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 photolabile benzphetamine analogue N-(p-azidobenzyl)-N-methyl-p-azidophenetylamine (N3-BP-N3) and its tritiated derivative were synthesized and used as photoaffinity ligands for P4501A1 substrate binding. The enzymatic activity of P4501A1 toward ethoxycoumarin was competitively inhibited by N3-BP-N3. After irradiation with UV light a radioactive photolysis product remained bound to P4501A1. After large scale labeling in the absence and in the presence of alpha-naphthoflavone, P-450 was digested with 1-p-tosyl-amino-2-phenylethyl chloromethyl ketone-treated trypsin and the resultant peptide fragments were separated with HPLC on a reverse-phase column. Six peptides with increased levels of incorporated radioactivity were detected and from a competition experiment in the presence of the inhibitor, four of them could be tentatively assigned as involved in substrate interaction. Amino acid sequences were determined and compared with the primary P-4501A1 sequence. N3-BP-N3 can bind amino acid residues through both ends of the molecule and, therefore, crosslinked peptides could be identified. Alignment of the primary structure of cytochrome P4501A1 with that of cytochrome P450102 revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and beta10-beta11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were predicted to be involved in reductase-P450 interaction.
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PMID:Identification and characterization of cytochrome P4501A1 amino acid residues interacting with a radiolabeled photoaffinity diazido-benzphetamine analogue. 865 89

Progesterone and some other steroids have been shown to induce a steroid 11alpha-hydroxylating enzyme system requiring cytochrome P450 in the filamentous fungus Rhizopus nigricans. In the present work, we attempted to find out whether the mycelial cytosol contained progesterone-binding sites (PBS) which could function as receptors for P450-inducing steroids and might, therefore, be included in the induction process. Two types of constitutive PBS, PBS-I and PBS-II, were identified in the cytosol pretreated with dextran-coated charcoal which removed the endogenous ligand. The protein nature of these binding activities was indicated by their susceptibility to trypsin and proteinase K digestion, heat denaturation, and their resistance to DNase. Progesterone binding was rapid, the maximal level being reached after 45 min of incubation at 22 degrees C. At this temperature, dissociation of progesterone from PBS-I proceeded with a t1/2 of 17 min and that from PBS-II with a t1/2 of 133 min. The apparent Kd of PBS-I determined by Scatchard analysis was 2.1-7.0 x 10(-9)M, and Bmax 36-218 fmol/mg protein. Bmax for PBS-II was >400 fmol/mg protein, whereas the value of Kd could not be determined accurately due to the sigmoidal nature of the association kinetics. The biological role of PBS-I in transcriptional regulation is suggested by the observation that this receptor-like protein contains a functional DNA-binding domain. A specific function of PBS-I in the induction of 11alpha-hydroxylase seems to be, however, questionable because of poor correlation between the affinity and the inducing capability of corresponding steroids.
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PMID:Identification and partial characterization of cytosolic progesterone-binding sites in the filamentous fungus Rhizopus nigricans. 865 7

Bovine adrenocortical cytochrome P450scc (CYP11A1) was selectively modified with diiodofluorescein iodoacetamide (DIFIA). Only Cys264 is labeled in the P450 polypeptide chain. The modification significantly affected the cholesterol-hydroxylating activity in the reconstituted system containing NADPH, adrenodoxin reductase, adrenodoxin, and soluble or membrane-bound P450scc. The inhibitory effect correlates with decreased affinity of cytochrome P450scc to intermediate electron carrier, adrenodoxin. Cytochrome P450scc is modified in liposomes and the modified membrane-bound protein is cleaved by trypsin forming two large fragments F1 and F2 corresponding to the N- and C-terminal regions of the molecule. The data indicate that the Cys264-containing region of the cytochrome P450scc molecule is exposed to the surface of protein globule, located outside of the membrane, and can participate in protein-protein interactions.
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PMID:[Selective chemical modification of cytochrome P450scc (CYP11A1) with diiodofluorescein iodoacetamide in the study of the role and topology of interdomain hinge of the hemoprotein molecule]. 915 54

Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494-512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501-504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501-L502-K503, is the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to beta-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. To our knowledge, this is the first report to localize the cumene hydroperoxide binding region in the cytochrome P450 active site.
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PMID:Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region. 943 87

The contractile actions of the proteinase-activated receptor-2-activating peptides (PAR2APs), SLIGRL-NH2 (SL-NH2), SLIGKV-NH2 (KV-NH2), trans-cinnamoyl-LIGRLO-NH2 (tc-NH2), and the PAR1-AP. TFLLR-NH2 (TF-NH2) as well as trypsin and thrombin were studied in endothelium-denuded and intact human umbilical vein (HUV) ring preparations. In HUV rings with, but not without an intact endothelium, PAR2APs caused a concentration-dependent contractile response, whereas LSIGRL-NH2 trypsin and PAR1APs were inactive. The contractile response was not affected by the endothelin ETA receptor antagonist, BQ123, the cyclooxygenase inhibitor, indomethacin, the leukotriene synthesis inhibitor, MK886, or the epoxygenase/P450 inhibitor, SKF-525A. Other pharmacological antagonists (prazosin, Losartan") were similarly inactive. The order of potencies of the PAR2APs to cause a contraction in the endothelium-intact preparation was: SL-NH2 > > KV-NH2 > or = tc-NH2. Using an endothelium-free rat aorta ring as a reporter tissue, surrounded with endothelium-intact HUV as a donor tissue in a 'sandwich assay,' we also monitored the ability of SL-NH2, TF-NH2, trypsin and thrombin to release either contractile (EDCF) or relaxant (EDRF) factors. In the 'sandwich assay' done in the presence of L-NAME (0.1 mM), the endothelium-intact HUV tissue (but not endothelium-denuded HUV) released a contractile factor (EDCF) in response to SL-NH2 (50 microM) but not to trypsin or LSIGRL-NH2. The SL-NH2-mediated release/action of the EDCF was not affected by BQ123, indomethacin, MK886 or SKF-525A. In the 'sandwich assay', trypsin (4-10 nM), SL-NH2, KV-NH2 and tc-NH2 caused the release of a relaxant activity (EDRF) from the endothelium-intact (but not the denuded) HUV preparation. The release of EDRF was blocked by 0.1 mM (omega)nitro-L-arginine-methylester (L-NAME). Neither thrombin (10 u ml(-1), 100 nM) nor TF-NH2 (50 microM) were active in this EDRF-release assay. The relative potencies of the PAR2 agonists for causing the release of EDRF in the HUV sandwich assay were: trypsin> >SL-NH2> >tc-NH2>KV-NH2. This order of potencies differed from the one observed for the same agonists in the HUV contraction assay (above) and in an intracellular calcium signalling assay, conducted with cloned human PAR2 that was expressed in cultured rat kidney KNRK cells: trypsin > > SL-NH2 = tc-NH2 > KV-NH2. We conclude that PAR2APs (but not PAR1APs) via a receptor distinct from PAR2, can cause a contractile response in endothelium-intact HUV tissue via the release of a diffusable EDCF, that is different from previously recognized smooth muscle agonists (e.g. prostanoid metabolites, endothelin, noradrenaline, angiotensin-II, acetylcholine).
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PMID:Endothelium-dependent contractile actions of proteinase-activated receptor-2-activating peptides in human umbilical vein: release of a contracting factor via a novel receptor. 988 72

Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH-cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17alpha was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17alpha was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 17alpha and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17alpha's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N- and C-terminal domains and the putative F-G loop were concluded to be in or near the membrane-binding domains of P450 17alpha.
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PMID:Membrane topology of guinea pig cytochrome P450 17 alpha revealed by a combination of chemical modifications and mass spectrometry. 1466 79

Previous studies have shown that the induction of P450 cytochrome 2E1 (CYP2E1) is associated with the loss of proteasomal activities. To correlate the loss of proteasomal activity with CYP2E1 induction, ethanol was fed intragastrically for 1, 3, 7, and 15 days. The maximum induction of CYP2E1 (3.5-fold) occurred after 15 days of ethanol feeding. However, there was no significant decrease in the 26 S chymotrypsin-like and trypsin-like activity over this period of time. When ethanol was given to rats for 1 month, CYP2E1 was significantly induced, and the proteasomal activity was significantly decreased. These results indicate that proteasomal activity was not directly affected by ethanol or CYP2E1 induction. Since 4-hydroxynonenal (4-HNE) concentration was significantly increased at 1 month of ethanol feeding, it was suspected that 4-HNE adduct formation with proteasome subunits could be the mechanism of proteasome inhibition. Using an antibody to 4-HNE adducted proteins in Western blot analysis of the 26 S proteasome fraction isolated from the liver of alcohol fed rats, one extra band appeared around 44 kDa. When the antibody to an ATPase Rpt4 was used to stain the stripped membrane, the same band that was detected with the 4-HNE antibody was detected with the Rpt4 antibody. An adduct of 4-HNE formed with the Rpt4 subunit of 26 S could impede the association of 19 S and 20 S and thus account for the observed decrease of proteasomal activity.
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PMID:The effect of ethanol-induced CYP2E1 on proteasome activity: the role of 4-hydroxynonenal. 1571 35

The effect of nonlamellar-prone lipids, diacylglycerol (DG) and phosphatidylethanolamine (PE), on the stability of human cytochrome P450 1A2 (CYP1A2) was examined. When 100% phosphatidylcholine (PC) in standard vesicles was gradually replaced with either DG or PE, the stability of CYP1A2 increased; the incubation time-dependent destruction of spectrally detectable P450, decrease of catalytic activity, reduction of intrinsic fluorescence, and increased sensitivity to trypsin digestion were significantly alleviated. The ternary system of PC/PE/DG increased the stability of CYP1A2 more, even at lower concentrations of each nonlamellar-prone lipid, than that of the binary lipid mixture (PC/nonlamellar lipid). By incorporating the nonlamellar-prone lipids, the CYP1A2-induced increase of the surface pressure of the lipid monolayer was much higher compared to that for 100% PC. Increased surface pressure indicates a deep insertion of the protein into lipid monolayers. Nonlamellar lipids also increased the transition temperature of CYP1A2 in thermal unfolding and reduced the incubation time-dependent detachment of membrane-bound CYP1A2 from vesicles. Taken together, these results suggest that nonlamellar lipids per se and/or the phase properties of the membrane containing these lipids are important in the enhanced stability of CYP1A2 and the concomitant maintenance of catalytic activity of the protein.
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PMID:Involvement of nonlamellar-prone lipids in the stability increase of human cytochrome P450 1A2 in reconstituted membranes. 1596 43

Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18O/16O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed.
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PMID:Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling. 1729 99

1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.
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PMID:Identification of covalent modifications in P450 2E1 by 1,2-epoxy-3-butene in vitro. 1729 33


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