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The mechanism of glycogen biosynthesis in the absence of added primers takes place at least in two different steps. The first step could be initiated by a new enzyme named glycogen intiator synthase that catalyzes the transfer of glucose from UDP-glucose to an acceptor protein. This step takes place in vitro only in the presence of some salts at high concentration. The complex product from this reaction has been isolated and demonstrated to act as a precursor for the synthesis of glycogen which takes place in the second step and is catalyzed by the already known glycogen synthase.

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.

Presents forthcoming papers for the "European Journal of Biochemistry". "A guanyloribonuclease mouse liver cytosol," by A. Pantazaki and colleagues; "Revised nonmenclature for eukaryotic DNA polymerases," by P.M.J. Burgers and colleagues; "The folding and solution conformation of penicillin G acylase," by C.D. Lindsay and colleagues.

Lists papers due for publication in the "European Journal of Biochemistry" after the January 16, 1984 issue. "Labelling and cross-linking of Escherichia coli penicillin-binding proteins with bis-Beta-lactam antibiotics"; "Sorbitol dehydrogenase. The primary structure of the sheep-liver enzyme"; "Extensive variations and basic features in the alcohol dehydrogenase-sorbitol dehydrogenase family".

The article presents information on various papers to be published in the "European Journal of Biochemistry." Some of them are, "Lipid Domains in Plasma Membranes From Rat Liver," by F. Schroeder, "Calcium Modulates Fatty Acid Dynamics in Rat Liver Plasma Membranes," by F. Schroeder, and C.S. Argilaga, and others.

The direct effects of insulin on hepatic triacylglycerol secretion are important because they may determine the degree of postprandial hyperlipidaemia, a known risk factor for the development of atherosclerotic lesions. Previous work from this laboratory, conducted on isolated perfused rat livers [Zammit, V.A., Lankester, D.J., Brown, A.M. & Park, B.S. (1999) Eur. J. Biochem. 263, 859-864], has indicated that the effect of insulin on hepatic triacylglycerol secretion is dependent on the prior physiological state of the donor animals. In this paper, we demonstrate that a switch in the direction of insulin action on hepatic partitioning of fatty acyl moieties towards triacylglycerol secretion also occurs in vivo between the fed, normoinsulinaemic state and the fasted or severely insulin-deficient states. The partitioning of fatty acids in the liver of awake, unstressed rats was studied using selective labelling of hepatic fatty acids during hyperinsulinaemic-euglycaemic clamps achieved through the use of hepatocyte-targeted liposome-encapsulated insulin preparations. The data show that, whereas in the fed, normoinsulinaemic state, insulinization of the liver raises the proportion of fatty acids directed towards secreted triacylglycerol, in the fasted or insulin-deficient states, insulin inhibits the partitioning of acyl moieties into secreted triacylglycerol. These data show that observations on the direction of insulin action on hepatic triacylglycerol secretion obtained using isolated perfused rat livers are reflected in the effects of the hormone on hepatic fatty acid partitioning in vivo. They offer an explanation for the positive relationship between chronic hyperinsulinaemia, hepatic VLDL-triacylglycerol secretion and hypertriglyceridaemia observed previously in insulin-resistant states.

Previously [Ainscow, E.K. & Brand, M.D. (1999) Eur. J. Biochem. 263, 671-685], top-down control analysis was used to describe the control pattern of energy metabolism in rat hepatocytes. The system was divided into nine reaction blocks (glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption) linked by five intermediates (intracellular glucose 6-phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential). The kinetic responses (elasticities) of reaction blocks to intermediates were determined and used to calculate control coefficients. In the present paper, these elasticities and control coefficients are used to quantify the internal regulatory pathways within the cell. Flux control coefficients were partitioned to give partial flux control coefficients. These describe how strongly one block of reactions controls the flux through another via its effects on the concentration of a particular intermediate. Most flux control coefficients were the sum of positive and negative partial effects acting through different intermediates; these partial effects could be large compared to the final control strength. An important result was the breakdown of the way ATP consumption controlled respiration: changes in ATP level were more important than changes in mitochondrial membrane potential in stimulating oxygen consumption when ATP consumption increased. The partial internal response coefficients to changes in each intermediate were also calculated; they describe how steady state concentrations of intermediates are maintained. Increases in mitochondrial membrane potential were opposed mostly by decreased supply, whereas increases in glucose-6-phosphate, NADH/NAD and pyruvate were opposed mostly by increased consumption. Increases in ATP were opposed significantly by both decreased supply and increased consumption.

The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus. Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6-phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels. The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6-phosphate concentration was due to the stimulation of glycogen breakdown and inhibition of glucose release.

Hepatic cell-specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis-acting elements located between positions -128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun. 220, 553-562]. A negative cis-acting element (+20 to +40) is located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor-3 (BRF-3) interacts with this. In this paper, we report the purification and characterization of BRF-3 from rat liver nuclear extracts. BRF-3 has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. Purified BRF-3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70-kDa and 67-kDa proteins have been found to hybridize specifically with labeled double-stranded oligonucleotide containing BRF-3 binding site in a South-Western blot. Double-stranded oligonucleotide containing mutations in the BRF-3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70-kDa and 67-kDa rat BRF-3 proteins were found to have 100% sequence homologies with DNA topoisomerase I. These data suggest that the 70-kDa and 67-kDa forms of BRF-3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may play an important role in transcriptional regulation of apoB.

The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I) over hepatic ketogenesis because its role in controlling this pathway during the neonatal period is of academic importance and immediate clinical relevance. Using hepatocytes isolated from suckling rats as our model system, we measured CPT I activity and carbon flux from palmitate to ketone bodies and to CO2 in the absence and presence of a range of concentrations of etomoxir. (This is converted in situ to etomoxir-CoA which is a specific inhibitor of the enzyme.) From these data we calculated the individual flux control coefficients for CPT I over ketogenesis, CO2 production and total carbon flux (0.51 +/- 0.03; -1.30 +/- 0.26; 0.55 +/- 0.07, respectively) and compared them with equivalent coefficients calculated by similar analyses [Drynan, L., Quant, P.A. & Zammit, V.A. (1996) Biochem. J. 317, 791-795] in hepatocytes isolated from adult rats (0.85 +/- 0.20; 0.23 +/- 0.06; 1.06 +/- 0.29). CPT I exerts significantly less control over ketogenesis in hepatocytes isolated from suckling rats than those from adult rats. In the suckling systems the flux control coefficients for CPT I over ketogenesis specifically and over total carbon flux (< 0.6) are not consistent with the enzyme being rate-limiting. Broadly similar results were obtained and conclusions drawn by reanalysis of previous data {from experiments in mitochondria isolated from suckling or adult rats [Krauss, S., Lascelles, C.V., Zammit, V.A. & Quant, P.A. (1996) Biochem. J. 319, 427-433]} using a different approach of control analysis, although it is not strictly valid to compare flux control coefficients from different systems. Our overall conclusion is that flux control coefficients for CPT I over oxidative fluxes from palmitate (or palmitoyl-CoA) differ markedly according to (a) the metabolic state, (b) the stage of development, (c) the specific pathway studied and (d) the model system.

Up until now, the primary structure of fatty-acid-binding proteins (FABPs) from the livers of four mammalian (rat, human, cow and pig) and three nonmammalian (chicken, catfish and iguana) species has been determined. Based on amino acid sequence comparisons, it has been suggested that mammalian and nonmammalian liver FABPs may be paralogous proteins that originated by gene duplication, rather than as a consequence of mutations of the same gene. In this paper we report the isolation and amino acid sequence determination of two FABPs from axolotl (Ambistoma mexicanum) liver. One of them is similar to mammalian liver FABPs (L-FABPs) and the other to chicken, catfish and iguana liver FABPs (Lb-FABPs). The finding of both L-FABP and Lb-FABP in a single species, as reported here, indicates that they are paralogous proteins. The time of divergence of these two liver FABP types is estimated to be of approximately 694 million years ago. The ligand-binding properties of axolotl liver FABPs were studied by means of parinaric-acid-binding and parinaric-acid-displacement assays. L-FABP binds two fatty acids per molecule but Lb-FABP displays a fatty-acid-conformation-dependent binding stoichiometry; L-FABP shows a higher affinity for fatty acids, especially oleic acid, while Lb-FABP has a higher affinity for other hydrophobic ligands, especially retinoic acid. In addition, the tissue-expression pattern is different, L-FABP is present in liver and intestinal mucosa while the expression of Lb-FABP is restricted to liver. Data indicate distinct functional properties of both liver FABP types.

Cytochromes P-450 are responsible for the biotransformation of drugs and other hydrophobic molecules by the liver. Several isoforms coexist which display an asynchronous onset during the perinatal period suggesting the involvement of multiple mechanisms of regulation. In this paper, we have shown that the CYP2E1 protein and its associated activity could not be detected in the fetal liver and rise during the first few hours following birth independently of the gestational age (between 25-40 weeks). During this period, the CYP2E1 RNA content remains fairly low: the stabilization of the low amount of existing CYP2E1 protein by endogenous ketone bodies could explain the early neonatal rise of the protein level. From 1 month to 1 year, the protein content gradually increases and is accompanied by the accumulation of CYP2E1 RNA, suggesting a transcriptional activation of the gene during the late neonatal period. We examined the methylation status of CpG residues in the 5' flanking region, first exon and first intron of CYP2E1 gene cleaved with HpaII/MspI. Genomic DNA from fetal liver shows several hypermethylated spots in the first-exon-first-intron region, which progressively disappear in neonatal samples. We conclude that during the neonatal period, the accumulation of hepatic CYP2E1 RNA is correlated with the degree of methylation at the 5' end of the CYP2E1 gene.

In this paper, we present the results of an investigation into the catalytic properties of CMP-Neu5Ac hydroxylase (Neu5Ac: N-acetylneuraminic acid) in high-speed supernatants of mouse liver. The enzyme was most active in Hepes/NaOH pH 7.4 and was markedly inhibited by relatively small increases in ionic strength, though the inhibition was abolished by desalting procedures. Several nonionic detergents could activate the hydroxylase to various degrees in a concentration-dependent manner. Ionic detergents and a number of phospholipids were, however, generally inert or inhibitory. The lack of inhibitory influence of a wide range of nucleotides revealed that CMP-Neu5Ac hydroxylase binds its sugar-nucleotide substrate with a high degree of specificity. Thus, even millimolar concentrations of several cytidine nucleotides elicited virtually negligible inhibition, though the reaction product, CMP-Neu5Gc (Neu5Gc: N-glycoloylneuraminic acid), was a weak inhibitor. The results also indicate that the enzyme is not regulated by any nucleotides or sugar-nucleotides. Dilution of high-speed supernatants with buffer gave rise to a decrease in the specific activity of the hydroxylase, implicating the involvement of more than one component in catalysis. Activity could be restored by the addition of a heat extract of the supernatant. The active principle in this extract was found to be a heat-stable protein with a molecular mass of about 17 kDa. Immunochemical studies allowed this protein to be identified as cytochrome b5 and it was shown that this electron carrier is essential for the activity of CMP-Neu5Ac hydroxylase. Inhibition studies using iron ligands and activation by exogenous iron salts suggest the involvement of a non-haem iron cofactor in the catalytic cycle of this hydroxylase. Cytochrome b5 may thus serve as an electron donor for this postulated cofactor.

The colon and lung amiloride-binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7-kb transcript codes for an 85-kDa protein, whereas the 1.2-kb transcript codes for a 25-kDa polypeptide. The 2.7-kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2-kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3′ terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter. Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5 =10 nM) > benzamil (K0.5 = 43 nM) > amiloride (K0.5 = 1.4 μM) ≈ ethylisopropylamiloride (K0.5 = 1.6 μM) for the long form, whereas it was phenamil (K0.5 = 68 nM) > amiloride (K0.5 = 3.2 μM) ≈ ethylisopropylamiloride (K0.5 = 4 μM) ≈ benzamil (K0.5 = 6.3 μM) for the short form. Although the binding proteins described here are distinct from the pore-forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride-binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride-binding protein, possibly associated with other proteins. The similarity between amiloride-binding proteins and a protein identified in seminal vesicles suggests that amiloride-binding proteins are the best members of a new family of epithelial-specific proteins. [ABSTRACT FROM AUTHOR]

Nucleoli, isolated from rat liver, synthesize in vitro high-molecular-weight RNA, the base composition and sedimentation pattern of which resembles that of ribosomal precursor RNA. In addition, RNA molecules larger than 45 S have been found. In this paper experiments are described which indicate that these large RNA molecules represent genuine transcription products and are not aggregates arising under the experimental conditions employed. This was established by comparing different extraction methods, by sedimentation analysis of the RNA after denaturation with formamide and by pulse-chase experiments. Hybridisation-competition studies showed that 45-S RNA competes with those rapidly sedimenting molecules to about 80-90%, thus providing evidence for the presence of ribosomal precursor RNA sequences in those long transcription products. Intact nuclei are able to synthesize in the presence of Mg2+ and α-amanitin RNA molecules larger than 45 S too, provided that the RNAase activity is suppressed effectively by the addition of cytoplasmic RNAase inhibitor. The significance of these results is discussed with respect to the initial transcript of the rDNA genes in rat liver nucleoli. [ABSTRACT FROM AUTHOR]

Aflatoxin B1 strongly inhibits protein synthesis in rat liver cells. We previously demonstrated that tiffs inhibition could be divided into two steps: up to 5 h aflatoxin blocks protein synthesis directly and specifically at the polysome level; beyond 7 h protein synthesis inhibition appears chiefly as a consequence of transcription impairment due to drug action. This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by Aflatoxin B1. We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972(Biochim. Biophys. Acta, 272, 451-462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis. Up to 5 h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2 h after dosing, each step of protein synthesis is blocked. Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin-treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin-treated rats synthesize proteins in vitro the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures. Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed. [ABSTRACT FROM AUTHOR]

Exposure of perfused rat livers to zymosan, arachidonic acid and phenylephrine, but not to latex particles, induces pronounced oxygen uptake, glycogenolysis and Ca2+ mobilization. The oxygen uptake induced by arachidonic acid and by zymosan remains elevated even after the agents have been removed. NaN3 was found to be much more effective in inhibiting the oxygen uptake induced by phenylephrine than that induced by zymosan or arachidonic acid. Glucose release induced by zymosan and by arachidonic acid reaches a maximum after about 2 min and then declines very rapidly even while the agents are still being infused. In contrast, glucose release induced by phenylephrine remains elevated for the duration of the infusion. Ca2+ fluxes induced by arachidonic acid are similar to those induced by phenylephrine in that efflux occurs when the agent is administered and influx occurs only when the agent is removed. This contrasts to the Ca2+ flux changes induced by zymosan where both Ca2+ effiux and Ca2+ influx occur even while zymosan is still being infused. Glucose release induced by zymosan is inhibited by bromophenacylbromide and nordihydroguaiaretic acid, but not by indomethacin. Indomethacin, however inhibits the arachidonic-acid-induced glucose release which is also inhibited by nordihydroguaiaretic acid but not by bromophenacylbromide. Indomethacin inhibits also the arachidonic-acid-induced Ca2+ flux changes whereas the zymosan- and the phenylephrine-induced Ca2+ flux changes are not inhibited by the cyclooxygenase inhibitor. The data presented in tbs paper suggest that in the perfused rat liver the zymosan-induced glycogenolysis, as well as the Ca2+ flux changes and glycogenolysis induced by arachidonic acid, are mediated by eicosanoids. [ABSTRACT FROM AUTHOR]

Nuclear RNP complexes, cytoplasmic mRNP particles and free and membrane-bound polysomes were prepared from rat liver and their tow-molecular-muss RNA components were analyzed on polyacrylamide/formamide gels. The separated small RNAs transferred to diazophenylthioether paper were hybridized to the nick-translated recombinant plasmid pA6 containing cDNA sequences for the low-Mr RNA called 7S(L) RNA. Nuclear RNP particles and free and membrane-bound polysomes were found to contain 7S(L) RNA. In the cytoplasm 7S(L) RNA could be identified as the major small RNA in 20-S cmRNP particles. [ABSTRACT FROM AUTHOR]

Apolipoprotein A-I, the major protein in mammalian high-density lipoprotein, acts as a cofactor for lecithin—cholesterol acyltransferase during the formation of cholesterol ester and as such, is thought to promote cholesterol efflux from peripheral cells to the liver. In this paper, we report the partial purification of rat liver apolipoprotein A-I mRNA by a polysome immunoadsorption technique, and its cDNA cloning. Isolation of two overlapping cDNA clones enabled us to derive the whole rate apolipoprotein A-I cDNA coding sequence. Comparison of the deduced protein sequence with its human counterpart reveals a striking homology between the prepropeptide precursors. Both mature protein amino-terminal regions are very homologous, suggesting that this particular domain could be involved in lipid/protein binding or lecithin—cholesterol acyltransferase activation. [ABSTRACT FROM AUTHOR]

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the enzyme horse liver alcohol dehydrogenase catalyzes a reaction between NAD+ and H2O2. The final isolated product was then called NADX because of its unknown structure. In this paper the results of spectroscopic investigations on this compound are described. They indicate that only the nicotinamide moiety of the original NAD+ molecule was modified by the action of hydrogen peroxide. From the ¹H and 13C nuclear magnetic resonance spectra of NADX the following structure was deduced: adenosine(5′)diphospho(5)-β-D-ribose-NH-CH=C(CHO)-CONH2. This structure is consistent with both ultraviolet and reactivity measurements performed on NADX. A tentative mechanism for the whole peroxidatic reaction pathway leading to NADX is finally proposed. [ABSTRACT FROM AUTHOR]

Liver and muscle aldolase display similar reaction mechanisms. Both the enzymes, by reacting with dihydroxyacetone phosphate, form an acid-labile intermediate which is in rapid equilibrium with an eneamine intermediate. Differences are found in the equilibrium concentration of the acid-labile intermediate, which represents approximately 25% of the total intermediates in the liver (this paper) and 60% in the muscle enzyme [E. Grazi and G. Trombetta, Biochem. J. 175, 361 (1978)] and in the rate of formation of the eneamine intermediate which is much slower in the liver enzyme. Furthermore, with liver aldolase, the rate by which the C-3H bond of dihydroxyacetone phosphate is cleaved is increased by 60 times in the presence of glyceraldehyde 3-phosphate. This, mechanistically, indicates that glyceraldehyde 3-phosphate is bound to the enzyme before the formation of the eneamine from dihydroxyacetone phosphate, and, physiologically, that in liver aldolase the gluconeogenetic activity is favoured over the glycolytic activity. [ABSTRACT FROM AUTHOR]

The mechanism of action of 3-hydroxy-3-methylglutaryl-CoA reductase solubilized from rat liver microsomes has been investigated. In the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate, an overall initial velocity study gave a linear intersecting pattern when both 3-hydroxy-3- methylglutaryi-CoA and NADPH were variable substrates. Adenosine 2′-monophospho-5′-diphos- phoribose, which was found to be a reversible inhibitor of reductase, inhibited the enzyme competitively with respect to NADPH, and uncompetitively with respect to 3-hydroxy-3-methylglutaryl-CoA. On the other hand, the inhibition of reductase by ML-236B (sodium salt), a specific reversible inhibitor of the enzyme isolated from the culture of a Penicillium (whose structure is given in the paper) is competitive with respect to 3-hydroxy-3-methylglutaryl-CoA and noncompetitive with respect to NADPH [Endo et al. (1976) FEBS Lett. 72, 323-326]. The reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate was subject to the substrate inhibition by NADPH attributed to the formation of a productive enzyme-NADPH complex. These results indicate that the two substrates bind to the enzyme effectively in an ordered manner; reductase first interacts with 3-hydroxy-3-methylglutaryl-CoA to make a binary complex, which, in turn, forms a ternary complex with one molecule of NADPH. Considered together with the results of product inhibition study, and assuming a hemithioacetal of mevaldate and CoA is an intermediate of the reductase reaction, a bi-uni-uni-ter-ping- pong mechanism is proposed as a model of the overall reaction. [ABSTRACT FROM AUTHOR]

A product of mitochondrial protein synthesis in rat liver mitochondria, characterized by a low molecular weight (Mr < 10000) and an unusually high hydrophobicity, has been identified as the dicyclohexylcarbodiimide-binding protein and as a peptide of the hydrophobic sector of the mitochondrial ATPase complex. The purified protein still possesses the ability to bind dicyclohexylcarbodiimide. [ABSTRACT FROM AUTHOR]

The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1-kinase, fructose-1,6-bisphosphatase, L-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2B and 2C exhibited activity towards all five substrates in vitro, although phosphatases-1 and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-1 and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2C contributes a significant percentage of the activity towards 6-phosphofructo-1-kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation.

Ribosomes have been shown to contain a proteolytic activity, characterized as an endopeptidase with serine in the active center. The enzyme has been given the name cathepsin R, following the recommendations of Barrett et al. (in a publication from the Cold Spring Harbor Laboratory, New York) for naming new proteinases. The present paper contains evidence that cathepsin R in rat liver ribosomes is present in a cryptic form. Upon dissociation of ribosomes to subunits (and to minor extent also by 0.5 M KC1 washes), the cryptic proteinase is released. Activation of the released cathepsin R is effected by equilibration with 2 M NaC1/0.05 M sodium acetate, pH 4.8. The molecular weight of free cathepsin R is 25 000-30 000.

The present paper shows that in the regenerating rat liver the membrane-bound-gamma-glutamyltransferase exists in two molecular forms. Depending on the state of proliferation, a sialic-acid-rich enzyme (in the fetal or regenerating liver) or a sialic-acid-poor enzyme (in the adult or quiescent liver) could be detected. In regeneration liver (24 h after 2/3 resection) only the sialic-acid-rich or fetal enzyme could be found. Since total enzyme activity (adult + fetal type) remained unchanged, it is assumed that the adult type of gamma-glutamyltransferase was modified by sialylation during the initial phase of liver regeneration. This process of sialylation was prevented by inhibitors of RNA or protein synthesis such as D-galactosamine, actinomycin D or cycloheximide, provided that the inhibitor (D-galactosamine) was given within the first 8 h after partial hepatectomy. Sialylation was not impaired by inhibitors of DNA synthesis, e.g. hydroxyurea or cytosine arabinoside. Administration of actinomycin D during a defined phase of proliferation (24 to 48 h after partial hepatectomy) stimulated the transfer of sialic acid to gamma-glutamyltransferase, a finding which describes for the first time the so-called 'superinduction' of a sialylation process.

Techniques allowing the recovery of large free and membrane-bound polysomes in high yield are reported. Subcellular fractions were prepared from rat liver homogenates as described in the preceding paper. Purified microsomal membranes (obtained from the post-lysosomal supernatant) were adjusted to 50 mM Mg(CH3COO)2 and treated with 2% Triton X-100 and 0.3% sodium deoxycholate in the presence of yeast RNA and cell sap, and polysomes were purified by overnight centrifugation through low-ionic-strength discontinuous sucrose gradients containing 2 mg/ml of cell sap proteins. Polysomes were isolated from the mitochondria/endoplasmic reticulum complex (fraction C) by treatment with 2% Triton X-100 and 0.5% sodium deoxycholate in the presence of 50 mM Tris-HCl, pH 7.6, 0.1 M KCl, 0.15 M NH4Cl and 50 mM Mg(CH3COO)2 and purified through sucrose layers of decreasing ionic strength containing 2 mg/ml of cell sap proteins. Analyses of polysomes in isokinetic sucrose gradients showed that the free polysome fraction and both membrane-bound polysome fractions had 14-15 ribosomes per mRNA at the maximum of absorbance. Experiments from which these methods were derived are described.

Exposure of perfused rat livers to zymosan, arachidonic acid and phenylephrine, but not to latex particles, induces pronounced oxygen uptake, glycogenolysis and Ca2+ mobilization. The oxygen uptake induced by arachidonic acid and by zymosan remains elevated even after the agents have been removed. NaN3 was found to be much more effective in inhibiting the oxygen uptake induced by phenylephrine than that induced by zymosan or arachidonic acid. Glucose release induced by zymosan and by arachidonic acid reaches a maximum after about 2 min and then declines very rapidly even while the agents are still being infused. In contrast, glucose release induced by phenylephrine remains elevated for the duration of the infusion. Ca2+ fluxes induced by arachidonic acid are similar to those induced by phenylephrine in that efflux occurs when the agent is administered and influx occurs only when the agent is removed. This contrasts to the Ca2+ flux changes induced by zymosan where both Ca2+ efflux and Ca2+ influx occur even while zymosan is still being infused. Glucose release induced by zymosan is inhibited by bromophenacylbromide and nordihydroguaiaretic acid, but not by indomethacin. Indomethacin, however inhibits the arachidonic-acid-induced glucose release which is also inhibited by nordihydroguaiaretic acid but not by bromophenacylbromide. Indomethacin inhibits also the arachidonic-acid-induced Ca2+ flux changes whereas the zymosan- and the phenylephrine-induced Ca2+ flux changes are not inhibited by the cyclooxygenase inhibitor. The data presented in this paper suggest that in the perfused rat liver the zymosan-induced glycogenolysis, as well as the Ca2+ flux changes and glycogenolysis induced by arachidonic acid, are mediated by eicosanoids.

Secretory products of cultured human blood monocytes contain a hepatocyte-stimulating factor which is able to induce the acute-phase proteins alpha 2-macroglobulin and fibrinogen in rat liver cells. Total RNA was isolated from unstimulated and lipopolysaccharide-stimulated human monocytes and translated in a reticulocyte lysate. The capability of the cell-free synthesized proteins to induce the acute-phase proteins alpha 2-macroglobulin and fibrinogen was assayed in rat hepatocyte primary cultures and in the rat hepatoma cell line Fao. The products translated from the mRNA of lipopolysaccharide-stimulated human monocytes induced mRNAs for alpha 2-macroglobulin and fibrinogen and therefore contain hepatocyte-stimulating factor. The translation products of unstimulated monocytes had no effect. A cDNA containing the coding sequence for interleukin-6 (B-cell stimulatory factor 2, interferon-beta 2/26-kDa protein, interleukin HP1) derived from human T-cells cloned into the transcription vector pGEM4 was transcribed in vitro. Translation of the isolated RNA in a reticulocyte lysate led to the synthesis of a protein of about 25 kDa. This cell-free synthesized interleukin-6 exhibited hepatocyte-stimulating activity measured by the induction of beta-fibrinogen mRNA in Fao cells. Using an antibody against interleukin-6, two proteins of 22 kDa and 23 kDa were immunoprecipitated from the culture medium of lipopolysaccharide-stimulated human monocytes. These two proteins were not synthesized by unstimulated monocytes. When total RNA from unstimulated human monocytes and lipopolysaccharide-stimulated human monocytes and lymphocytes was subjected to Northern analysis and hybridized with the interleukin-6 cDNA, a strong hybridization signal corresponding to an RNA of about 1300 bases was detected only in the RNA from lipopolysaccharide-stimulated human monocytes, indicating that human monocytes express the interleukin-6 gene after stimulation. The data presented in this paper strongly suggest that hepatocyte-stimulating factor from human monocytes and interleukin-6 from T-cells are identical.

In this paper experiments are reported which show evidence for a relation between quaternary structure and catalytic activity of cytoplasmic NADP-linked isocitrate dehydrogenase from beef liver. The inactivation of the enzyme occurring upon dilution and the plots of the catalytic activity versus the enzyme concentration indicate that the monomeric species is catalytically inactive and that the monomer-dimer equilibrium is shifted towards the dimer upon binding of the substrate magnesium isocitrate complex. The association of the enzyme following binding of the substrate takes place at a rate comparable with that of the enzymatic reaction, which results in a 'hysteretic' behaviour of the enzyme. The possibility is discussed that slow changes in quaternary structure can give rise to a physiological regulation of the enzymatic activity.

In this paper, human low-density lipoprotein (LDL), rat chylomicron remnants and very-low-density lipoproteins of beta-mobility from cholesterol-fed rabbits (beta VLDL) have been shown to bind strongly to a protein present in solubilised liver membranes of rats, rabbits and dogs by ligand blotting with biotin-modified lipoproteins. This binding protein was identified as the LDL-receptor on several criteria. First, binding of the lipoproteins to the receptor was saturable and Ca2+-dependent; secondly, the apparent relative molecular mass of the binding protein (ranging from 128,000 in the rabbit, 145,000 in the rat to 147,000 in the dog) was similar to that of the purified bovine LDL receptor. Finally, binding activity was greatly increased in the livers of rats treated with oestrogen in pharmacological doses and absent from the liver of Watanabe heritable hyperlipidaemic (WHHL) rabbits that have a genetic defect in the LDL receptor. Some binding was also observed to a high-molecular-mass protein present in solubilised liver membranes of rats and rabbits, which, in rabbits at least, shared antigenic determinants with rabbit apoB and was not likely to be related to the LDL receptor as it was present in equal amounts in normal and WHHL rabbits. No evidence was obtained for a specific chylomicron remnant binding protein, distinct from the LDL receptor, whose activity could be detected in solubilised liver membranes by ligand blotting although a variety of solubilisation and fractionation conditions were employed.

The association of imidazole and auramine O to native horse-liver alcohol dehydrogenase [Zn(II)LADH] and active-site specifically cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10-phenanthroline and 2,2'-bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal-bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s-1 and 200 s-1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d-d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate-limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal-bound water molecule) are not responsible for the step characterized by kmax. We propose the rate-limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.

At a concentration of 1 mM, fructose 1-phosphate stimulated about twofold, and glucose 6-phosphate inhibited by about 30%, the phosphorylation of 5 mM glucose in high-speed supernatants prepared from rat liver or from isolated hepatocytes, but did not affect, or barely so, the activity of a partially purified preparation of glucokinase. Anion-exchange chromatography of liver extracts separated glucokinase from a fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive inhibitor of that enzyme. This inhibitor could be further purified by chromatography on phospho-Ultrogel. It was destroyed by trypsin and was heat-labile. It inhibited glucokinase competitively with respect to glucose and its inhibitory effect was greatly reinforced by fructose 6-phosphate although not by glucose 6-phosphate. Fructose 1-phosphate relieved the enzyme of the inhibitory effect of the regulator and antagonised the effect of fructose 6-phosphate in a competitive manner. It is concluded that the regulator plays a role in the physiological control of the activity of glucokinase, particularly with respect to the stimulatory effect of fructose in isolated hepatocytes (see preceding paper in this journal).

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.

Aflatoxin B1 strongly inhibits protein synthesis in rat liver cells. We previously demonstrated that this inhibition could be divided into two steps: up to 5 h aflatoxin blocks protein synthesis directly and specifically at the polysome level; beyond 7 h protein synthesis inhibition appears chiefly as a consequence of transcription impairment due to drug action. This paper confirms the foregoing results and represents an attempt to localize the translational step inhibited in vivo by aflatoxin B1. We used the simulation study developed by Li, Kisilevsky, Wasan and Hammond, 1972 (Biochim. Biophys. Acta, 272, 451-462) to determine precisely the site inhibited in vivo after drug intoxication. This analysis is based on two parameters: the kinetics of polysome labeling to follow the nascent peptide synthesis, and the kinetics of supernatant labeling to follow the completed protein synthesis. Up to 5 h after dosing, aflatoxin specifically inhibits the elongation and/or termination steps during protein synthesis; after longer periods of time inhibition occurs essentially at the initiation step. When the intracellular concentration of aflatoxin is too high, particularly 2 h after dosing, each step of protein synthesis is blocked. Polypeptide synthesis by the postmitochondrial supernatants isolated from aflatoxin-treated animals is impaired in the same proportion as protein synthesis in vivo. The damage caused by aflatoxin is mostly observed on microsomes. However, purified polysomes isolated from aflatoxin-treated rats synthesize proteins in vitro to the same extent as those from controls. These results suggest that aflatoxin metabolite(s) are bound to polysomes with noncovalent bonds. These active metabolites are probably lost during polysome isolation procedures. Finally, relationships between protein metabolism and aflatoxin carcinogenesis are discussed.

The present paper reviews recent advances which have been made in studies, predominantly in rat liver, on the nutritional and hormonal regulation of gene expression of lipogenic enzymes (acetylCoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase). [ABSTRACT FROM AUTHOR]

We have identified a mannosidase in rat liver that releases α1 → 2, α1 → 3 and α1 → 6 linked manose residues from oligosaccharide substrates, MannGlcNAc where n = 4-9. The end product of the reaction is Manα1 → 3[Manα1 → 6]Manβ1 → 4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X-114, by anion-exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110-kDa subunit, has a pH optimum between 6.1 and 6.5 and a Km of 65 μM and 110 μM for the Man5GlcNAc-oligosaccharide or Man9GlcNAc-oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the α1 → 2 linked residue followed by hydrolysis of α1 → 3 and α1 → 6 linked residues. The purified enzyme shows no activity against p-nitrophenyl-α-mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1-deoxymannojirimycin at concentrations 50-500-fold higher than required for complete inhibition of Golgi-mannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases. [ABSTRACT FROM AUTHOR]

A major inducible form of heine oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pre-treated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase. NADPH -cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 ± 0.5 μM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 ± 0.8 μM ; and the Km for NADPH was 6.1 ± 0.4 μM (all values mean ± SD, n = 3). Oxygen concentration as low as 15 μM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4: at 37 C, the apparent Vmax was 580 ± 44 nmol biliverdin · (mg protein)-1 · min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heine to non-biliverdin products and led to nearly stoichiometric conversion of heine to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin > tin protoporphyrin > zinc protoporphyrin > manganese protoporphyrin > cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 μM) (and several other porphyrins) and metallic ions (100 μM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 μM and totally at 15 μM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from eDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2. It is suggested that this region plays a critical role in heme binding and in catalyzing the heme oxygenase reaction. [ABSTRACT FROM AUTHOR]

1. Spectral changes following the addition of hydroperoxides to isolated hepatocytes and to perfused rat liver were observed. Cytochrome b5 is the major, if not the only, hemoprotein exhibiting redox changes under these conditions: cytochrome b5 is oxidized by added hydroperoxides, e.g. tert-butyl or cumene hydroperoxides. No spectral changes attributable to cytochrome b5 were obsewed with tert-butanol. 2. The effect is present also when the mitochondrial respiratory chain is inhibited by antimycin A, and it is not observable with isolated mitochondria. On the other hand, the oxidation of cytochrome b5 by hydroperoxides is readily demonstrable in microsomal fractions in presence of NADH. 3. Spectral evidence for a participation of the other microsomal hemoprotein, cytochrome P-450, in the hydroperoxide-linked effects was not obtained. Thus, in hepatocytes from phenobarbitalpretreated rats, no formation of cytochrome P-420, no displacement of a type I substrate, hexobarbital, and no major steady state redox change of cytochrome P-450 was detectable. However, when cytochrome P-450 was dithionite-reduced, an oxidation of this cytochrome occurred upon subsequent hydroperoxide addition. 4. Hydrogen peroxide addition to hepatocytes also leads to a lower steady-state degree of reduction of cytochrome b5. Evidence is provided with hepatocytes from rats pretreated with 3-amino1,2,4-triazole that H2O2 generated intracellularly, e.g. from added glycolate, also causes a detectable oxidation of cytochrome b5. 5. The mechanism of these hydroperoxide effects remains to be established, and it is not clear whether cytochrome b5 reacts directly or indirectly. However, it is suggested that these effects may be of significance for the further study of cytochrome-b5-linked metabolic pathways. [ABSTRACT FROM AUTHOR]

Six types of nuclease activities were found to be concentrated in the large granule fraction isolated from rat liver homogenates by differential centrifugation. Analysis by density equilibration shows that three nucleases are associated with mitochondria: an alkaline ribonuclease (pH optimum 8.8), an alkaline deoxyribonuclease (pH optimum 7.6) and an enzyme acting on polyriboadenylate (pH optimum 7.5). When the outer mitochondrial membrane is ruptured in hypotonic medium, the three mitochondrial nucleases are partially solubilized. Solubilization is however obtained by addition of KCl to the suspension medium. It is concluded that mitochondrial nucleases are localized in the intermembrane space but that an adsorption to the outer face of the inner mitochondrial membrane occurs in sucrose 0.25 M. The mitochondrial localization of alkaline ribonuclease, alkaline deoxyribonuclease and polyadenylate accounts for at least 80% of the activity of liver homogenate; nevertheless, an excess of these enzymes is present in the microsomal fraction. Although no definitive conclusion can be reached for the significance of this observation, it is shown by density equilibrium analysis that these nucleases are not associated either with ribosomes or with the membranes which are the major component of the microsomal fraction. [ABSTRACT FROM AUTHOR]

Embryonic and adult chicken liver galactosyltransferases behave differently on DEAE-Sepharose chromatography. After solubilization, two embryonic activities (one transfers galactose in a β1&-4 linkage to asialo-agalacto-α1-acid glycoprotein and to N-acetylglucosamine; the other transfers galactose in a β-3 linkage to asialo-ovine submaxillary mucin) elute after the bulk of the protein, and after free glucose. The same two enzymes in adults elute more rapidly, almost coincident with the bulk of the protein, and before free glucose. The difference in elution patterns occurs when the column buffer contains 0.1 M NaCl. Without salt, both embryonic and adult transferases bind to the column, but with 0.5 M NaCl, the embryonic and adult transferases elute identically, and with the bulk of the protein. After treatment with neuraminidase, the embryonic transferase activities elute significantly earlier on a DEAE-Sepharose column in the presence of 0.1 M NaCl. The embryonic forms migrate more rapidly than do the adult forms on cellulose acetate electrophoresis, but neuraminidase treatment renders both enzyme forms immobile in this system. Neuraminidase treatment also inhibits the binding of the embryonic transferases to a wheat-germ-agglutinin-Sepharose column. Kinetically, the embryonic and adult transferases are indistinguishable. [ABSTRACT FROM AUTHOR]

Possible mechanisms are evaluated for the acute regulation of the hamster brown-fat mitochondrial proton conductance pathway which is active during non-shivering thermogenesis. Isolated mitochondria are incubated under conditions designed to approximate to the non-thermogenic state, and the effect of the steady infusion of fatty acids or acyl derivatives upon respiration, membrane potential and membrane proton conductance is monitored continuously. Fatty acids increase the proton conductance with no detectable threshold concentration, allowing the generated acyl carnitine to be rapidly oxidized. The extent of depolarization and of respiratory increase is a function of the rate of infusion. Immediately infusion is terminated the conductance decreases, the mitochondria repolarize and respiration returns to the initial rate. Infusion of acyl-CoA and acylcarnitine cause only a slight depolarization or respiratory increase after high concentrations of these derivatives have accumulated. Any factor which decreases the rate of conversion of fatty acid to acyl-CoA potentiates the conductance increase. An effect of acyl-CoA upon chloride permeability is not specific to brown-fat mitochondria. Fatty acids infused into rat liver mitochondrial incubations produced a small conductance increase, comparable to that of acyl-CoA or acylcarnitine. It is concluded that fatty acids are the most plausible acute regulators of the proton conductance. The relation to the brown-fat-specific 32000-Mr protein is discussed. [ABSTRACT FROM AUTHOR]

Addition of ammonium ions to isolated rat hepatocytes stimulated the rate of synthesis of pyrimidines. Isolation and quantification of pyrimidine nucleotides, orotic acid an the acid-hydrolyzed product of carbamoylaspartic acid by non-exchange chromatography and high-pressure liquid chromatography show a marked simulation in the incorporation of [14C]bicarbonate in incubations with added ammonium ions. The incorporation into total uridine nucleotides (σUMP) was increased twofold in the presence of 5 mM ammonium ion, and approximately eightyfold into orotic acid. There was a parallel increase in labelling a carbamoylaspartic acid from undetectable to a level similar to that of orotic acid. The specific activity of urea formed during the incubations did not change during incubations or in the presence of ammonium ions confirming that the change in labelling of pyramidine was not due to a change in the specific activity of precursor. Despite the stimulation in incorporation of label into pyrimidines, there was no increase in the hepatcyte content of σUMP, which was 11.5 µmol/g dry weight, although the orotic acid content increased from 0.09 µmol/g dry weight in the absence of added ammonium ions (but in the presence of 2 mM L-glutamine) to 8.6 µmol/g dry weight with 5 mM ammonium ion. The stimulated incorporation of [14C]becarbonate in the presence of 5 mM and 10 mM ammonium ion was shown to be due to a stimulated synthesis of carbamoyl phosphate, since greater than 80% of label in the uracil ring was present at position C-2. Incubation of hepatocytes in basal medium (Eagles) containing 2.5% foetal calf serum and 20 mM bicarbonate showed that there was a significant stimulation of pyrimidine synthesis with 1 mM ammonium completely reversed by 5 mM L-ornithine and was partially reversed by 1 mM L-ornithine. Evidence for a contribution of the urea cycle carbamoyl phosphate synthetase to pyrimidine synthesis is discussed. [ABSTRACT FROM AUTHOR]

Characterization of polysomes from estrogenized chicken liver revealed that very-low-density lipoprotein II (VLDL&subII, serum albumin and vitellogenin mRNAs are differently packed with ribosornes during translation in vivo. The ribosome density per number of nucleotides is high for VLDL&subII mRNA. intermediate for serum albumin mRNA and low for vitellogenin mRNA. This difference in ribosomal load is maintained throughout the period of hormone effect. The differential utilization observed for vitellogenin and VLDL&subII mRNAs partly explains the large difference in molar production rate between these yolk protein precursors. Translation properties and efficiency of the three hepatic mRNAs were also compared in the mRNA-depleted reticulocyte lysate. Elongation of the nascent chains for vitellogenin and serum albumin proceeded in a discontinuous fashion. Initiation in vitro was studied at varying ionic strengths., in the presence of aurintricarboxylic acid, and at suboptimal hemin concentrations. VLDL&subIImRNA expression is by far the most resistant to 7-methylguanosine 5'- triphosphate (m[sup7]GTP) and high salt concentrations, vitellogenin mRNA the least. This behavior resembles the differential utilization of the mRNAs in vivo. The putative structural basis of these differences is discussed. [ABSTRACT FROM AUTHOR]

The results of steady-state kinetic measurements on the initial rate of the peroxidatic reaction between β-NAD+ and hydrogen peroxide, catalyzed by horse liver alcohol dehydrogenase, at pH 7 are described. A simple sequential mechanism is deduced from graphical analysis of the data plotted according to Eadie-Augustinsson-Hofstee primary plots and the values of the true kinetic parameters KmNAD, KmH2O2 and V are estimated from the corresponding secondary plots. Ethanol has been found to compete with hydrogen peroxide for the same enzyme active site. During the catalytic process a progressive inactivation of the enzyme occurs caused by H2O2. The rate law of this process is quantitatively described at pH 7 both in the absence and in the presence of NAD+. The coenzyme has been found to protect the enzyme against inactivation by H2O2, which oxidized essential cysteine residues. The results obtained from the study of both catalytic and inactivating processes are finally rationalized on the basis of a general mechanistic scheme. [ABSTRACT FROM AUTHOR]