Liver Of Adult Male Rats Research Articles

Characterization studies of a rat hepatie cytosolic androgen-binding protein

A rat hepatic cytosolic [3H]methyltrienolone (R1881) binding protein was studied under various conditions. This protein was also compared with the male-specific high capacity--low affinity estrogen-binding protein derived from the same cytosolic fraction. Analysis of the R1881 binding protein in adult (60-85 days old) male rat liver cytosol indicated the presence of a high affinity--low capacity binding site (Kd = 0.3 nM; Bmax = 5.9 fmol/mg) and a lower affinity--higher capacity component (Kd = 10.4 nM; Bmax = 131 fmol/mg). The latter component was eliminated by addition of triamcinolone or cortisol to the assay mixture. Steroid binding to the high affinity R1881 site was specific for testosterone, dihydrotestosterone, androstenedione, and mibolerone, with a moderate specificity to cyproterone acetate, flutamide hydroxide, and estradiol. Saturation studies indicated that these steroids were binding to the same or a similar high affinity component except for flutamide hydroxide which produced nonsaturable displacement. The high affinity site had no specificity for progesterone, diethylstilbestrol, or cortisol. Like the high capacity--low affinity protein, this protein was not present in the immature, adult, or 10-day ovariectomized adult female. However, unlike the high capacity--low affinity protein, it was present in low quantities in the immature male. In addition, castration of the adult for 18 h, 4 days, or 10 days or hypophysectomy for 10-17 days did not have a significant effect on the high affinity component compared with the controls. Testosterone administration to these animals did not alter this protein binding. These studies indicate that a specific, high affinity--low capacity androgen-binding protein exists in rat hepatic cytosol. Furthermore, this protein shows age and sex dependency, but its presence is not affected by altering gonadal or hypophyseal factors in the adult male.

Binding characteristics of epidermal growth factor receptors in male and female rat liver cell membrane preparations

The binding characteristics of epidermal growth factor (EGF) receptors were studied in cell membrane preparations from adult male ( n = 14) and female ( n = 13) rat livers. Results indicate a significant lower (about 65%) number of EGF receptors in female preparations. The possibility that the decrease in EGF receptors was only a reflection of an excess free EGF in female preparations was ruled out by means of acid extraction, ultrafiltration, and measurement of EGF in the acid extracts. In view of the known role of EGF in cell differentiation, it may be important to recognize that the number of its receptors, at least in liver preparations, is markedly different between sexes.

Transformation of primary cultured and co-cultured adult rat liver cells by 3'-methyl-4-dimethylaminoazobenzene.

Under various conditions of culture and carcinogen treatment, the transformation of liver cells by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) was studied. Primary liver cell (PLC) cultures from adult male rats and co-cultures with PLCs of ARL-D8 cells of a liver epithelial-like clear cell line from adult female rats were treated with 0.24 mM 3'-Me-DAB for 6 days. Four of 8 carcinogen-treated PLC cultures contained cells with marker chromosomes, and 3 of the 8 cultures contained gamma-glutamyltranspeptidase (GGT)-positive cells. Three of 5 carcinogen-treated co-cultures contained cells with marker chromosomes, and 2 of the 5 co-cultures contained GGT-positive cells. Pure cultures of ARL-D8 cells were treated for 6 or 12 days with 3'-Me-DAB (0.24 mM)-containing-medium perfused through the liver of adult male rats in situ. In the 6-day treatment, none of 5 carcinogen-treated cultures showed chromosomal abnormality or cytochemically exhibited GGT activity. However, in the 12-day treatment, 2 of the 5 carcinogen-treated cultures contained cells with marker chromosomes, and 2 of the 5 cultures contained GGT-positive cells. None of the control cultures exhibited chromosomal abnormality or GGT-positive cells. In summary, transformation markers increased in ARL-D8 cells when they were co-cultured with PLCs.

Characterization of a [ 3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17β-hydroxy-17α-methyl-estra-4, 9, 11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [ 3H] R 1881 macromolecular complex which sediments in the 8–9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [ 3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [ 3H] R1881 was recovered by thin layer chromatography. Characteristics of this [ 3H] R1881-8S binding protein include high affinity ( K d = 2.3 ± 41 nM) and low binding capacity (18.8 ± 3.3 fmol/mg cytosol protein), precipitability in 0–33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [ 3h] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a K d = 2.8 nM for [ 3H] R1881, and is androgen specific (testosterone > 5α-dihydrotestosterone > estradiol > progesterone > cyproterone acetate > di-ethylstilbestrol > dexamethasone > triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.

Post-transcriptional regulation of rat liver gene expression by glucocorticoids

We have investigated the mechanisms whereby glucocorticoids control the expression of specific genes in the livers of adult male rats. Construction and differential screening of a cDNA library representing normal rat liver polysomal poly(A)+ RNAs allowed selection of probes for hormonally regulated genes. The mechanism of this regulation was analysed by studying the changes in relative abundance of the RNA sequences homologous to four selected recombinants in RNA from subcellular fractions of liver, and comparing them with that of albumin mRNA. The relative abundance of these four RNA sequences increased to varying degrees in the nucleus, whilst that of three of them was concomitantly depleted in polysomal RNA when circulating levels of glucocorticoids were negligible, i.e. 14 days after adrenalectomy. One of the sequences was identified as alpha 2U-globulin mRNA. Within 2 hours of injecting Dexamethasone (a synthetic glucocorticoid) into rats that had been adrenalectomised 14 days previously, the relative abundances of alpha 2U-globulin RNA in nuclear and polysomal RNA returned to those found in normal rat liver. The data indicate that reduced glucocorticoid levels lead to sequence specific retention of RNA in the nucleus and that the RNA retained is released to the cytoplasm following glucocorticoid injection. Our results provide an example, for the first time, of glucocorticoid regulation of gene expression at the post-transcriptional level of nucleo-cytoplasmic transport.

Effect of valproic acid on zinc metabolism in the rat

Previous studies have suggested that the mechanism of valproic acid (VPA) hepatotoxicity may involve a drug-induced Zn deficiency. To test this hypothesis, the uptake of 65Zn or tissue Zn concentration was determined in plasma, liver, bone, kidney, and brain of adult male rats, administered parenteral VPA according to one of 3 schedules: 750 mg/kg; 500 mg/kg; and 100 mg/kg/day × 7 days. Histopathological changes in liver and weight loss were observed in rats 5 days after administration of VPA (750 mg/kg). The plasma Zn level in VPA toxic rats was significantly depressed compared to saline-injected controls, although the Zn content of liver and bone was unaffected. Furthermore, tissue uptake of 65Zn was not altered in rats 6 h after receiving VPA 500 mg/kg or after chronic administration at 100 mg/kg/day. On the basis of the present study, there is no evidence that Zn deficiency is induced by hepatotoxic doses of VPA in rats.

Estrogen Binding Protein Activity in Morris Hepatoma 7777 Compared With Normal Rat Liver

Estrogen binding protein activities were determined in the cytosol from adult male Buffalo rat liver and Morris hepatoma 7777. Estrogen receptors were prepared using the protamine sulfate precipitation technique of Chamness. The ability of various unlabeled steroids competing with [3H]estradiol was examined to establish the binding specificity. Estradiol binding in Morris hepatoma 7777 cytosol was greatly decreased compared with that present in hepatic cytosol prepared from normal rat liver. The receptor concentration expressed as femtomoles per milligram of cytoplasmic protein was 31.1 +/- 2.9 SD for normal rat liver and 0.41 +/- 0.88 SD for the hepatoma. Gel filtration chromatography revealed the presence of an estrogen binder in hepatoma cytosol which was not present in either normal liver or in the protamine sulfate precipitates of hepatoma cytosol. The molecular weight, binding specificity, and precipitation of this protein by specific antiserum suggests that it is alpha-fetoprotein.

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone.

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.

Sexual differentiation of rat liver carbonic anhydrase III

Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.

Neonatal phenobarbital administration results in increased cytochrome P 450-dependent monooxygenase activity in adult male and female rats

The effects of neonatal exposure to phenobarbital during the first five days after birth on the enzymatic activity of the adult male and female rat liver P 450-dependent monooxygenase system were investigated. Although liver weight per 100 grams of body weight and total hepatic microsomal protein content were not altered in adult rats treated neonatally with phenobarbital, both sexes did show significant increases in cytochrome P 450 content, cytochrome P 450 reductase activity, cytochrome c reductase activity, ethoxycoumarin-O-deethylase activity and in the activity of a specific glucuronyl-transferase. Several of these activities were increased to a larger extent in the females, suggesting that females may be more sensitive to this phenomenon.

Differential regulation of male rat liver glutathione S-transferases : Effects of orchidectomy and hormone replacement

The effects of orchidectomy and hormone replacement on glutathione S-transferase activities in adult male rat liver were investigated. Due to the overlapping yet distinct substrate specificities of the hepatic glutathione S-transferases, we measured activity in cytosol toward four substrates: 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, trans-4-phenylbut-3-en-2-one and p-nitrobenzyl chloride. Orchidectomy resulted in a decrease in transferase activity toward 1-chloro-2,4-dinitrobenzene, trans-4-phenylbut-3-en-2-one and p-nitrobenzyl chloride to 76, 64 and 70% of control. In contrast, transferase activity toward 1,2-dichloro-4-nitrobenzene was increased to 137% of control. To determine the role of specific androgens in the hormonal dependence of the glutathione S-transferases, rats were subcutaneously implanted for 4 weeks with either blank or steroid-filled sustained-release capsules at the time of orchidectomy. Transferase activities toward 1-chloro-2,4-dinitrobenzene or p-nitrobenzyl chloride were increased to control levels by testosterone but not by any of its 5α-reduced metabolites. Transferase activity toward trans-4-phenylbut-3-en-2-one was increased to control level by either dihydrotestosterone or 5α-androstan-3-α, 17β-diol. Activity toward 1,2-dichloro-4-nitrobenzene was decreased to control level by all of the androgens studied, testosterone, dihydrotestosterone, 5α-androstan-3β,17β-diol or 5α-androstan-3α,17β-diol. Thus, the hepatic glutathione S-transferases are under separate control and are differentially regulated by testosterone and its 5α-reduced metabolites.

Multihormonal control of microsomal 5 alpha-reductase activity in cultured adult female rat hepatocytes.

The activity of microsomal 5α-reductase, active on 3-oxo-4-ene-steroids, is much higher in adult female than in adult male rat liver. To examine the hormonal control of this enzyme under defined in vitro conditions, adult female rat hepatocytes were isolated by collagenase perfusion and cultured onto collagen membranes. The isolated hepatocytes form a nonreplicating monolayer after 24 h when cultured with or without serum, although the addition of fetal calf serum (FCS) or charcoal-extracted FCS improves hepatocyte plating efficiency. The charcoal-extracted FCS also significantly increases the 5α-reductase activity in cells cultured for 48 h. Time-course studies on 5α-reductase activity in isolated cells show that the activity in isolated cells is significantly lower than that in the intact animal. The activity is stable for 48 h in culture and then decreases. In cells cultured for 48 h, insulin significantly increases 5α-reductase activity at all doses tested (0.005–50 μg/ml), whereas estradiol induces h...

Ribonucleic Acid Metabolism in Rat Liver During Long-Term Adaptation to Malnutrition

Ornithine decarboxylase (ODC) and nucleolar DNA-dependent RNA polymerase (RNA polymerase I) activities increased in the liver of young adult male rats fed a 6% casein diet (malnourished) for 1 week when compared with rats fed a 25% casein diet (control). ODC activity increased progressively and reached a peak after 3 weeks of malnutrition and then decreased to control values by 5 weeks. RNA polymerase I reached peak activity 1 week after malnutrition was imposed, decreasing thereafter to control values by 3 weeks. At 4 and 5 weeks, RNA polymerase I activity in malnourished animals was lower than control. Nucleoplasmic DNA-dependent RNA polymerase activity remained unchanged in the first 2 weeks of malnutrition and decreased thereafter to values significantly lower than control. The data confirm our previous observations of cyclical changes during prolonged malnutrition and suggests a process of “biochemical adaptation” to malnutrition in which the organism enhances essential metabolic processes to maintain cellular homeostasis to the detriment of less essential functions like systemic growth.RNA polymerase malnutrition RNA metabolism polyamines ornithine decarboxylase

Delta-aminolevulinic acid dehydratase assay.

gamma-Aminolevulinic acid (ALA) dehydratase catalyzes the synthesis of porphobilinogen (PBG) from two molecules of ALA. A semimicro method for the colorimetric determination of ALA dehydratase is presented and applied to various tissues. The enzyme activity in adult male rat liver was 2.22 and 1.94 mumol PBG formed/g liver/h for homogenates assayed with or without dithiothreitol, respectively. The assay was linear for at least 2.5 h and for up to 2.5 mg tissue per assay. The Km for ALA was 4.0 X 10(-4)M and the pH optimum was 6.2-6.4. The effects of activators and inhibitors on enzyme activity are discussed.

Albumin secreted by rat liver bypasses Golgi apparatus cisternae.

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [3H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60-90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elements of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.

Testosterone-induced, sulfonamide-resistant carbonic anhydrase isozyme of rat liver is indistinguishable from skeletal muscle carbonic anhydrase III

Three isozymes of carbonic anhydrase, CA I, CA II and CA III, have been characterized from the tissues of a variety of vertebrate species. CA I (low-activity, sulfonamide-sensitive form) has been found mainly in red cells but also occurs in other tissues (e.g., rumen epithelium, caecum, colonic mucosa, pituitary gland, ciliary body), CA II (high-activity, sulfonamide-sensitive form) is found in red blood cells and in a majority of other tissues, and CA III (low-activity, sulfonamideresistant form) has been reported to occur largely in red skeletal muscle; cf. [l-7]. Although all 3 isozymes catalyze the reversible hydration of CO*, hydrolyze certain ester linkages, and are selectively inhibited by heterocyclic sulfonamides such as acetazolamide (Diamox), their relative activities and degrees of sulfonamide inhibition can vary considerably. For example, the CA II isozymes have the highest CO2 hydrase and esterase (toward p-nitrophenyl acetate) activities and the highest affinities for sulfonamides, followed by the CA I isozymes, while the CA III isozymes exhibit markedly lower CO2 hydrase and esterase activities and are only weakly inhibited by sulfonamides [l-4]. Early studies on carbonic anhydrase activity in rat liver homogenates [8,9] showed that its CO* hydrase activity was poorly inhibited by acetazolamide compared to the strong inhibition of rat red cell carbonic anhydrase (presumably CA I and CA II) activities. A partial characterization of a carbonic anhydrase isolated from livers of adult male rats [IO] showed that the specific CO* hydrase activity of this form was -1% that of the high-activity CA II isozyme purified

Testicular neonatal imprinting of sex dependent differences in hepatic foreign compound metabolism in the rat.

Abstract A dimethylated chlorocyclodiene epoxide (DME) ∗ is metabolized by adult male rat liver enzymes to produce two g.l.c. detectable metabolites M1 and M2. Liver enzymes from adult female rats metabolize DME producing only metabolite M1 in detectable quantities. This apparent qualitative sex difference in the metabolism of DME is first manifest between the ages of 35 and 45 days. Liver enzymes from rats of both sexes younger than 35 days metabolize DME in a qualitatively similar manner. The actions of the testes in the development of this apparent qualitative sex difference are exerted between the ages of 7 and 14 days. Castration of male rats at and before the age of 7 days results in the expression of a typically feminine pattern of metabolism of DME by liver enzymes in adult life. Castration of male rats after the age of 14 days does not prevent adult liver enzymes from exhibiting a basically masculine pattern of DME metabolism. At ages between 7 and 14 days castration has a graded effect on the metabolism of DME displayed by liver enzymes in adult life. These results suggest that testicular androgens do not play a direct role at puberty and in adulthood in the expression of the ability of liver enzymes to produce metabolite M2, but that the apparent qualitative sex dependent difference in DME metabolism is determined primarily by neonatal imprinting by testicular androgens.

Effects of acute hepatic ischemia on the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of rat liver

The purpose of this study was to establish when alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of the livers of rats become observable after initiation of acute hepatic ischemia. Ischemia was initiated by clamping the vascular supply to the left and median lobes of the livers of adult male rats. The animals were killed at various times thereafter (up to 6 h, and in certain instances, 24 h) and microsomal membrane fractions were prepared from each. The patterns of the polypeptides and phosphopolypeptides of these fractions were analysed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, using staining with Coomassie blue to analyse the polypeptides and radioautography to analyse 32P-labelled phosphopolypeptides. Alterations of the polypeptide pattern were apparent in the fractions from animals killed at 4 h and after; prior to this time point, subtle alterations, at most, could be distinguished. Effects of acute ischemia on the pattern of phosphopolypeptides of the microsomal membrane fraction were studied after phosphorylation in vivo (produced by intraperitoneal injection of [32P]phosphoric acid) and in vitro (using [gamma-32P]ATP as phosphate donor). No marked changes in the phosphopolypeptide pattern produced by phosphorylation in vivo were observed until 6 h after clamping, by which time a diminution of the radioactivity in the majority of the phosphopolypeptides was evident. However, noteworthy alterations of the pattern of phosphopolypeptides produced by phosphorylation in vitro were observable in the membrane fractions from animals subjected to 2 h of ischemia. Overall the study provides a base line delineating the time sequence during which alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of rat liver microsomal membranes become evident following the onset of acute hepatic ischemia and reveals that gross alterations of the polypeptide patterns of these membranes and of certain other subcellular fractions are not an early occurrence following this severe type of injury. The possible utility of the application of phosphorylation in vitro for detecting early alterations in membrane structure following cell injury is suggested.

Fluorophosphate-sensitive esterases in mammalian liver: the radioautographic localization and measurement of fluorophosphate-reactive sites in adult rat liver.

Fluorophosphate-reactive (FPR) sites in the adult male rat liver, tentatively identified as esterase active centers, were localized and measured using the combined techniques of quantitative electron microscope radioautography and morphometric analysis with the light and electron microscope. The FPR sites were measured in liver which had been prefixed by perfusion with 1.5% glutaraldehyde and reacted with 10(-4) M tritium-labeled diisopropyl fluorophosphate (3H-DFP). Under the experimental conditions 64-67% of the esterase activity in fresh liver was retained for reaction with the 3H-DFP, which is known to bind irreversibly to the active sites of certain esterases. In light and electron microscope radioautographs the developed silver grains were concentrated over the cytoplasm of hepatocytes. A low concentration occurred over erythrocytes. All other areas in the liver had a concentration of grains resembling the background concentration. Quantitative measurements of grain density in the electron microscope radioautographs revealed the highest density, after correcting for radiation spread, in cytoplasmic granules (mainly cytolysomes). The grain densities over the rough and smooth endoplasmic reticulum and associated structures were also equal to or above the average hepatocyte grain density. Due to the large fractional volume of endoplasmic reticulum per hepatocyte (58% of cell volume) and the fraction of the liver occupied by hepatocytes (79% of liver volume) the majority of FPR sites in the liver occurred in the rough and smooth endoplasmic reticulum and associated structures. The average numbers of FPR sites were calculated per micrometer3 of hepatocyte (5.0 x 10(5) sites/micrometer3) and per unit volume of each significantly labeled organelle. In addition, the number of FPR sites per hepatocyte (2.5 X 10(9) sites/cell), per cm3 liver (4.1 X 10(17) sites/cm3) and in the total liver of an average 100 g male rate (2.2 X 10(18) sites/total liver) were also calculated.

Diurnal rhythm in rat liver 5‐aminolevulinate synthetase activity

Abstract 5‐Aminolevulinate synthetase activity was measured in livers of adult male rats by means of an improved assay. The enzyme exhibited a diurnal rhythm with a maximum during dark‐time. Inductions by allylisopropylacetamide during diurnal increase and decrease of enzyme activity resulted in the same amount of additionally formed enzyme.