Biochimica et Biophysica Acta (BBA) - Specialized Section on Nucleic Acids and Related Subjects最新文献

We tested the ability of a number of synthetic pyrimidines to inhibit degradation of uracil and thymine by rat-tissue supernatants. Among nine tissues tested, only liver preparations showed significant pyrimidine-degrading activity, and among 46 compounds tested, only 5-substituted uracils, analogs of thymine, showed appreciable inhibition of pyrimidine degradation. Despite their structure, all active compounds were more effective inhibitors of uracil than of thymine degradation; similarly, uracil and thymine showed reciprocal inhibition, but thymine was considerably more effective. It was concluded that inhibition occurred only during the initial reductive step in pyrimidine degradation, that reduction of both uracil and thymine was catalyzed by the same enzyme (dihydrouracil dehydrogenase (4,5-dihydrouracil: NADP oxidoreductase, EC 1.3.1.2)), and that inhibition resulted from substrate competition for the active site on the enzyme.

Leaves of Brassica pekinensis plants contain 83-S ribosomes which occur free in the cytoplasm rather than in chloroplasts. A 68-S class of ribosomes present in 20–35% the amount of the 83-S, occurs largely if not entirely in chloroplasts. Both classes may occur as polyribosomal aggregates which can be temporarily preserved in leaf extracts using polyvinyl sulphate as a nuclease inhibitor. The major environmental influence on polyribosome levels in leaves is a diurnal cycle in which light is the predominant factor. Polyribosomes decrease in amount during the night and reach their lowest level before dawn. 2 or 3 h after sunrise 80–90% of the 83-S ribosomes may be in polyribosomes. If the natural dark period is extended for 2–3 h no polyribosomes can be detected. When such plants are exposed to sunlight a rise in polyribosomes and a drop in 83-S ribosomes can be detected within 4 min. This light-dependent increase in polyribosomes appears to be associated, at least in part, with the synthesis of new RNA.

• 1.

  1. Addition of the valine antagonist, α-amino-β-chlorobutyric acid, to rabbit reticulocytes produces a rapid inhibition of hemoglobin synthesis with a subsequent increase in labelling of protein of the ribosomal fraction.

• 2.

  2. At appropriate antagonist: valine ratios, the incorporation of valine itself occurs at the ribosomal level, but is inhibited in hemoglobin. This indicates that the block in hemoglobin synthesis is caused by substitution of the analogue for valine at specific valine sites, and it is suggested that the loci of these substitutions may be the Val-Val residues on the precursor protein of the β-chain.

• 3.

  3. The antagonist also inhibits [¹⁴C]isoleucine incorporation into hemoglobin, but unlike other amino acids, isoleucine does not accumulate in protein of the ribosomal fraction. This observation is discussed in relation to the characteristics of isoleucine incorporation into reticulocyte proteins and the location of residues of this amino acid near the NH₂-terminal end of the α-chain of rabbit hemoglobin.

• 4.

  4. Synthesis of both the α- and β-chains of hemoglobin is blocked by the antagonist and there is no evidence for the preferential synthesis and release of either chain.

• 5.

  5. Abortive protein formed in the presence of the antagonist is rapidly degraded into trichloroaceeric acid-soluble products.

11 Aminoacyl RNA's of Escherichia coli were prepared using the homologous (E. coli) aminoacyl RNA synthetases (amino acid-RNA ligases). Their chromatographic behavior was compared to those aminoacyl RNA's prepared using three heterologous (mouse liver, maize, Neurospora crassa) synthetases. In some cases the products of two enzyme preparations with a single amino acid and a single s-RNA mixture are chromatographically separable, indicating a chemical difference in the s-RNA molecules selected by the enzymes from different sources.

The relative order of chromatographic mobilities of the 11 aminoacyl RNA's from each organism, homologously aminoacylated, were shown to vary.

The administration in vivo of 3-methylcholanthrene (MC) increases microsomal and ribosomal amino acid incorporation in vitro in rat liver. The microsomal amino acid incorporating system from MC-treated rats has the same cofactor requirements as does the normal preparation. Both preparations exhibit optimal incorporation at the same concentration of MgCl₂, GSH, and GTP and both preparations are equally sensitive to puromycin inhibition. The MC-induced increase in amino acid incorporation is due at least in part to an increase in the number of active microsomal incorporation sites and an apparent increase in the messenger-RNA content of the microsomes. The rate of loss of messenger RNA during a preincubation is the same in normal and MC microsomes. After removal of messenger RNA by preincubation, l-[¹⁴C]phenylalanine incorporation is completely dependent on added polyuridylic acid. In the presence of saturating levels of polyuridylic acid, the preincubated MC microsomes incorporate greater amounts of l-[¹⁴C]phenylalanine than do preincubated normal microsomes.

The MC effect on amino acid incorporation is paralleled by an MC-induced increase in the activity of liver benzpyrene hydroxylase. Administration of actinomycin-D prevents the stimulatory effect of MC on microsomal amino acid incorporation and on enzyme activity. The possible relationship between the effects of MC on the enzyme forming system and carcinogenesis are discussed.