Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

Estimation of the dermal carcinogenic activity of petroleum fractions using a modified Ames assay

The Ames Salmonella/microsomal activation mutagenesis assay has been adapted to improve sensitivity to complex hydrocarbon mixtures produced by the refining of petroleum. Extraction of oil samples with dimethyl sulfoxide produces aqueous-compatible solutions that more easily interact with the tester bacteria. These extracts, therefore, produce higher revertant values than do equivalent volumes of oil delivered neat or dissolved in organic solvent. Parallel increases in the liver microsomal S-9 concentration further improve the sensitivity of the assay, allowing detection of mutagenicity in otherwise inactive samples. The effect of increased microsomal fraction from rodent liver is apparently attributable to the higher levels of activating enzymes rather than to the concomitant increase in the overall hydrophobicity of the test system. The modified assay has been used to rank thirteen petroleum-derived oils and a corn oil control for relative mutagenic activity. This ranking closely correlates (r = 0.97) with potency rankings of the same samples previously determined from dermal carcinogenicity bioassays.Abbreviations DMSO dimethyl sulfoxide - S-9 Microsomal fraction from rodent liver - 2-AA 2-aminoanthracene - BaP benzo(a)pyrene - NADP nicotinamide adenine dinucleotide phosphate - DMF dimethyl formamide - EGDE ethylene glycol dimethyl ether     

The use of rec-bacteria for testing of carcinogenic substances.

A series of rec-Escherichia coli strains were tested for their sensitivity to four known carcinogenic compounds by examination of a zone of inhibited bacterial growth around a central well containing the test chemical. The mutants recA-, recB-, recC-, and recA- recB- recC- were all more sensitive to the mutagens than the parental strain AB1157. The recB- recC- strain was examined with a larger series of compounds and was found to respond to many of the substances in a similar way as the Salmonella typhimurium strains of Ames but with some notable exceptions. Nitrosamines, with rat liver microsomal activation, could be detected at lower levels and a group of aromatic amino compounds failed to react with these rec-E. coli. An unusual feature of these rec-mutants is their sensitivity to mixtures of nitrosamines and 2-acetyl amino-fluorene in the absence of microsomal activation.     

Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions

A highly sensitive spectrophotometric assay was developed for measuring flavin-containing monooxygenase activity using methimazole (N-methyl-2-mercaptoimidazole) as the substrate. With the procedure described, flavin-containing monooxygenase activity can be accurately measured in whole cell homogenates without interference due to NADPH oxidase activities. The effects of detergents and octylamine on female mouse liver flavin-containing monooxygenase activity were characterized for whole homogenates and microsomes prepared under conditions which tend to cause or minimize microsomal aggregation. A small activation was observed with 0.2% (v/v) Emulgen 913 with nonaggregated microsomes; higher levels of detergents gave maximal activity with aggregated microsomes. Variations in the activity of the female mouse liver enzyme with nutritional state and time of day were evaluated. Higher specific activities were observed in homogenates and microsomes of livers from fed animals than from livers of 24-h starved animals, and higher specific activities were present in samples from livers of animals sacrificed in late afternoon than in the early morning. In the period where activity increased in fed animals (i.e., the AM to PM transition), a portion of flavin-containing monooxygenase was more resistant to thermal inactivation. Other properties are described which suggest structural differences for at least a portion of the flavin-containing monooxygenase. The possibility that these differences may be related to turnover of the flavin-containing monooxygenase is discussed.     

Cytosolic activation of 2-aminoanthracene: implications in its use as diagnostic mutagen in the Ames test.

The metabolic activation of 2-aminoanthracene to mutagens in the Ames test was investigated using hepatic S9, microsomal and cytosolic fractions from control and Aroclor 1254-treated rats as activation systems. Microsomal and S9 preparations from control animals could activate 2-aminoanthracene, but the efficiency of activation was suppressed by pretreatment of animals with Aroclor 1254. Cytosolic fractions from Aroclor 1254-treated rats could readily activate the promutagen more readily than microsomes. The cytosolic activation of 2-aminoanthracene required NADPH and could not be accounted for by possible microsomal contamination. The molybdenum oxygenases appear not to contribute to the cytosolic activation of this promutagen. It is concluded that (a) the microsomal activation of 2-aminoanthracene is catalysed more effectively by enzyme systems other than the P450 I family and (b) an enzyme system capable of activating this carcinogen in vitro is present in the hepatic cytosol. The implications of these findings in the use of 2-aminoanthracene as a positive control in the Ames test are discussed.     

Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic and AMP allosteric sites of microsomal HMG-CoA reductase kinase

The nucleotide analogue 5-p-fluorosulfonylbenzoyladenosine reacts with rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is eleven times higher (K1 = 0.107 min-1) than the rate constant for inactivation (K2 = 0.0094 min-1). Mg-ATP protects preferentially against inactivation, while Mg-AMP at a low concentration (7.5/0.05 mM) protects preferentially against loss of the AMP activation capacity. Oppositely, Mg-ADP at a low concentration (7.5/0.05 mM) hardly protects against loss of AMP activation capacity. We conclude that microsomal reductase kinase has distinct sites for activation and catalysis.     

Mutagenicity of antiamebic and anthelmintic drugs in the Salmonella typhimurium microsomal test system

4 amebicides (chloroquine diphosphate, diiodohydroxyquin, iodochlorohydroxyquin and dehydroemetine) and 6 anthelmintics (bephenium hydroxynaphthoate, 4-hexylresorcinol, mebendazole, niclosamide, pyrantel pamoate and pyrvinium pamoate) were tested for mutagenicity in the Salmonella typhimurium microsomal test system. Frameshift mutations were induced by dehydroemetine and niclosamide following activation by microsomal enzymes, while pyrvinium pamoate induced both frameshift and base-pair substitution mutations with or without metabolic activation. The urine of mice treated with dehydroemetine or pyrvinium pamoate showed no mutagenic activity. However, urine obtained from mice treated with niclosamide was mutagenic in strains TA98 and TA1538. The fluctuation assay showed chloroquine diphosphate to be mutagenic in TA1537, a strain which detects frameshift mutations.     

Metabolic activation of 10-aza-substituted benzo[a]pyrene by cytochrome P450 1A2 in human liver microsomes

We previously reported that 10-azabenzo[a]pyrene (10-azaBaP), a 10-aza-analog of BaP and an environmental carcinogen, showed greater mutagenicity than BaP in the Ames test using pooled human liver S9. To investigate the cytochrome P450 (CYP) isoform involved in the activation of 10-azaBaP to the genotoxic form, the mutagenicity of 10-azaBaP using nine individual donors' and pooled human liver microsome preparations was compared with each CYP activity. Induced revertants by 2.5 nmol per plate 10-azaBaP with 0.5 mg per plate human liver microsomal protein showed a large inter-individual variation (42-fold) among the nine donors. The number of induced revertants highly correlated with the CYP1A2-selective catalytic activity from each microsome preparation, and no correlation was observed with other CYP isoform-selective catalytic activities. Moreover, recombinant human CYP1A2 contributed to the mutagenicity of 10-azaBaP more markedly than recombinant human CYP1A1. These results suggest that CYP1A2 may be the principal enzyme responsible for the metabolic activation of 10-azaBaP in human liver microsomes. With regard to the proposal that BaP may be activated by human CYP1A1, our results suggest that the nitrogen-substitution at position-10 of BaP may cause the CYP enzyme-specificity in metabolic activation to change from CYP1A1 to CYP1A2.     

3-Methoxy-4-aminoazobenzene, a unique carcinogenic aromatic amine as a substrate for cytochrome-P-450-mediated mutagenesis

Rat liver microsomal enzyme(s) that catalyze mutagenic activation of a carcinogenic aminoazo dye, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), was studied by virtue of the Salmonella typhimurium TA98 assay using o-aminoazotoluene (OAT) as the control. Male Wistar rats were pretreated with phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyl (PCB), and the liver microsomal activities for mutagenic activation of 3-MeO-AAB and OAT were examined. In agreement with the reported results on several carcinogenic aromatic amines, MC pretreatment resulted in greater activation of microsomal activity in the OAT mutagenesis (about a 4-fold increase as compared to the untreated control) than did PB (1.5-fold increase). By contrast, the mutagenic activation of 3-MeO-AAB is found to be more efficiently catalyzed by those enzyme(s) that are induced by PB pretreatment (4-fold increase) than by those that are induced by MC (1.8-fold increase). The induced enzymes that principally mediate the mutagenic activation of these azo dyes are indicated to be cytochrome P-450s, because the mutagenic activation was strongly inhibited by addition of cytochrome P-450 inhibitors such as 2-diethylaminoethyl-2,2-diphenylvalerate (SKF 525A) and 7,8-benzoflavone. These data suggest that 3-MeO-AAB is a unique carcinogenic aromatic amine as a substrate for mutagenic activation via catalysis of those cytochrome P-450s that are induced by PB pretreatment.     

Mutagenicity testing on chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene.

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.     

In vitro microsome-mediated aflatoxin B1-DNA binding and its inhibition by cytosol of various organs of the hamster and quail

We studied thein vitro activation of aflatoxin B1 (B1) by microsomes and its inactivation by the cytosol of various quail and hamster organs, using B1-DNA binding as an index. The microsomal activity of the liver to bind B1 to DNA was not largely different between the two species and was higher than that of the other organs examined in either species. The microsomal activity of the kidney and lung was very low in the quail compared with the hamster, indicating the very small contribution of the lung and kidney microsomes to the activation of B1 in birds. Only the hamster liver cytosol showed strong inhibition of microsome-mediated B1-DNA binding.     

Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.     

Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

Acetaminophen (APAP), a widely used over-the-counter analgesic, is known to cause hepatotoxicity when ingested in large quantities in both animals and man, especially when administered after chronic ethanol consumption. Hepatotoxicity stems from APAP activation by microsomal P450 monooxygenases to a reactive metabolite that binds to tissue macromolecules, thereby initiating cellular necrosis. Alcohol consumption also causes the induction of P450IIE1, a liver microsomal enzyme that in reconstitution studies has proven to be an effective catalyst of APAP oxidation. Thus, elevated microsomal P450IIE1 levels could explain not only the known increase in APAP bioactivating activity of liver microsomes after prolonged ethanol ingestion but also the enhanced susceptibility to APAP toxicity. We therefore examined the role of P450IIE1 in human liver microsomal APAP activation. Liver microsomes from seven non-alcoholic subjects were found to convert 1 mM APAP to a reactive intermediate (detected as an APAP-cysteine conjugate by high-pressure liquid chromatography) at a rate of 0.25 +/- 0.1 nmol conjugate formed/min/nmol microsomal P450 (mean +/- SD), whereas at 10 mM, this rate increased to 0.73 +/- 0.2 nmol product/min/nmol P450. In a reconstituted system, purified human liver P450IIE1 catalyzed APAP activation at rates threefold higher than those obtained with microsomes whereas two other human P450s, P450IIC8 and P450IIC9, exhibited negligible APAP-oxidizing activity. Monospecific antibodies (IgG) directed against human P450IIE1 inhibited APAP activation in each of the human samples, with anti-P450IIE1 IgG-mediated inhibition averaging 52% (range = 30-78%) of the rates determined in the presence of control IgG. The ability of anti-P450IIE1 IgG to inhibit only one-half of the total APAP activation by microsomes suggests, however, that other P450 isozymes besides P450IIE1 contribute to bioactivation of this compound in human liver. Of the other purified P450 isozymes examined, a beta-naphthoflavone (BNF)-inducible hamster liver P450 promoted APAP activation at rates even higher than those obtained with human P450IIE1. The extensive APAP-oxidizing capacity of this hamster P450, designated P450IA2 based upon its similarity to rat P450d and rabbit form 4 in terms of NH2-terminal amino acid sequence, spectral characteristics, immunochemical properties, and inducibility by BNF, agrees with previous reports concerning the APAP substrate specificity of the rat and rabbit P450IA2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system.

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.     

HGF ameliorates a high-fat diet-induced fatty liver

Hepatocyte growth factor (HGF) has various effects especially on epithelial cells. However, the precise role of HGF on lipogenesis is still not fully understood. A high-fat diet was administered to HGF transgenic mice and wild-type control mice in vivo. Furthermore, recombinant human HGF (rhHGF) was administered to HepG2 cell line in vitro. We performed an analysis regarding the factors relating to lipid metabolism. An overexpression of HGF dramatically ameliorates a high-fat diet-induced fatty liver. HGF transgenic mice showed an apparently reduced lipid accumulation in the liver. The activation of microsomal triglyceride transfer protein (MTP) and apolipoprotein B (ApoB) accompanying higher triglyceride levels in the serum were found in HGF transgenic mice on a normal diet. Interestingly, this upregulation of the MTP activation became more apparent in the high-fat diet. In addition, the administration of rhHGF stimulated MTP and ApoB expression while reducing reduced the intracellular lipid content in HepG2 cell line. However, this induction of MTP and ApoB by HGF was clearly inhibited by PD98059 (MAPK inhibitor). In conclusion, the data presented in this study indicated that HGF ameliorates a high-fat diet-induced fatty liver via the activation of MTP and ApoB.     

Analytical and biological analyses of test materials from the synthetic fuel technologies. I. Mutagenicity of crude oils determined by the Salmonella typhimurium/microsomal activation system.

We have assayed the mutagenicity of crude industrial products and effluents with the Salmonella/microsomal activation system. Test materials (crude products from coal-conversion processes and natural crude oils) were initially fractionated into primary classes by liquid--liquid extraction and then further fractionated by column chromatography. Prescreening was accomplished over a wide concentration range with the Ames tester strains. Active fractions (mainly the neutral fractions containing polycyclic aromatic hydrocarbons and certain basic fractions) can be identified, and dose--response relationships can be established. Standard values are expressed as revertants/mg of the test material assayed with frameshift strain TA98 including metabolic activation with rat-liver preparations. Total mutagenic activity of synthetic fuel samples was consistently higher than that of natural crude "controls." Activities of subfractions are roughly additive and presumably reflect the mutagenic potential of the whole test material. These results are being extended to other genetic assays. Chemical identification is carried out along with the bioassays.     

THE RELATION BETWEEN THE INTRACELLULAR RIBONUCLEIC ACID DISTRIBUTION AND AMINO ACID INCORPORATION IN THE LIVER OF THE DEVELOPING CHICK EMBRYO

The RNA-P and DNA-P content of the nucleus and the RNA-P content of the whole cell of the livers of 8- to 20-day chick embryos and of adult fowls have been determined. The DNA-P content of the liver nuclei was slightly higher in the 8- and 10-day embryo than in all the other stages examined. A significant decrease in the RNA content of the cell occurred during embryonic development. The RNA content of the adult cell was the same as that of the 14- to 16-day embryo. The proportion of the cellular RNA contributed by the nucleus also decreased during development. In respect to both nuclear RNA content and distribution of RNA between nucleus and cytoplasm, the adult resembled the 8- to 12-day embryo. Examination of the fine structure of the cell showed that, as development progressed, free ribosomes decreased in number and the rough membranes increased. Slices of 8-, 14-, and 20-day embryonic livers and of adult livers were incubated with ¹⁴C-leucine, and the amount of labeled amino acid incorporated into whole tissue protein and into the proteins of the subcellular fractions was measured. Embryonic liver incorporated ¹⁴C-leucine 15 to 30 times more rapidly than adult liver. The microsomal protein was always more highly labelled than the protein in any other subcellular fraction; however, in the 8-day embryonic and the adult liver the proportion of total counts found in the nuclear fraction was considerably higher than in the 14- or 20-day embryonic liver. The significance of an apparent correlation between the proportion of the cell's RNA contributed by the nucleus and the proportion of total counts in the nuclear fraction is discussed.     

Partial hepatectomy of marmoset: clinical and pathological effects and utility in microsomal enzyme analysis.

Liver biopsy based on a partial hepatectomy technique (shearing) was performed in 10 common marmosets (Callithrix jacchus). This is a preliminary study to evaluate the effects of drugs on hepatic microsomal enzymes: cytochrome P-450 and T4 uridine diphosphate glucuronyl transferase (T4-UDPGT), by comparing post-treatment values with pre-treatment values individually with a limited number of animals. The effects of the biopsy on clinical findings and liver pathology were evaluated during the first 5 post-surgical weeks. Although the plasma aspartate aminotransferase (AST) activities tended to decrease from 1 to 4 weeks post-surgery, no abnormality was noted in clinical sign, body weight, the hematocrit value or other blood chemical values. At necropsy, adhesion of the sheared site of the liver to the parietal peritoneum or the small intestine was evident in 2 of the 4 marmosets. Microscopic examination revealed focal fibrosis in the liver, but it was localized around the sheared site. Based on the above results, it was concluded that liver biopsy must be performed more than one month before administration of the drug to be tested. The biopsy samples and the whole liver samples obtained at autopsy were subjected to analysis of microsomal protein content, cytochrome P-450 content and T4-UDPGT activity. In comparison with the values from the whole liver samples, those from the biopsy samples showed no significant difference. Furthermore, there was a significant correlation rather than difference between matched values. This suggested that partial hepatectomy is a useful method for obtaining pretreatment values in liver biochemistry to evaluate the effects of drug-treatment in individual animals.     

Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.     

Rate of microsomal protein metabolism in the mouse liver

NaH14CO3, a poorly reutilized biosynthesis precursor, was used to study the rate of whole microsomal protein degradation in mouse liver. The use of the precursors, however, does not prevent the reutilization of labeled amino acids on phenobarbital administration. To avoid reutilization, a new method has been developed. It was shown that phenobarbital injections have no effect on the degradation rate of the whole microsomal protein. The effect of amidopyrine, a monooxygenase microsomal system substrate, on the rate of whole microsomal protein degradation was examined. An experimental model was developed, in which the monooxygenase microsomal system substrate does not exhibit the properties of its inducer. Amidopyrine administration to mice simultaneously with phenobarbital induction has no effect on the degradation rate of the whole microsomal protein.