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

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The monoclonal antibody, 3/4/2, which was raised against purified rat cytochrome P450 isoenzyme 1A1 (CYP1A1) binds to cytochromes P4501A in many species. It was shown by immunoblotting that the antibody binds to CYP1A1 in microsomal fractions prepared from rat, mouse, rabbit, hamster and human. The antibody also binds to cytochrome P450 isoenzyme 1A2 in microsomal fractions prepared from rabbit and human, but not rat or mouse. Using purified isoenzymes in an enzyme-linked immunosorbent assay it was found that the affinity of binding to the two rabbit hydrocarbon-inducible isoenzymes is reduced compared with that for rat CYP1A1. Binding is not affected by denaturation of the antigens. The effects of chemical and enzymatic treatments on rat CYP1A1 showed that the epitope contains a trypsin-sensitive site that includes arginine, but lacks lysine. The epitope does not contain methionine, cysteine, aspartic acid or glutamic acid residues. In addition, digestion of the protein with cyanogen bromide produces a fragment of Mr 20,000 which contains the antibody binding site. By comparing the cross-reactivity of the antibody with the primary structures of CYP1A1 and 1A2 from the rat, mouse, rabbit and human, and by considering the results of the chemical and enzymatic treatments, it was possible to deduce the likely location and structure of the binding site of 3/4/2 on members of the CYP1A subfamily. It is concluded that the epitope for this antibody is Phe-Arg-His-Ser-Ser-Phe, which lies at positions 380-385 in rat CYP1A1. Further, it is predicted from a model of the tertiary structure of eukaryotic cytochrome P450 that a part of this binding site lies within a helix in the native protein.
...
PMID:Identification of the epitope of a monoclonal antibody which binds to several cytochromes P450 in the CYP1A subfamily. 137 49

Acetaminophen is metabolized by cytochrome P450 to a reactive metabolite that covalently binds to proteins and this binding correlates with the hepatotoxicity. The major protein adduct was previously reported to be a 55 kDa protein that was detected on Western blots using antisera specific for 3-(cystein-S-yl)acetaminophen. In this study, the 55 kDa protein was isolated using a combination of ion exchange fast flow chromatography, hydroxyapatite HPLC and anion exchange HPLC. Amino acid sequences of 8 internal peptides from a trypsin digestion of the 55 kDa protein were found to have 97% homology with the deduced amino acid sequence from a cDNA that corresponds to a 56 kDa selenium binding protein. This is the first report of a specific protein to which a metabolite of acetaminophen covalently binds.
...
PMID:A metabolite of acetaminophen covalently binds to the 56 kDa selenium binding protein. 154 Jan 79

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.
...
PMID:Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation. 172 52

Cytochromes P450 beta NF-A, beta NF-B, and beta NF-C were purified from beta-naphthoflavone-treated adult hens. Cytochrome P450 beta NF-A, however, appeared at two places in the purification scheme. They were designated as cytochromes P450 beta NF-A1 and beta NF-A2 for property comparison. The cytochromes beta NF-A1 and beta NF-A2 were induced by both phenobarbital and beta-naphthoflavone treatment and were similar to P450 PB-A (previously purified from phenobarbital-induced hen livers) in molecular weights, isoelectric pH, spectral properties, behavior on chromatography columns, catalysis of substrates, immunological cross-reactivity on Ouchterlony plates and by immunoblotting, and NH2-terminal amino acid sequence. However, P450 PB-A differed from beta NF-A1/beta NF-A2 in peptide pattern after partial proteolysis by alpha-chymotrypsin and Staphylococcus aureus V8 protease, and complete digestion of 125I-labeled cytochromes by trypsin. The cytochrome P450 PB-A also differed from beta NF-A1/beta NF-A2, in that its antibodies cross-reacted with P-450 of normal, PB-, and beta-NF-induced rabbit liver microsomes. The cytochromes beta NF-B and beta NF-C, although immunochemically cross-reactive with each other, were distinct enzymes on the basis of molecular weights, spectral characteristics, isoelectric pH, peptide pattern on partial proteolysis, tryptic peptide pattern, cross-reactivity of their antibodies with other species, and NH2-terminal amino acid sequence. The most notable difference between beta NF-B and beta NF-C was that the anti-beta NF-C IgG completely inhibited O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin by beta-NF-induced microsomes. These activities increased 40- to 50-fold in beta-NF-induced microsomes as compared to only 2- to 4-fold in PB-treated hens. The amino-terminal sequences of beta NF-B and beta NF-C were different from those of mammalian and other nonmammalian species.
...
PMID:Purification and characterization of cytochrome P450 isozymes from beta-naphthoflavone-induced adult hen liver. 217 27

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.
...
PMID:Human colon tumor cell line LS174T drug metabolizing system. 234 45

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.
...
PMID:[Mechanisms of mixed-function oxidase system breakdown in the hepatic endoplasmic reticulum. The role of membrane phospholipid peroxidation]. 671 28

This study was conducted to determine whether a factor responsible for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in rat liver microsomes is involved in iron reduction by cooperation with NADPH-cytochrome P450 reductase. Under anaerobic conditions, NADPH-dependent reduction of ferric pyrophosphate in microsomes was not dependent on cytochrome P450 levels and was not inhibited by carbon monoxide (CO). All of the iron complexes with chelators such as adenosine 5'-diphosphate, pyrophosphate, nitrilotriacetate, oxalate or citrate were reduced in microsomes, although in the reconstituted system containing purified NADPH-cytochrome P450 reductase little or no iron reduction was found. A cytochrome P450-free fraction from a cholate-solubilized preparation of microsomes after passage through a laurate sepharose column was required for reduction of iron pyrophosphate in the reconstituted system leading to lipid peroxidation. The iron reduction was not inhibited by CO and was destroyed by heat treatment or trypsin digestion of the fraction. All iron complexes were reduced in the presence of the fraction, using a reducing equivalent of NADPH via NADPH-cytochrome P450 reductase. The results indicate that a heat-labile component, which is probably a protein distinct from cytochrome P450, is associated with iron reduction responsible for lipid peroxidation in microsomes.
...
PMID:A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation. 776 Jun 89

At low levels of phenobarbital induction two forms of isoenzyme 2 (LM2; CYP2B4) were obtained during purification of cytochrome P450 from rabbit liver microsomes. At high levels of induction only one form (LM2A) was present. Although the two purified forms (LM2A and LM2B) were very similar they differed in: (a) peak elution on CM-Sepharose, (b) wavelength maximum of the reduced P450-CO spectrum, and (c) metabolism of several substrates, where the activities of LM2B ranged from 0.6 to 2.65 times that of LM2A. A third LM2 fraction (2C) was isolated from untreated rabbit liver and, although homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, appeared to be a mixture of LM2B and a form of P450 LM2 other than LM2A. LM2A was not found in the untreated rabbit liver microsomes. On CM-Sepharose the elution of fraction 2C overlapped that of LM2B. The apparent molecular weight and immunoresponse to anti-LM2A IgG were the same for fraction 2C as for LM2A and LM2B. Peptide mapping using trypsin showed no difference between LM2A and LM2B, but consistently revealed at least one extra band with fraction 2C. After CNBr cleavage and high-pressure liquid chromatography separation of the LM2A and LM2B fragments the peptide beginning with Pro(347) of LM2A (peak 4A) eluted 1/2 min later than that of LM2B (peak 4B) indicating a difference in the fragments, although partial NH2-terminal amino acid sequences and molecular masses were the same. The corresponding CNBr fragment of fraction 2C splits into two peaks (4C:1 and 4C:2) with retention times corresponding to 4B and 4A, respectively. The mass of 4C:1 was the same as that of 4B, while the mass of 4C:2 markedly differed from that of 4A and 4B. Both fragments had the same partial NH2-terminal amino acid sequence as 4A and 4B. After comparing the physiochemical properties as well as catalytic activities of these isolated and purified LM2 forms with the cDNA-expressed forms 2B-B0, 2B-B1, 2B-B2, and 2B-Bx [see R. Ryan et al. (1993) Arch. Biochem. Biophys. 304, 454-463], the data suggest that LM2A is the form designated as 2B-B0 (LM2), LM2B is 2B-Bx, and fraction 2C is a mixture containing 2B-B1 and 2B-Bx. This is the first isolation and identification of the three isozymic LM2 proteins from rabbit liver.
...
PMID:The isolation and comparison of multiple forms of CYP2B from untreated and phenobarbital-treated rabbit liver microsomes. 784 Jun 28

A proline-rich region is present following the signal-anchor sequence in the amino-terminal portion of all known microsomal cytochrome P-450s. To assess the functional significance of the proline residues in this region, we systematically altered these residues of cytochrome P450(M1) (P450 2C11); one, two, and three proline residues out of the five in the region were exchanged for alanine residues. The wild-type and the mutated proteins were expressed in the fission yeast Schizosaccharomyces pombe under the control of nmt1 promoter. The wild-type and the mutated proteins were all highly expressed in the yeast cells (5-9% of the total membrane protein). The expressed wild-type P450(M1) showed a typical carbon monoxide difference spectrum of P-450 and the activity of testosterone hydroxylation, whereas all the mutated proteins constructed in the present study showed no characteristic P-450 spectrum, suggesting that the substitution of the proline residues in this region resulted in a defect of proper heme incorporation. Furthermore, the mutated proteins in which more than one proline residues had been exchanged were more sensitive to trypsin digestion than the wild type. From these results, we propose that the proline residues in the proline-rich region are crucial for the formation of the correct conformation of microsomal P-450 molecules.
...
PMID:Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s. 811 16

Chemical modification of cytochrome P450 was used to study the involvement of lysine and arginine residues in the interaction between cytochrome P450 and NADPH-cytochrome P450 reductase. Acetylation of 2.2 and 8.5 mol of lysine/mole of P450 by acetic anhydride led to 38.7 and 95% reductions, respectively, in benzphetamine demethylation activity by NADPH-dependent reconstituted P450/reductase complex, while modification of up to 8.5 mol of lysine/mol of P450 did not inhibit cumene hydroperoxide-supported P450-dependent benzphetamine demethylation. Acetylation of lysine residues by acetic anhydride does not grossly disturb the P450 protein conformation as revealed by absolute, CO-difference and fluorescence spectral studies. Modification of P4502B1 by acetic anhydride did not affect its substrate binding ability either. Lysine residues of P4502B1 putatively involved in the interaction with reductase have been identified by radiolabeling of lysine residues with [14C]acetic anhydride followed by trypsin digestion, HPLC separation, and amino acid microsequencing. Radiolabeled lysines occur at positions 251, 384, 422, 433, and 473. Modification of arginine residues in P4502B1 with phenylglyoxal and 2,3-butanedione seemed to have no significant effect on the benzphetamine demethylation activity of P4502B1 either reconstituted with reductase and NADPH or supported by cumene hydroperoxide. Studies of incorporation of [14C]phenylglyoxal showed no concentration- or time-dependent incorporation of phenylglyoxal into the P4502B1. These results support the hypothesis of a predominant role of lysine residues of P450 in the electrostatic interaction with NADPH-cytochrome P450 reductase.
...
PMID:Role of lysine and arginine residues of cytochrome P450 in the interaction between cytochrome P4502B1 and NADPH-cytochrome P450 reductase. 832 89


1 2 3 4 Next >>

---