Further Characterization of the Phosphate Moiety of the Adenovirus Type 2 DNA‐Binding Protein

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Abstract
The adenovirus type 2 DNA-binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in MW from 72,000 to 70,000. The dephosphorylated protein binds to single-stranded and double-stranded DNA as well as does the phosphorylated protein. Controlled chymotrypsin treatment cleaves the DNA-binding protein into 2 subspecies of MW about 45,000 and 25,000. The 45,000-MW polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25,000-MW polypeptide contains all the phosphate groups and shows no binding to DNA. Isoelectric focusing gels show heterogeneity of the DNA-binding protein and 15 subspecies with different charges can be observed after partial dephosphorylation by alkaline phosphatase. After extensive dephosphorylation, 2 or 3 basic species with a MW around 70,000 are observed. Quantitative immunoprecipitation from cells labeled to equilibrium with inorganic 32PO4 gives a molar ratio of phosphate to protein of 4-7 and direct chemical determination of the phosphate residues yields 4 mol Pi/mol protein. These results suggest that there exist subspecies of the protein moiety of the adenovirus DNA-binding protein. The DNA-binding protein isolated from infected cells after a short pulse of [35S]methionine has a MW which corresponds to that of the dephosphorylated protein. After a chase period the MW increases to 72,000, but alkaline phosphatase treatment converts it to a species with the same MW as the newly synthesized DNA-binding protein, indicating that the modification of the protein is due to phosphorylation.