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)

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.
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PMID:Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes. 2 1

Two common environmental pollutants, DDT and a polychlorinated biphenyl (PCB) mixture (Clophen A-50), were administered ip to rats in discrete single doses (160 and 100 mg/kg, respectively) and in combination. All the enzyme activities studied were enhanced by DDT and PCB. the overall drug hydroxylation reactions, and their components, achieved maximal induction in 1 wk. The cytochrome P-450 content of microsomes was increased nearly fourfold and NADPH-cytochrome c reductase activity was enhanced twofold by both compounds. p-Nitroanisole O-demethylase was increased sevenfold by PCB and fourfold by DDT, aryl hydrocarbon hydroxylase threefold by PCB and 1.7-fold by DDT. After 2 wk the activities began to decline. Distinct increases in enzyme activities were still detectable 1 month after a single dose. Epoxide hydratase and UDPglucuronosyltransferase activities were also enhanced in 1 wk (epoxide hydratase 2.5-fold by both compounds, UDPglucuronosyltransferase tenfold by PCB in trypsin-activated microsomes but only threefold by DDT). The disappearance of induction in epoxide hydratase was slower than in the monooxygenases, and UDPglucuronosyltransferase still showed a trend toward increased activity 4 wk after the administration. The DDT-enhanced UDPglucuronosyltransferase activity slightly returned toward the control level. Glutathione S-transferase differed from the microsomal enzymes in that it was already elevated 1 day after the administration of both DDT and PCB. Its activity was only doubled, but the increased activity remained at almost the same level through the whole 1 month period.
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PMID:Long-term effects of single and combined doses of DDT and PCB on drug-metabolizing enzymes in rat liver. 41 32

Male rats were fed a cholesterol-free diet for 5 weeks, followed by a 2% cholesterol diet for 4 weeks. Another group of rats was continuously fed a cholesterol-free diet. A third group was fed standard pelllets during the whole experiment. Hepatic microsomal protein and cholesterol contents and drug-metabolizing enzyme activities were measured. The cholesterol-rich diet increased microsomal protein content and this increase disappeared after trypsin digestion of microsomal membranes. Microsomal cholesterol content was enhanced three-fold by cholesterol feeding. Cytochrome P-450 concentration, NADPH cytochrome c reductase and aryl hydrocarbon hydroxylase activities showed only minor changes following cholesterol feeding. The p-nitroanisole O-demethylase and ethoxycoumarin deethylase activities were doubled by cholesterol in comparison to cholesterol-free diet. Trypsin digestion activated the UDP-glucuronosyltransferase enzyme eight- to ten-fold on a protein basis. Trypsin treatment increased the cholesterol activation of UDP-glucuronosyltransferase when compared to the activity in native microsomes. The data suggest that dietary cholesterol regulates the cholesterol content of microsomal membranes. The activities of drug-metabolizing enzymes are also altered, possibly due to the compositional changes of the membranes.
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PMID:Dietary cholesterol-induced enhancement of hepatic biotransformation rate in male rats. 70 59

Rats were fed cholesterol, cacao butter, or olive oil diets to determine the effect of dietary lipids on the rate of drug biotransformation in the liver and duodenum. The cholesterol rich diet maintained the hepatic aryl hydrocarbon hydroxylase activity at the same level as did the standard diet. Rats fed olive oil and cacao butter diets showed lower hepatic aryl hydrocarbon hydrorylase activity. The p-nitroanisole O-demethylase activity was doubled in hepatic microsomes of rats fed the high cholesterol diet when compared to rats fed the standard diet. The hepatic uridine diphosphate glucuronosyltransferase activity showed different patterns depending on the in vitro treatment of the microsomal membranes. If the enzyme activity was assayed from the native, untreated microsomes, an increase in the measurable uridine diphosphate glucuronosyl transferase activity was found in rats having cholesterol rich diet. After the in vitro activation of membrane-bound uridine diphosphate glucuronosyltransferase by trypsin, the increase in measurable activity was 10 fold in the group fed the standard diet, 6 fold in group fed cholesterol, 4 fold in group fed cacao butter, and 3 fold in group fed olive oil. Trypsin digestion of microsomes increased the measurable uridine diphosphate glucuronosyltransferase activity less in rats fed diets rich in neutral fats than those fed the standard diet. In the duodenal mucosa, lipid diets decreased the activities of drug hydroxylation and glucuronidation.
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PMID:Dietary fats and properties of endoplasmic reticulum: II. Dietary lipid induced changes in activities of drug metabolizing enzymes in liver and duodenum of rat. 80 76

A combined effect of cholesterol and polychlorinated biphenyls (PCBs) on the microsomal drug hydroxylation and glucuronidation in the liver of the rat was studied. PCBs, Clophen A-50 and A-60, having an average chlorination degree of 50 and 60% affect the structure of microsomal membranes. It was found that Clophen A-60 increased the binding of trypsin- and digitonin-sensitive proteins to the membranes. Also it was found that PCBs enhanced the phsopholipid content of microsomes. PCBs increased the activity of hepatic NADPH cytochrome c reductase about 1.5-fold. Aryl hydrocarbon hydroxylase activity doubled with Clophen A-50 and quadrupled with Clophen A-60. Hepatic UDPglucuronosyltransferase activity was doubled with both PCBs. The enhancement in hepatic aryl hydrocarbon hydroxylase and in UDPglucuronosyltransferase was found to be lower in the presence of high cholesterol level in the diet when compared to earlier results. This is supposed to be due to the membraneous effects of cholesterol.
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PMID:Enhancement of hepatic drug biotransformation rate by polychlorinated biphenyls in rats fed cholesterol-rich diet. 81 Sep 23

DDT was administered to the guinea pig, mouse and rat either ig or ip and to the hamster ig in order to investigate variations in the response of hepatic and duodenal drug-metabolizing enzymes to DDT. The intragastric dose (160 mg/kg) was found to produce gastric bleeding and severe tremor in rats and mice but not in other rodents. The hepatic aryl hydrocarbon hydroxylase activity and cytochrome P-450 concentration decreased after the ig administration of DDT to rats, mice and guinea pigs but in hamsters the activiy of aryl hydrocarbon hydroxylase and cytochrome P-450 concentration increased 12 hr after the dosage. The aryl hydrocarbon hydroxylase activity decreased also in the duodenal mucosa of the rat after the ig administration of DDT. The ip dose had no effects on the hepatic or duodenal monooxygenase system in 12 hr. The UDPglucuronosyltransferase activity was slightly lowered in hepatic microsomes of the rat and mouse after the ig dose of DDT, but the decrease was more profound when measured after in vitro trypsin digestion of microsomes. The trypsin digestion activated the hepatic UDPglucuronosyltransferase in all the species studied, i.e., guinea pig, hamster, mouse and rat (3-, 3-, 5-, and 8-fold, respectively). The duodenal UDPglucuronosyltransferase activity was not affected by DDT administration in any of the species studied. The results suggest that the acute toxic effects of DDT are species-dependent and the administration route is important in DDT toxicity. The hydroxylation step in drug metabolism is more sensitive to DDT than the glucuronidation step.
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PMID:Effect of administration route on DDT on acute toxicity and on drug biotransformation in various rodents. 81 74

In a previous publication (Narhi, L. O. and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a soluble 119,000-dalton P-450 cytochrome (P-450BM-3) that was induced by barbiturates in Bacillus megaterium. This single polypeptide contained 1 mol each of FAD and FMN/mol of heme and, in the presence of NADPH and O2, catalyzed the oxygenation of long-chain fatty acids without the aid of any other protein. We have now utilized limited trypsin proteolysis in the presence of substrate to cleave P-450BM-3 into two polypeptides (domains) of about 66,000 and 55,000 daltons. The 66-kDa domain contains both FAD and FMN but no heme, reduces cytochrome c in the presence of NADPH, and is derived from the C-terminal portion of P-450BM-3. The 55-kDa domain is actually a mixture of three discrete peptides (T-I, T-II, and T-III) separable by high performance liquid chromatography. All three contain heme and show a P-450 absorption peak in the presence of CO and dithionite. The major component, T-I (Mr = 55 kDa), binds fatty acid substrate and has an N-terminal amino acid sequence identical to that of intact P-450BM-3, an indication that this domain constitutes the N-terminal portion of the 119-kDa protein. T-II (54 kDa) is the same as T-I except that it is missing the first nine N-terminal amino acids and does not bind substrate. T-III (Mr = 53.5 kDa) has lost the first 15 N-terminal residues and does not bind substrate. Since trypsin digestion of P-450BM-3 carried out in the absence of substrate yields T-II and T-III but no T-I, it appears that 1 or more residues of the first nine N-terminal amino acids of this protein are intimately involved in substrate binding. Although both the heme- and flavin-containing tryptic peptides retain their original half-reactions, fatty acid monooxygenase activity cannot be reconstituted after proteolysis, and the two domains, once separated, show no affinity for each other. In most respects, the reductase domain of P-450BM-3 more closely resembles the mammalian microsomal P-450 reductases than it does any known bacterial protein.
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PMID:Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. 310 60

In cell cultures previously treated with homologous interferon, the magnitude of antiviral activity and the degree of stimulation of aryl hydrocarbon hydroxylase induction appear to be directly related. In a highly purified mouse interferon preparation, the factor stimulating hydroxylase induction and the factor directing antiviral activity are inactivated by heating to 70 C or by treating with trypsin. Also, both activities demonstrate species specificity.
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PMID:Stimulation of aryl hydrocarbon hydroxylase induction in cell cultures by interferon. 434 23

The splake, a popular game fish, is a crossbreed which must be reared in nurseries. The fish are marked under anesthesia for later study. We analyze the effect of a common anesthetic, tricaine (MS-222), on the metabolism of foreign compounds in the liver and duodenum of the splake. In the liver and to some extent in the duodenum, aryl hydrocarbon hydroxylase and epoxide hydrase activities were reduced during treatments. The ethoxycoumarin O-deethylase activities were not affected in either the liver or duodenum. Tricaine significantly decreased the hepatic UDP-glucuronosyltransferase activity. The decrease was greater when the aglycone used was p-nitrophenol than with methylumbelliferone. A similar effect was also found after trypsin treatment of the microsomes. No significant decrease in the UDP-glucuronosyltransferase activity was detected in the duodenal mucosa. This was the case when both p-nitrophenol and methylumbelliferone were used as aglycones.
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PMID:Tricaine (MS-222) induced modification on the metabolism of foreign compounds in the liver and duodenal mucosa of the splake (Salvelinus fontinalis X Salvelinus namaycush). 627 38

The environmental influence of various drugs on the epoxide hydrolase with styrene oxide (EHSO) or benzo(a)pyrene-4,5-oxide (EHBPox) as substrate and the aryl hydrocarbon hydroxylase (AHH) activity was studied in monolayer cultures of human fetal hepatocytes (HFH) obtained at legal abortions. Hepatocytes were isolated by trypsin treatment of liver fragments and primary HFH cultures were maintained in Eagle's minimum essential medium supplemented with 15% newborn calf serum. The HFH were plated on culture dishes and allowed to 'settle' for one day before adding various drugs (in 1 microliter dimethylsulfoxide/ml) or solvent only and assay 1-2 days later. The basal AHH activity [assayed with 3H-benzo(a)pyrene as substrate] varied between 2 and 8.4 pmoles/min/mg protein and the basal EHSO activity was 0.3-4.9 nmoles/min/mg protein (n = 6) after one or two days' culture. The corresponding activity of EHBPox was 0.23-1.48 nmoles/min/mg protein (n = 5). Exposure of cultures to 2 mM phenobarbital (Pb), 2.5-25.0 microM benzanthracene (BA), 0.1 mM trans-stilbene oxide (TSO), or 5 microM beta-naphtoflavone (beta NF) resulted in a 1.2-3.7-fold induction of EHSO. Induction of EHBPox was also observed with Pb, beta NF, BA and TSO as inducers. Pb gave a dose-dependent induction of both EH at 0.1, 1.0 and 2.0 mM. Our results demonstrate that EH and AHH activities in HFH cultures are inducible by classical in vivo inducers. Although difficult to prove, it is plausible that such induction takes place also in intrauterine life.
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PMID:Human fetal liver cultures: basal activities and inducibility of epoxide hydrolases and aryl hydrocarbon hydroxylase. 653 14


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