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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.

This paper describes the fragments obtained by digestion with RNAase T1 from rat liver serine-tRNA1 as well as from a mixture of very lipophilic serine-tRNAs. From the data obtained by these digest as well as from the digestion with pancreatic RNAase the nucleotide sequence of the anticodon of serine-tRNA1 can be constructed. Evidence from different nucleotide sequences in other serine-tRNAs is presented [ABSTRACT FROM AUTHOR]

1. Tryptic digest, s of the [14C]carboxymethylated derivatives of the t, hree isoenzymes EE, ES and SS of horse liver alcohol dehydrogenase have been compared in "fingerprint" experiments. 2. Eight peptide spots present in the digest of the carboxymethylated EE enzyme were not detected in the digest of the carboxymethylated SS enzyme; and seven spots found in the latter were not discovered in the former. No other differences were noticed. The ES derivative yielded both types of spots but in reduced amounts, It is concluded that the E- and S-types of subunits are very similar and that the ES isoenzyme is a hybrid molecule. 3. From the carboxymethylated SS and ES isoenzymes the S-chain peptides that, differ from their counterparts in the E-chain were prepared and their structures analysed. They were compared to the known structures [ 1 — 3] of the corresponding E-chain peptides. It is concluded that all the differences between the two sets of peptides are accounted for by amino acid changes at only six positions along the protein chains of the E- and S-types of subunits, and an ancestral geneduplication is suggested. The differences at five positions (17, 94, 101, 110 and 366) are amino acid exchanges compatible with one-base mutations, while the nature of the sixth difference (position 115) is not fully established. 4. The six differences make the S-chain more hydrophobic and three units of charge more positive than the E-chain. These properties fit the solubilities and electrophoretic mobilities of the three isoenzymes. The difference in substrate specificity between the E- and S-chains might be explained by a direct participation in the substrate binding site of some of the residues exchanged, but, other explanations cannot be excluded. [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]

Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms. After 2500-5000-fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl-cellulose columns and were purified 2500-5000-fold, 800-1000-fold and 600-1000-fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose-lysine as a competitive inhibitor of arginase, shows only one main form, however. Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea, the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin-layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants. [ABSTRACT FROM AUTHOR]

The object of this paper is to analyse the effects of the coenzymes NAD(P)+ and NAD(P)H, and the effectors ADP and GTP, on the polyhexameric structure of pig-liver dehydrogenase. The linear polymerisation model proposed by Eisenberg for the native quaternary structure of this protein is valid with any effector; the observed variations of the degree of polymerization are explained by the modification of the apparent association constant of the hexamers. The appendices I and II define the free and associated areas and give, the theoretical foundations of the variation of the association constant of hexamers in terms of the binding of the ligands to the protemers. The increase in the degree of polymerization of the glutamate dehydrogenase with the binding of NAD(P)H is explained by a higher affinity of the coenzymes for the protomers which have an associated area compared to the protomers which have a free area. No variation is observed with NAD(P)+, ADP, or GTP alone. The formation of the protein · GTP · NAD(P)H ternary complex leads to a complete depolymerization when the two ligands are in saturating concentrations. The systematic study of the variations of polymerization in terms of increasing concentration of NAD(P)H at constant concentration of GTP, or in terms of increasing concentration of GTP at constant concentration of NAD(P)H shows that the interaction between the two opposite protomers of two consecutive hexamers is responsible for the sigmoidal shape of the depolymerization curves. The reversibility of this effect by ADP is assigned to a competition between the binding of ADP and the binding of GTP. [ABSTRACT FROM AUTHOR]

An enzyme, exhibiting NAIDPH-dependent oxidoreductase activity towards alicyclic ketones has been extracted from pig liver and purified 122-fold with respect to the protein contained in the crude extract after centrifugation at 54000×g. General properties, ultraviolet spectrum, stability, kinetic constants (V and Km) for NADPH and for typical substrates are reported. The molecular weight of the enzyme was estimated at 500000 by gel-filtration. The enzyme is HS(HB)-specific with respect to coenzyme. Alicyclic ketones can be conveniently used to measure the activity at all stages of purification. The topography of the active site responsible for oxidoredutase activity has been investigated by use of rigid alicyclic ketones such as trans-decal-1-ones as probes. In the accompanying paper it is shown (1) that the biological function of the whole enzyme complex is that of a fatty acid synthetase and (2) that the oxidoreductase activity can be ascribed to its 3-oxoacyl-acyl-carrier protein reductase component. [ABSTRACT FROM AUTHOR]

This paper deals with investigations in isolated perfused rat livers on tryptophan-oxygenase a under various experimental conditions. Enzyme-activity Showed a linear rise with amounts of tryptophan (0; 125 and 250 mg of tryptophan/kg) in the perfusate. Adrenalectomized and sham-operated animals have been compared and activity in the adrenalectomized rats were significantly lower in all cases. A significant decrease in the substrate induced increase of tryptophan-oxygenase-activity occured 12 h after adrenaleetomy. Substrate-concentration was 250 mg/kg in these experiments. The influence of substrate and cortisol on tryptophan-oxygenase-activity has been investigated 7 days after operation, Combined application of both substrate and steroid resulted in no difference to sham-operated animals when compared to substrate only in the same concentration. On the other hand, in livers from adrenlectomized rats values were significantly higher with combined application than in livers of adrenalectomized animals which received substrate only. The substrate-dependent rise in trsyptophan-oxygenase-activity could not be influenced by actinomycin D in contrast to to he cortisol effect which was significantly inhibited under these conditions. Both "inducers" were inhibited by cycloheximid. These inhibition-experiments suggest and confirm two different mechanisms of the substrate-activation and steroid-induction of tryptophan-oxygenase. [ABSTRACT FROM AUTHOR]

This paper reports the partial purification and characterization of a new type of arginase from chicken liver. The enzyme can be demonstrated only in less than 10% of a fasting chicken population, and has never been found in fed animals. The new arginase differs with respect to several properties (chromatographic behaviour, sedimentation coefficient, Km for arginne) from the arginase normally found in chicken liver and is more like the ureotelic arginase isolated from rat liver. [ABSTRACT FROM AUTHOR]

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.

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]

Smooth and rough endoplasmic reticulum and free ribosomes from rat liver each show cyclicAMP-stimulated protein kinase activity utilizing exogenous substrates. The protein kinases of smooth and rough endoplasmic reticulum can be divided into two classes, one which is extracted by ionic media, and one more firmly attached enzyme fraction which is solubilized by Triton X-100. The protein kinase of free ribosomes is extracted by ionic media. All the three microsomal fractions support an endogenous phosphorylation of proteins which is only slightly stimulated by cyclic AMP. The endogenous phosphorylation shows two pH optima at pH 6.5 and 8.5. The phosphate is incorporated into seryl and threonyl residues of several protein species. Two major phosphoproteins are present in both smooth and rough endoplasmic reticulum, while a third major phosphoprotein is present only in the smooth fraction. There are also several minor phosphoproteins in the two fractions. The endogenous phosphorylation is initially rapid, especially in smooth and rough endoplasmic reticulum where it reaches a maximum after 10—15 rain incubation. The endogenously phosphorylated microsomal fractions also support an endogenous dephosphorylation, which is rapid initially, but which leaves approximately 60% of the phosphoryl groups unhydrolyzed. Like the protein kinase the proteins of smooth and rough endoplasmic reticulum which can undergo endogenous phosphorylation can be divided into two classes, one which is extracted in ionic media and one more tightly bound which is solubilized by Triton X-100. Protein kinase, cyclic-AMP-binding material and protein substrates extracted from smooth and ruogh endoplasmic reticulum by salt or detergent all show recoveries substantially exceeding 100%, suggesting that these activities are partly masked while associated with membrane material. [ABSTRACT FROM AUTHOR]

The rates of synthesis and degradation of glucose-6-phosphate dehydrogenase in the liver of male C57BL mice are followed by a modified immunochemical method. Mice are given labeled amino acids for a predetermined period, and the labeled enzyme is isolated from liver homogenates with goat anti-enzyme, followed by rabbit anti-goat IgG serum. The precipitates are either counted directly or analyzed on acrylamide gels. The rate of glucose-6-phosphate dehydrogenase synthesis is found to be independent of the state of induction or repression of the animal. The synthesis of the enzyme constitutes 0.20-0.25% of the synthesis of all soluble liver proteins. This rate is maintained even during periods of most rapid formation or disappearance of enzyme activity. Quantitative immunoprecipitation shows that similar amounts of precipitable antigen are present in induced and non-induced liver homogenates. The results indicate that the large reversible increase in activity of glucose-6-phosphate dehydrogenase, observed when animals are transferred from a high-fat to a fatless diet, does not involve the synthesis of a new enzyme protein, and that mechanisms involving modulation of existing enzyme molecules ought to be considered for the adaptation of this enzyme in mouse liver.

A simple optical method for the assay of Δ4-3β-hydroxysteroid dehydrogenase activity in cell-free preparations of rate liver was designed. This test is based on the fact that under the chosen conditions no reduction of the Δ4-bond occurs. Thus the Δ4-3β-hydroxysteroids dehydrogenase activity can be directly measured by the production of Δ4-3-ketosteroids, the sum of which in turn may be quantitated by their isonicotinic acid hydrazone formation. Δ4-3β-Hydroxysteroid dehydrogenases are localized in both cytoplasmic and microsomal fractions, exhibiting higher activity with NAD than with NADP. Microsomal enzyme activities show a significant sex difference which is more pronounced with NADP as coenzyme than with NAD (male:female activity ratios 5.8 with NADP, 1.9 with NAD). No sex differences were observed in the cytoplasmic enzyme activity. These results indicate the existence of two NAD-dependent enzyme activities of which the microsomal activity shows sexual differences. On the basis of the male:female activity ratios a further separate NADP-dependent microsomal enzyme activity can also be distinguished. [ABSTRACT FROM AUTHOR]

This paper describes the fragments obtained by digestion with RNAase T1 from rat liver serine-tRNA1 as well as from a mixture of very lipophilic serine-tRNAs. From the data obtained by these digest as well as from the digestion with pancreatic RNAase the nucleotide sequence of the anticodon of serine-tRNA1 can be constructed. Evidence from different nucleotide sequences in other serine-tRNAs is presented [ABSTRACT FROM AUTHOR]