Biosynthesis of Protamine in Trout Testis

Abstract

Abstract A cell-free protein-synthesizing system from rainbow trout liver can initiate de novo synthesis in the presence of exogenous messenger RNA if the system is previously incubated for 60 min. Polyuridylic acid and polyguanylic uridylic acid can be used as synthetic messenger RNA to test for the ability of the system to reinitiate synthesis. Low molecular weight RNA isolated from protamine stage testis microsomes actively stimulates the incorporation of [14C]arginine and [35S]methionine into acid-insoluble material. Starch gel and polyacrylamide gel fractionation show that both isotopes are incorporated in vitro into protamine and histone. The protamine product was characterized by separately incorporating [14C]arginine, [14C]proline, and [14C]serine into in vitro synthesized material and carrying out ten sequential Edman degradations. The order of appearance of labeled amino acids during this process was consistent with the known sequence of rainbow trout protamine. Most of the in vitro [35S]methionine incorporation into protamine was found to be NH2-terminal. Electrophoresis of a tryptic digest of this material showed that methionine occurred in a basic peptide with the same mobility as that previously found (Wigle, D. T., and Dixon, G. H. (1970) Nature 227, 676) with in vivo methionine-labeled protamine. Methionine appears to be involved both in the in vitro and in vivo initiation of protamine synthesis. A substantial proportion (45%) of the [35S]methionine incorporated into histone was also found to be NH2-terminal.