and reconstitution of Ah receptor transformation in vitro: role and partial characterization of a cytosolic factor(s). 165 20

The periplasmically located cytochrome c553i of Paracoccus denitrificans was purified from cells grown aerobically on choline as the carbon source. The purified protein was digested with trypsin to obtain several protein fragments. The N-terminal regions of these fragments were sequenced. On the basis of one of these sequences, a mix of 17-mer oligonucleotides was synthesized. By using this mix as a probe, the structural gene encoding cytochrome c553i (cycB) was isolated. The nucleotide sequence of this gene was determined from a genomic bank. The N-terminal region of the deduced amino acid sequence showed characteristics of a signal sequence. Based on the deduced amino acid sequence of the mature protein, the calculated molecular weight is 22,427. The gene encoding cytochrome c553i was mutated by insertion of a kanamycin resistance gene. As a consequence of the mutation, cytochrome c553i was absent from the periplasmic protein fraction. The mutation in cycB resulted in a decreased maximum specific growth rate on methanol, while the molecular growth yield was not affected. Growth on methylamine or succinate was not affected at all. Upstream of cycB the 3' part of an open reading frame (ORF1) was identified. The deduced amino acid sequence of this part of ORF1 showed homology with methanol dehydrogenases from P. denitrificans and Methylobacterium extorquens AM1. In addition, it showed homology with other quinoproteins like alcohol dehydrogenase from Acetobacter aceti and glucose dehydrogenase from both Acinetobacter calcoaceticus and Escherichia coli. Immediately downstream from cycB, the 5' part of another open reading frame (ORF2) was found. The deduced amino acid sequence of this part of ORF2 showed homology with the moxJ gene products from P. denitrificans and M. extorquens AM1.
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PMID:Isolation, sequencing, and mutagenesis of the gene encoding cytochrome c553i of Paracoccus denitrificans and characterization of the mutant strain. 165 73

The orientation of cytochrome b6 in the thylakoid membrane and the question of whether the number of membrane spanning helices is an even or odd number was tested through the relative trypsin susceptibility of epitopes (Asp-5 to Gln-14) and (Ile-205 to Leu-214) at the NH2 and COOH termini, respectively, of the 214-residue cytochrome b6 polypeptide. A structure of the cytochrome with an even number of helices and the NH2 and COOH termini on the stromal side of the membrane was inferred from the following: 1) cleavage of cytochrome b6 by trypsin added to thylakoids occurs by removal of both of the exposed NH2- and COOH-terminal epitopes. The epitopes at the termini were more sensitive to trypsin after prior treatment of thylakoids with carboxypeptidase A, indicating that these epitopes are shielded on the stromal side of the membrane by the COOH termini of other proteins. 2) Both epitopes were more trypsin-sensitive in thylakoid membranes than was cytochrome f that is only sensitive to trypsin acting on the lumen side of the membrane. 3) The NH2- and COOH-terminal epitopes of cytochrome b6 were also more sensitive to trypsin added to thylakoid membranes than were the oxygen-evolving complex 16- and 33-kDa proteins that are completely located on the lumen side. 4) The order of trypsin susceptibility was reversed in inside-out membranes, where the cytochrome NH2- and COOH-terminal epitopes were less sensitive than the 16- and 33-kDa proteins. The decreased relative sensitivity of the cytochrome b6 epitopes occurs in spite of a greater absolute sensitivity of these epitopes to trypsin in inside-out membranes. 5) The greater absolute sensitivity can be explained by a 4-helix model that includes trypsin-sensitive sites on the lumen side.
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PMID:Thylakoid membrane protein topography. Location of the termini of the chloroplast cytochrome b6 on the stromal side of the membrane. 169 78

The cytochrome d complex is a two-subunit, membrane-bound terminal oxidase in the aerobic respiratory chain of Escherichia coli. The enzyme catalyzes the two-electron oxidation of ubiquinol and the four-electron reduction of oxygen to water. Previous work demonstrated that the site for ubiquinol oxidation was selectively inactivated by limited proteolysis by trypsin, which cleaves at a locus within subunit I. This work is extended to show that a similar phenomenon is observed with limited chymotrypsin proteolysis of the complex. The cleavage patterns are similar whether one uses the purified oxidase in nondenaturing detergent or reconstituted in proteoliposomes or uses spheroplasts of E. coli as the substrate for the proteolysis. Hence, the protease-sensitive locus is periplasmic in the cell. Fragments resulting from proteolysis were characterized by N-terminal sequencing and by immunoblotting with the use of a monoclonal antibody of known epitope within subunit I. The data indicate that inactivation of the ubiquinol oxidase activity results from cleavage at specific residues with a hydrophilic region previously defined as the Q loop. This domain has been already implicated in ubiquinol oxidation by the use of inhibitory monoclonal antibodies. Electrochemical and HPLC analysis of the protease-cleaved oxidase suggests no global changes in either the quaternary or tertiary structure of the enzyme. It is likely that the Q loop is directly involved in forming a portion of the ubiquinol binding site near the periplasmic surface of the membrane.
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PMID:Proteolysis of the cytochrome d complex with trypsin and chymotrypsin localizes a quinol oxidase domain. 170 10

Monoclonal antibodies (MAbs) were prepared against native cytochrome f (cyt f) isolated from turnip leaves. The two MAbs obtained, designated MAb-JB2 and MAb-ED4, were Western blot positive to purified turnip cytochrome f and also reacted with inside-out (ISO) but not right-side-out (RSO) spinach thylakoid membranes. MAb-ED4 reacted with a covalent adduct formed by crosslinking cyt f and plastocyanin (PC), whereas MAb-JB2 did not. In contrast, MAb-JB2 reacted with the isolated cyt b6/f complex but MAb-ED4 did not. These results indicate that MAb-JB2 binds to cyt f at or near the PC binding site on f, whereas MAb-ED4 binds to a portion of cyt f which is not exposed in the cyt b6/f complex. The location of the epitopes in the primary sequence of cyt f was determined by trypsin hydrolysis, HPLC separation of tryptic peptides, and ELISA identification of the purified peptides. The molecular weights of the purified peptides, determined by gel exclusion chromatography, were found to be 5040 and 3130 Da for MAb-JB2 and MAb-ED4, respectively. Amino acid sequencing showed that the first eight amino acids of the MAb-ED4 positive peptide were L-D-Q-P-L-T-S-N. These results suggest that the 3130-Da peptide has 28 amino acids extending from Leu 223 to Arg 250. This peptide is located on the N-terminal (lumen) side of the postulated membrane-spanning sequence. The first eight amino acids of the MAb-JB2-positive peptide were N-I-L-V-I-G-P-V. This sequence and the peptide molecular weight indicate that the epitope for MAb-JB2 is located within a 44-amino acid peptide extending from Asn 111 to Arg 154.
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PMID:The use of monoclonal antibodies to study the structure and function of cytochrome f. 171 77

Monospecific polyclonal rabbit antibodies to a purified form of haem oxygenase of chick liver, showing sequence similarity to mammalian haem oxygenase-1, were raised and used to study characteristics of the oxygenase. The antibodies inhibited activity of the purified oxygenase, but not other enzyme components (NADPH:cytochrome reductase and biliverdin reductase) of the standard assay mixture of haem oxygenase. In addition, the antibodies inhibited activity of haem oxygenase in microsomes (microsomal fractions) from Cd(2+)-treated chick liver, spleen, testis and brain. Western (immuno-) blots of microsomal proteins of selected organs from chick, rat and man, and homogenates of chick-embryo liver-cell cultures, probed with the antibodies, showed a major protein with a molecular mass of 33-34 kDa and a lower-molecular-mass protein (28-29 kDa) of variable intensity. Studies with trypsin and selected proteinase inhibitors established that the smaller peptide was a proteolytic product of the larger. Treatment of chick-embryo liver-cell cultures with CdCl2, a potent inducer of haem oxygenase, increased the degree of proteinase-mediated cleavage of the 33 kDa protein to the lower-molecular-mass form. These results indicate that, under at least some conditions, such cultures should be homogenized in the presence of trypsin inhibitor to prevent proteolytic degradation of the enzyme and allow maximal expression of haem oxygenase activity. The antibodies also reacted with haem oxygenase from spleen, testis and brain of both chicks and rats, and the spleen of humans. A method for quantifying the amount of haem oxygenase protein was developed with use of slot-blots and laser densitometry; linearity was observed from 0 to 5 ng of haem oxygenase protein per slot, and the method was applied to sonicated cultured chick-embryo liver cells treated with Cd2+ (0.3 mM) or iron plus glutethimide. In both cases, increases in enzyme activity were of similar magnitude to increases in amounts of enzyme protein. Approximate amounts of haem oxygenase protein in microsomes of several organs from intact animals could also be estimated by the use of slot-blot-laser densitometry, and the amounts measured were increased by the addition of purified haem oxygenase to the microsomal preparations. Results of these studies indicated that haem oxygenase-1 could be detected in microsomes from all chick or rat organs studied, including testis and brain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Immunochemical studies of haem oxygenase. Preparation and characterization of antibodies to chick liver haem oxygenase and their use in detecting and quantifying amounts of haem oxygenase protein. 195 81

The catalytic role of subunit IV, the Mr 17,000 protein, in the chloroplast cytochrome b6-f complex was established through trypsinolysis of the complex under controlled conditions. When purified chloroplast cytochrome b6-f complex, 1 mg/ml, in 50 mM Tris-succinate buffer (pH 7.0) containing 1% sodium cholate and 10% glycerol is treated with 80 micrograms of trypsin at room temperature for various lengths of time, the activity of the cytochrome b6-f complex decreases as the incubation time increases. A maximal inactivation of 80% is reached at 7 min of incubation. The trypsin inactivation is accompanied by the destruction of the proton translocation activity of the complex. No alteration of absorption and EPR spectral properties was observed in the trypsin-inactivated complex. Subunit IV is the only subunit in the cytochrome b6-f complex that is digested by trypsin, and the degree of digestion correlates with the decrease of electron transfer activity. The binding of azido-Q to subunit IV of the complex decreases as the extent of inactivation of the cytochrome b6-f complex by trypsin increases. The residue molecular mass of trypsin cleaved subunit IV is about 14 kDa, suggesting that the cleavage site is at lysine 119 or arginine 125 or 126. When the thylakoid membrane was assayed for cytochrome b6-f complex activity, very little activity was observed; and the activity was not sensitive to trypsinolysis. Upon sonication, activity and sensitivity to trypsinolysis was greatly increased, suggesting that subunit IV protrudes from the lumen side of the membrane.
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PMID:The catalytic role of subunit IV of the cytochrome b6-f complex from spinach chloroplast. 201 49


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