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)

Rat liver microsomes have previously been shown to contain hemoproteins having molecular weights of 53,000, 50,000, and 45,000. The 45,000-dalton hemoprotein, which is induced in rat liver microsomes by pretreatment of animals with phenobarbital, is resistant to proteolysis by trypsin. This characteristic was used to purify it from the other microsomal hemoproteins. In the procedure used, a sodium cholate-solubilized microsomal fraction from phenobarbital-pretreated rats was treated with trypsin and chromatographed on Sephadex G-100 to separate the hemoprotein from preolytic degradation products. The hemoprotein thus isolated was homogenous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified spectrally as a cytochrome P-420 hemoprotein. This hemoprotein was free of cytochrome b5 and NADPH-cytochrome c reductase activity. Antibody prepared against the protease-treated cytochrome P-420 hemoprotein will not cross-react with the 53,000- and 50,000-dalton hemoproteins. This was assessed by three criteria. First, immunoprecipitation studies were conducted with detergent-solubilized partially purified cytochrome P-450 preparations isolated from the liver microsomes of control and phenobarbital- and 3-methylcholathrene-retreated rats. The antibody immunoprecipitated only the 45,000-dalton hemoprotein from these partially purified cytochrome P-450 preparations, each of which contains all three hemoproteins. Second, the antibody demonstrated specificity with regard to the microsomal hydroxylation reactions it would inhibit in a reconstituted hydroxylation system containing partially purified cytochrome P-450 (448) fractions isolated from the liver microsomes from phenobarbital- or 3-methylcholathrene-pretreated rats. The antibody would inhibit benzphetamine-N-demethylation catalyzed by both cytochrome P-450 fractions but would not inhibit benzpyrene hydroxylation catalyzed by either. Third, agglutination and complement fixation assays were performed to assess the binding of the antibody to liver microsomes isolated from control and phenobarbital- or 3- methylcholanthrene-pretreated rats. These studies demonstrated that the antibody binds preferentially to the liver microsomes isolated from phenobarbital-pretreated rats, in which the 45,000-dalton hemoprotein has been shown to be induced. It is hypothesized that there are very significant structural and catalytic differences among the cytochrome P-450 hemoproteins.
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PMID:Multiplicity of cytochrome P-450 hemoproteins in rat liver microsomes. Preparation and specificity of an antibody to the hemoprotein induced by phenobarbital. 80 94

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

The T1 variety of tyrosinase is present in both particulate and soluble or readily solubilized forms in the pigmented hypodermis (hair bulbs) of C57BL mice and Harding-Passey mouse melanoma. Trypsin treatment of 35,000g supernatants containing the microsomal (small granule) fraction of gentle homogenates of hair bulbs and melanoma results in significantly increased T1 activity within polyacrylamide gels. Similar treatment of 100,000g supernatants results in a slight increase in T1 activity. Addition of Triton-X or DOC to 35,000g supernatants of hair bulb and melanoma homogenates followed by centrifugation at 100,000g results in a marked enhancement of T1 when the latter supernatants are treated with trypsin. In the absence of trypsin treatment, T1 activity is comparable to nondetergent-treated controls. A slow-moving dopa-reactive band (Ts) is found in electropherograms of the nontrypsinized 100,000g supernants of detergent-treated 35,000g supernatants. It is absent in those treated with trypsin. The slow-moving enzyme appears to give rise to T1 molecules when eluted from acrylamide gels and even to a greater extent when elution is combined with trypsin treatment prior to reelectrophoresis. In mammals, tyrosinase apparently is not derived by a proteolytic activation of protyrosinase.
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PMID:Action of trypsin and detergents on tyrosinase of normal and malignant melanocytes. 81 38

Microsomes were prepared from the liver, kidney and lung of phenobarbital or 20-methylcholanthrene treated and control rats with the conventional ultracentrifugation and calcium aggregation methods. The two methods were compared as to the yield of microsomal protein, amount of cytochrome P-450/448 and activity of UDP-GLUCURONOSYLTRANSFERASE, BENZOPYRENE HYDROXYLASE AND P-NITROANISOLE O-demethylase. The absolute amount of cytochrome P-450/448 (nmol/g wet weight), as well as the enzymatic activities dependent on it (nmol produced/g wet weight) did not differ significantly in any tissue of either treated or control animals nor did that of UDPglucuronosyltransferase. However, the ultracentrifugation method resulted in a slightly smaller yield of the hepatic microsomal protein and a correspondingly higher yield of cytochrome P-450/448 per mg protein as well as higher specific enzymatic activities of both the consecutive drug biotransformation reactions studied. The specific activity of UDPglucuronosyltransferase in digitonin treated microsomes was twice as high in the conventional microsomes as in the calcium aggregated microsomes; no differences was found in the trypsin treated microsomes. The specific activity of the hepatic benzpyrene hydroxylase of the benzpyrene treated animals in the calcium harvested microsomes was 55 per cent of that in the ultracentrifugated microsomes.
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PMID:UDP glucuronosyltransferase and mixed function oxidase activity in microsomes prepared by differential centrifugation and calcium aggregation. 82 31

A 4% cholesterol diet fed to rats for four weeks was found to increase the phospholipid and cholesterol contents and the activities of drug metabolizing enzymes in rat liver microsomes. Microsomes from rats on a high cholesterol diet were able to enhance the fluorescence of membrane bound 1-anilinonaphthalene 8-sulphonate (1,8-ANS) and ethidium bromide more than microsomes from rats on a standard diet. In the case of 1,8-ANS, the enhanced fluorescence was found to be due to the increased affinity of the molecules for microsomes. In the case of ethidium bromide the fluorescence increased partly because of the larger amount of binding sites and partly because of the enhanced quantum yield of the molecules. P-nitrophenol was found to compete with 1,8-ANS for the same binding sites in microsomes. On the other hand, 1,8-ANS lowered the rate of drug metabolism when present in the incubation mixture. In vitro treatments of microsomes with trypsin, phospholipase A or digitonin altered the binding properties of 1,8-ANS and ethidium bromide to microsomes. It is concluede that the binding sites of 1,8-ANS in microsomes are important for the activity of drug-metabolizing enzymes. The mechanisms of dietary cholesterol in enhancing the drug metabolism and the role of microsomal phospholipids in regulating the activity of drug-metabolizing enzymes are discussed.
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PMID:Dietary cholesterol caused modification in the structure and function of rat hepatic microsomes, studied by fluorescent probes. 82 81

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].
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PMID:Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein. 84 74

1. Heavy microsomal fraction (HM) of rabbit skeletal muscle obtained by differential centrifugation between 8 000-30 000 g and consisting of sarcoplasmic reticulum (SR) vesicles contains variable amounts of glycogen and reveals some activity of phosphorylase b. The monomer of this enzyme of mol. wt. about 100 000 co-migrates in SDS-polyacrylamide gel electrophoresis with the main SR protein--Ca2+,Mg2+--dependent ATPase. 2. The highest specific activity of phosphorylase and the highest content of glycogen is present in the light microsomal (LM) fraction (30 000-100 000 g). 3. Contrary to the ATPase, phosphorylase b is released from the microsomal fraction by treatment with EDTA and is resistant to trypsin. 4. Both HM and LM fractions can be further fractionated on continuous sucrose density gradient at high speed. Main fraction of HM consists of highly purified SR vesicles. The second, small fraction of HM is identical with the main fraction of LM and consists of two populations: vesicles of structure and properties different from those of SR vesicles, and the particles of a complex of glycogen with some glycolytic enzymes.
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PMID:Sarcoplasmic reticulum vesicles and glycogen-protein particles in microsomal fraction of skeletal muscle. 87 37

A protein fraction which has a high affinity for polyribosomes was isolated from rough microsomal membranes of rat liver. The mode of polyribosome binding to this fraction (R-fraction) was studied by using CsCl equilibrium centrifugation and compared with that for stripped rough microsomal membranes. The following were found. (1) The polyribosome-binding cpacity of the R-fraction was heat-labile and sensitive to trypsin, and was suppressed by increasing KCl concentration and addition of 0.1 mM-aurintricarboxylic acid. (2) Of the four subfractions obtained by gel filtration of the R-fraction on a Sephadex G-200, only the R1-fraction, eluted at the void volume, showed a high affinity for polyribosomes. The polyribosome-binding capacity of the R1-fraction decreased with time on storage at 4 degrees C. (3) The R1-fraction contained three major proteins with mol. wts. 108,000, 99,000 and 65,000.
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PMID:Isolation and characterization of membrane proteins responsible for attachment of polyribosomes to rough microsomal fraction of rat liver. 88 Feb 37

1. The amount of cytochrome b5 was not changed significantly by a single injection of cobalt (60 mg/kg body weight) or by daily injection of cobalt (30 mg/kg body weight) for 4 days or 8 days. On the other hand, the amount of cytochrome P-450 was depressed strongly by both cobalt treatments. 2. The incorporations of [3H]leucine as well as 5-amino[3H]levulinate into cytochrome b5 in cobalt-treated animals were almost the same as those in the controls 5 h after injections of the radioisotopes, whereas the radioactivity of heme labelled with 5-amino[3H]levulinate in microsomal residues after trypsin digestion, which would consist mainly of cytochrome P-450, was higher in the controls than in cobalt-treated animals after 5 h.
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PMID:Effect of cobalt on the synthesis of liver microsomal cytochromes. 88 9


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