Murine mammary tumor virus (MuMTV) labeled in vivo with [32P]orthophosphate and 3H-amino acids was analyzed on cylindrical sodium dodecyl sulfate (SDS)-polyacrylamide gels. Of all the MuMTV polypeptides (gp47, gp34, p27, p23, p16, and p12), only two, p23 and p27, appeared to co-migrate with labeled phosphate. The MuMTV structural proteins were fractionated by hydrophobic chromatography (which completely separates p23 from p27) and the resulting fractions were analyzed by both cylindrical and slab gel SDS-polyacrylamide gel electrophoresis to determine the 32P label associated proteins and for the resistance or sensitivity of the label to RNase and protease. The results of these studies indicated that both p23 and p27 contained phosphate. Fractionation of MuMTV polypeptides performed in an A 5m agarose column in the presence of guanidine-HCl yielded homogenous preparations of p27, which was also found to be associated with phosphate. The major phosphoamino acid of both p23 and p27 was found to be phosphoserine. Immunoprecipitation with monospecific anti-p27 serum showed that p23 is antigenically unrelated to p27. Thus, it appears that MuMTV contains two distinct phosphoproteins. A comparison of the 32P and 3H labels of both p23 and p27 showed that more phosphate per amino acid residue is associated with p23 than with p27, suggesting that p23, although a minor polypeptide, is the major phosphorylated component of the virus.
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