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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]

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 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]

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]

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]