Kinetics of Enzymatic Modification of the Protamines and a Proposal for Their Binding to Chromatin

Trout testis maturation is characterized by the complete replacement of the histones by the protamines. During this process, the protamines are extensively modified by enzymatic phosphorylation of their seryl residues. In addition, a methionyl residue is transiently incorporated at the NH2 terminus of newly synthesized protamine. However, these modifying groups are removed by the time that spermatid maturation is complete. To study the kinetics of these transformations, trout testis cells were incubated with [3H]arginine, inorganic [32P]phosphate, and [35S]methionine for varying times and the labeled protamines were separated by starch gel electrophoresis. At least six modified bands (one methionyl-(P0M), three phosphoryl-(P1, P2, P3), and two phospho methionyl-(P1M, P2M) derivatives of unmodified protamine (P0)) were observed. Analysis of radioactivity showed that within 5 to 10 min, newly synthesized protamine (P0M) is phosphorylated to P1M and P2M. Methionine is then removed to form P1 and P2. Phosphorylation of P1 and P2 continues, and labeled arginine is found in P3 after 5 to 10 hours. Very little arginine label was seen in unmodified protamine (P0) in these in vitro studies. However, 5 to 10 days after injection of [3H]arginine, label was found in unsubstituted protamine (P0) derived from the sequential dephosphorylation of P3. These data suggest that pools of each modified species of protamine exist; molecules within a given pool are modified at random by the appropriate enzyme and become part of another pool. The phosphorylation and dephosphorylation of protamine appears to be unidirectional. The long lapse (5 to 10 days) between appearances of [3H]arginine in newly synthesized protamine (P0M) and in fully mature, unmodified protamine may be a consequence of the label having to pass through the various pools of protamine. The controlled phosphorylation of protamine may be important in the correct binding of protamine to DNA, while the dephosphorylation of protamine is related to the controlled condensation of the spermatid chromatin.