Biosynthesis of liver catalase in rats treated with allylisopropylacetylcarbamide. II. Double-labeling of catalase with (14C)leucine and delta-(3H)aminolevulinic acid.

Double-labeling of liver catalase [EC 1.11.1.6] with [14-c]leucine and delta-[3H]aminolevulinic acid was carried out both in vivo and in vitro using rats treated with allylisopropylacetylcarbamide (Sedormid). These radioactive precursors were incorporated into catalase at a lower rate than in normal rats. In particular, the incorporation of 3H was remarkably inhibited. The results suggest that the administration of Sedormid can inhibit synthesis of the protein moiety of catalase, and possibly interfere with the binding of heme to the catalase protein.     

Effect of cobalt on the synthesis and degradation of hepatic catalse in vivo

1. The administration of CoCl(2) to rats caused a decrease in hepatic catalase activity as well as a decrease in the amount of catalase protein as measured by immunological assay. The mitochondrial enzyme decreased progressively over 2 days, whereas the cytosol enzyme decreased over 12h and then remained essentially unchanged for 2 days after a single injection of cobalt. 2. Incorporation of [(14)C]glycine into catalase haem was dramatically decreased by a single injection of cobalt, but that into catalase protein remained essentially unaltered. 3. Incorporation of [(3)H]leucine into liver protein increased in rats in a steady state receiving a daily injection of cobalt, which was in contrast with a marked inhibition observed in 5-amino[(3)H]laevulinate incorporation. 4. The initial rate of [(3)H]leucine incorporation into mitochondrial and cytosol catalase did not alter or was slightly depressed in the cobalt-treated animals, whereas the incorporation of 5-amino[(3)H]laevulinate into mitochondrial and cytosol catalase was conspicuously decreased, indicating that haem synthesis was limiting catalase formation. 5. The degradation rate of catalase protein, as measured by a double-labelling method, was not changed by the cobalt treatment.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : IV. Biochemical Pathway of Catalase Synthesis

Early events in the biosynthesis of liver catalase were studied on female rats receiving [³H]leucine or [³H]δ-aminolevulinic acid or a mixture of [³H]leucine with [¹⁴C]δ-aminolevulinic acid by intraportal injection. Catalase antigen was selectively separated from homogenates by immunoprecipitation, both without and after partial purification of the enzyme. Label from both precursors appeared first in immunoprecipitable material which was lost upon purification of catalase; the label subsequently became associated with material indistinguishable from catalase. Kinetic analysis of the results indicates that the nonpurifiable material identified by early labeling consists of two distinct biosynthetic intermediates, the first lacking heme and representing about 1.6% of the total catalase content or 13 µg/g liver, the second containing heme and representing about 0.5% of the total catalase content or 4 µg/g liver. The first intermediate migrates at the same rate as catalase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and therefore has a monomeric molecular weight of about 60,000.     

Comparison of the effect of acetone and isopropanol on lipid metabolism in rats]

Isopropanol and acetone administered to rats in conditions leading to a similar blood acetone level differ markedly in their effects on lipid metabolism. Isopropanol administration determines a fatty liver, which is mainly related to a defect in hepatic lipoprotein synthesis. Acetone administration gives only raise to a slight increase in the liver triacylglycerol level. It does not alter the [1-14C] palmitate, [1-14C] glycerol or [U-14C] leucine incorporation into blood lipoproteins. Acetone does thus not appear to play a preminent role in the isopropanol induced fatty liver which seems to be related mainly to a direct action of the alcohol itself.     

Effect of thiamine on the activity of the pyruvate dehydrogenase complex in rat liver following stimulation of lipogenesis

It is shown that thiamine administration to rats (250 micrograms per 100 g of mass) who were given high-carbohydrate diet (lipogenesis intensification) after fasting inhibits an increase in the pyruvate dehydrogenase activity in the liver homogenate and mitochondria usual under these conditions. This is observed when determining total activity of the pyruvate dehydrogenase complex and activity of its first component--pyruvate dehydrogenase estimated from the ferricyanide reduction and [1-14C] CO2 formation from [1-14C] pyruvate. Fasting animals and animals whom thiamine was administered against a background of lipogenesis intensification revealed a higher ability of the liver tissue to synthesize acetoin as compared with the control group and animals with the intensified lipogenesis without thiamine administration.     

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : V. Intracellular Pathway of Catalase Synthesis

The subcellular distribution of the biosynthetic intermediates of catalase was studied in the livers of rats receiving a mixture of [³H]leucine and [¹⁴C]δ-aminolevulinic acid by intraportal injection. Postnuclear supernates were fractionated by a one-step gradient centrifugation technique that separates the main subcellular organelles, partly on the basis of size, and partly on the basis of density. Labeled catalase and its biosynthetic intermediates were separated from the gradient fractions by immunoprecipitation, and the distributions of radioactivity were compared with those of marker enzymes. The results show that catalase protein is synthesized outside the peroxisomes, but rapidly appears in these particles, mostly still in the form of the first hemeless biosynthetic intermediate. Addition of heme and completion of the catalase molecule take place within the peroxisomes. During the first 15 min after [³H]leucine administration, more than half of the newly formed first intermediate was recovered in the supernatant fraction, where it was found to exist as an aposubunit of about 60,000 molecular weight.     

Limited myogenic response to a single bout of weight-lifting exercise in old rats

The purpose of the present study was to compare themyogenic response of hindlimb muscles in young (14-20 wk of age)and old (>120 wk of age) rats with a single exhaustive bout of heavyresistance weight lifting. [³H]thymidine and[¹⁴C]leucine labeling were monitored for up to2 wk after the exercise bout to estimate serial changes in mitoticactivity and the level of amino acid uptake and myosin synthesis.Histological, histochemical, and immunohistochemical[anti-5-bromo-2'-deoxyuridine and myogenic determinationgenes (MyoD)] analyses of whole muscles and analysis ofmuscle-specific gene expression (MyoD) using Western blotting andRT-PCR were performed. Old rats showed significant muscle atrophy and alower exercise capacity than young rats. Exercise-induced muscledamage, as assessed in histological sections, and increases in serumcreatine kinase activity were evident in both young and old exercisedgroups. Mitotic activity was increased in young, but not old, rats 2 days after exercise. There was a biphasic increase in[¹⁴C]leucine uptake during the 14 dayspostexercise (peaks at 1-4 and 10 days) in young rats: only thefirst peak was observed in old rats. There was a lower uptake of[¹⁴C]leucine in the myosin fraction and animpaired expression of MyoD at the protein (immunohistochemistry andWestern blotting) and mRNA (RT-PCR) levels in old rats throughout thepostexercise period. These results demonstrate a reduced reparativecapability of muscle in response to a single bout of exercise in oldcompared with young rats.

     

Effect of insulin and cortisol on oxidation of (1-14C)glucose, (6-14C)glucose, (1-14C)palmitate and (1-14C)leucine in the tissues of swine during the neonatal period

The intensity of [1-14C]glucose, [6-14C]glucose, [1-14C]palmitate and [1-14C]leucine oxidation and the effect of insulin and hydrocortisone on this process were studied in the brain, duodenum mucosa, liver and skeletal muscle of 1- and 5-day old piglets in vitro. Most of the studied substrates are oxidized in the tissues of 5-day piglets more intensively. Insulin stimulates oxidation of [1-14C]glucose, [6-14C]glucose and [1-14C]leucine in the brain and duodenum mucosa in 1- and 5-day old piglets, while in the liver and skeletal muscle--only in 5-day old piglets. Hydrocortisone administration enhances oxidation of [1-14C]leucine in most of the studied tissues in 1-day piglets and oxidation of [1-14C]glucose and [6-14C]glucose--in 5-day piglets. Both hormones produce no essential influence on the intensity of [1-14C]palmitate oxidation in the studied tissues of piglets or somewhat weaken it.     

Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney

1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [(14)C]leucine and [(14)C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [(14)C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [(14)C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [(14)C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [(3)H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.     

Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.     

Degradation of peroxisomal catalase and urate oxidase of rat liver.

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes.

After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats.     

Similarities between the biochemical actions of cycasin and dimethylnitrosamine

1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [(14)C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of (14)C from l-[U-(14)C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.     

Degradation of peroxisomal catalase and urate oxidase of rat liver

Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [³H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes.     

Effects of acute CS2 intoxication on liver protein and drug metabolism

Rats pretreated with phenobarbitone and their controls were exposed to 0.15% atmospheric CS₂ for 2 h. Liver protein metabolism and microsomal drug metabolizing enzyme activities were analyzed 1, 4 and 46 h later. In the pretreated rats the [¹⁴C]leucine uptake was at first inhibited, then liver RNA tended to increase. This increase was followed by a decline in the [¹⁴C]leucine uptake, while RNA content diminished to the control level. In the control rats (not pretreated with phenobarbital) the effects of CS₂ on liver protein metabolism were less; only at 4 h after the exposure was liver RNA increased and [¹⁴C]leucine uptake slightly stimulated. In the pretreated rats CS₂ had decreased microsomal P-450 by about 50% at 1 and 4 h, and the activity of 7-O-dealkylase of ethoxycoumarin had decreased even more. The measurable UDPglucuronosyltransferase of the liver microsomes of the pretreated rats had increased by 26% at 1 h and by 80% at 4 h after the CS₂ exposure. The in vivo activation of microsomal UDPglucuronosyltransferase may result from a stimulated lipid peroxidation of reticuloendothelial membranes by CS₂ metabolites in vivo, as suggested by the diene conjugation spectra. In control rats CS₂ depressed only the ethoxycoumarin deethylase activity. All the microsomal changes caused by CS₂ exposure were restored within 46 h.It is suggested that the different action of CS₂ on the liver protein metabolism of rats pretreated with phenobarbitone and controls results from the different stage of protein turnover in the two groups, i.e. in the barbituratetreated animals there is a phase of increased protein synthesis and accordingly the protein turnover is more sensitive to the action of CS₂.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

Induction of hepatic drug metabolizing enzymes by DL-methionine in rats

A single intraperitoneal injection of DL-methionine (500 mg/kg body wt.) to adult male Wistar rats was shown to significantly induce all the components of the hepatic microsomal mixed function oxidase system such as NADPH cytochrome C reductase activity, cytochromes P-450 and b₅, as well as activities of drug metabolizing enzymes such as aminopyrine demethylase and uridine 5′ -diphosphate-glucuronosyltransferase. Combined administration of nicotinamide (250 mg/kg body wt.) and DL-methionine (500 mg/kg body wt.) was shown to bring about an additional increase (25-30%) in the activities of these enzymes as compared to their induction on independent administration of the two endobiotics. In rats bearing Yoshida sarcoma (ascites) tumour as well as in normal rats injected with serum from tumour bearing animals, the decreased activities of hepatic mixed function oxidases could be restored to their normal levels by administration of DL-methionine (500 mg/kg body wt.) to these rats. Whereas actinomycin D (1 mg/kg body wt.) had no effect on the increased incorporation of [¹⁴C] labelled leucine into microsomal proteins following administration of nicotinamide, the enhanced incorporation of the label following DL-methionine administration was completely inhibited by the same dose of actinomycin D. Administration of cycloheximide (0·5 mg/kg body wt.) to rats could completely inhibit the increased incorporation of [¹⁴C] leucine into hepatic microsomal proteins following independent administration of nicotinamide and DL-methionine. Similar inhibitory pattern with actinomycin D and cycloheximide was also demonstrated in case of induction of NADPH cytochromeC reductase activity by both these endobiotics.