Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

Abstract

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of (14)C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of (14)C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105 degrees for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[(14)C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.

Evidence for the absence of the terminal adenine nucleotide at the amino acid-acceptor end of transfer ribonucleic acid in non-lactating bovine mammary gland and its inhibitory effect on the aminoacylation of rat liver transfer ribonucleic acid

“…It has been shown however that the pH 5 enzyme fraction prepared from non-lactating bovine mammary gland is incapable of forming aminoacyl-tRNA complexes (Herrington & Hawtrey, 1969a). This enzyme fraction was also found to inhibit markedly the aminoacylation of tRNA in the rat liver system as well as the transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in rat liver polyribosomes.…”

Incorporation of 14C-Leucine into Proteins of the 25,000g Supernatant of the Aorta

“…The shape of the reaction velocity curve is different from that of hyperbolic curves which usually are seen in purified systems (1,5,6,10,13). This may be attributed to hydrolysis by proteolytic enzymes present in crude enzyme preparations (14) used in experiments pre sented here.…”

Effects of Fasting on the Activity of Cytosol Factors Engaged in Protein Biosynthesis in Mouse Hepatocytes

“…1). When polysomes derived from livers of mice fed ad (12). 2 Radioactivity measured according to (15).…”

“…Acylation o f tRNA in vitro Acylation of tRNA was measured by a method based on that of Herrington and Hawtrey (12). The incubation mixture does not include ribosomes, and so is not capable of synthesiz ing protein.…”