UBC Theses and Dissertations
Biosynthesis of protamine in trout Salmo gairdnerii testis Ling, Victor
At a late stage of spermatogenesis a sperm-specific protein, protamine, is synthesized in the testis of salmonid fish and progressively replaces histones in combination with DNA. Protamine has a molecular weight near 5,000 and contains 2/3 of its total amino acid residues as arginine. Studies on the biosynthesis of protamine have been made on the rainbow trout (Salmo gairdnerii) testis. Successive column chromatography on Bio-Gel P-10 and CM- cellulose has been employed to isolate and characterize newly synthesized labelled protamine. Newly synthesized protamine is phosphorylated and is eluted earlier from the CM-cellulose column than mature protamine. However, the two forms of protamine chromatograph coin-cidentally when newly synthesized protamine is first treated with alkaline phosphatase. Protamine is separated into three components on CM-cellulose and the amino acid compositions of the components are very similar. The relative amounts of the components present in the testis nuclei are different at different stages of spermatogenesis and the synthesis of each component appears to be independently controlled. This suggests that the components, while chemically very similar, are the products of separate structural genes and may have different functions. By pulse labelling testis cell suspensions for different lengths of time and analyzing the amount of ¹⁴C- protamine found in the cytoplasm and in the nucleus, the site of protamine synthesis can be shown to be in the cytoplasm. Further, a cell-free, isolated post-mitochondrial cytoplasmic fraction can incorporate ¹⁴C-arginine into whole protamine molecules, while both an isolated nuclear fraction and high-speed supernatant were relatively inactive. This indicates that protamine synthesis occurs on cytoplasmic microsomes. Sedimentation analysis of pulse-labelled testis ribososes indicates that protamine is synthesized on a class of small polysomes, the disomes, sedimenting at 120S. While dimeric ribosomes investigated in various tissues have been shown to be inactive artefacts formed during isolation, the disomes in trout testis have been demonstrated to be a functional class of polysomes. They are not dissociable at 1 mM Mg⁺⁺ ion concentration, are not the breakdown product of larger polysomes, nor are they produced by interaction with free protamine. These disomes contain the major quantity of nascent protamine and increase in number in the testis cells during the active protamine synthesizing stage of development. The probable function of protamine is for the packaging of DNA into the sperm head. The phosphorylation of protamine and the protamine components may serve to regulate this packaging process.
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