UBC Theses and Dissertations
Studies on protamine Ingles, Charles James
Spermatogenesis in fish is a remarkable example of a rapid and extreme form of cellular differentiation. Protamines, the small basic proteins associated with DNA in the spermatozoa of certain fish, are perhaps the most characteristic of the sperm-specific macromolecules. A study of the synthesis of these unusual proteins was initiated using suspensions of cells from the testes of both naturally maturing Pacific salmon (Oncorhynchus spp.) and immature Salmo gaird-nerii in which spermatogenesis had been induced by the injection of salmon pituitary extracts. Protamine, easily characterized by polyacrylamide gel electrophoresis, was only present in the acid extract of testis tissue at a late stage of spermatogenesis. The incorporation of arginine-H³ into protamine was studied in this testis cell suspension system. The inhibition of protamine synthesis by puromycin, cycloheximide and chloramphenicol indicated that in spite of their small size and unusual amino acid composition, protamines were synthesized by a mRNA, tRNA, ribosomal system Actinomycin D, however, did not inhibit this synthesis of protamine. Protamines from several different species of salmonoid fish were purified by a two step chromatography procedure utilizing gel filtration on Sephadex G-25 and Biogel P-10. The amino acid compositions of these protamines were compared, the N and C-terminal amino acids of the Salmo gairdnerii protamine determined, and fractionation of the components of Oncorhynchus tshawytscha (chinook salmon) protamine, by chromatography on both carboxymethyl Sephadex and alumina, attempted. During the late stages of maturation, the testis cells from S. gairdnerii, which are synthesizing protamine, have been shown to incorporate phosphate-P³² into both histone and protamine fractions. The site of this phosphorylation of protamine was identified as seryl residues, and a number of radioactive phospho-peptides were isolated from the tryptic digest of protamine-P³². The major phospho-peptide was identified as val-serP-arg; however phosphorylation of other serine sequences in protamine was indicated by the presence of the peptides (ala,serP,arg)arg, (ser,serP)arg and arg-serP-serP-arg. This phosphorylation of protamine proceeded independently of the amino acid incorporation during synthesis of protamine; however, parallel incorporation of phosphate-P³² and serine-C¹⁴ into protamine showed that all newly incorporated seryl residues were phosphorylated. Since the total content of o-phosphoserine in protamine fell during the later stages of testis maturation, a rapid turnover of phosphate in protamine was indicated. This view was confirmed by the almost complete absence of phosphate in protamine prepared from mature spermatozoa of naturally spawning salmonoid fish. The phosphorylation of protamine may play a role both in the synthesis and intracellular transport of protamine, and in the control of gene expression in these testis cells.
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