Studies on protein biosynthesis in cell-free systems from vicia faba (l)

Abstract

Selected aspects of the mechanism of protein synthesis on plant 80S ribosomes were investigated using amino acid incorporating systems isolated from the plumules of germinated seeds and the developing cotyledons of Vicia faba (L). Complete and transfer amino acid incorporating systems were characterised using ribosomes and enzyme fraction prepared in a variety of ways. The soluble fraction obtained from the developing cotyledons was partially resolved by chromatography on Sepharose 4B into two complementary fractions required for peptide chain elongation. Chromatography on Sephadex-G200 failed to resolve the enzyme fraction into complementary fractions. Further purification of the partially resolved fractions was eliminated by the extreme lability of the fractions. An attempt was made to demonstrate the synthesis of the storage globulin, legumin, by a microsomal system from 60 day developing cotyledons. In addition to the expected [(^35)S] methionyl tryptic peptides a number of additional fragments were obtained suggesting that peptides other than the constituent peptides of legumin were synthesised in the endogenous mRNA directed system. The possible nature of these products is discussed. Chromatography on BD-cellulose or DEAE-cellulose was used to resolve the tRNA(^MET) species present in the total tRNA from developing cotyledons. In both cases two major tRNA(^MET) species were resolved, one of which appeared to function as a chain initiator as demonstrated by data from AUG dependent binding, the AUG dependent reaction with puromycin, and N-terminal analyses of the products of Poly(AUG), Poly(UG) or endogenous mRNA directed incorporation. In addition BD-cellulose chromatography partially resolved a third tRNA(^MET) species probably equivalent to the initiator tRNA of cell organelles. Although this system contains an active transformylase activity, the cytoplasmic initiator tRNA is not formylatable in contrast to that of animal and microbial systems.