Further characterization of the phosphate moiety of the adenovirus type 2 DNA-binding protein.
The adenovirus type 2 DNA-binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in molecular weight from 72000 to 70000. The dephosphorylated protein binds to single-stranded and double-stranded DNA as well as the phosphorylated protein does. Controlled chymotrypsin treatment cleaves the DNA-binding protein into two subspecies of Mr about 45000 and 25000. The 45000-Mr polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25000-Mr 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 two or three basic species with a molecular weight around 70000 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 molecular weight which corresponds to that of the dephosphorylated protein. After a 'chase' period the molecular weight increases to 72000, but alkaline phosphatase treatment converts it to a species with the same molecular weight as the newly synthesized DNA-binding protein, indicating that the modification of the protein is due to phosphorylation.
D000260:Adenoviruses, Human / Q000032:analysis*; D000469:Alkaline Phosphatase; D002918:Chymotrypsin; D004247:DNA; D004265:DNA Helicases / Q000302:isolation & purification; D004277:DNA, Single-Stranded; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification; D010727:Phosphoric Diester Hydrolases; D011863:Radioimmunoassay
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D010750:Phosphoproteins; D004247:DNA; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D002918:Chymotrypsin; D004265:DNA Helicases