Histone Kinase Activity of Buffalo Sperm Chromatin

Abstract

Phosphorylation of buffalo sperm chromatin proteins under optimum conditions (8 mM Mg²⁺, pH 8.0, and at 30°C) using [γ-³²P]ATP and endogenous protein kinase activity was linear for 15 min incubation time and up to 330 μg protein. The ³²P transferred from [γ-³²P]ATP was located in protein as a phosphoester bond. Fractionation with 1.2 M NaCl-4 M urea-0.2 M 2-mercaptoethanol-l mM PMSF followed by acid treatment solubilized 87% of the total chromatin proteins termed “sperm histones.” The remaining 21% nonhistone protein was tightly bound to DNA. Follow-up of the label showed 91% of the ³²P in sperm histone and 9% with DNA-associated proteins. Histone kinase activity was solubilized with 0.35 M NaCl, which extracted 70% of the initial enzyme activity associated with chromatin. Of the different histones tested as substrates, histone kinase phosphory-lated only histone H₃ and, therefore, is highly specific for arginine rich histone. It also phosphory-lates the acidic protein, casein. Cyclic AMP at concentrations up to 50 μM had no effect on the phosphorylation of histone H, Phosphoamino acid analysis using histone H₃ as the substrate showed serine to be the acceptor amino acid for phosphoester link.

Key Words:

• arginine-rich histone (H₃)
• histone kinase
• phosphorylation of buffalo sperm chromatin
• serine residue
• sperm histone