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Comparative Studies on the Endogenous Phosphorylation of Microsomal Membrane Derived from Morris Hepatoma, Eight-Day-Old Rats, and Host Livers in the Presence of Micromolar Levels of Mg²⁺ Ion¹

McArdle Laboratory for Cancer Research, The University of Wisonsin, Madison, Wisonsin 53706

When microsomes from normal and control livers were incubated with adenosine [-³²P]triphosphate in the absence of added metal ions, two intrinsic polypeptides with molecular weights of 145,000 and 130,000 were selectively phosphorylated (K. S. Lam and C. B. Kasper, J. Biol. Chem., 255: 259–266, 1980). Microsomes derived from 8-day-old rats catalyzed the same reaction, but the extent of ³²P incorporation into the M.W. 130,000 polypeptide was greatly diminished. Similar studies using microsomes derived from various Morris hepatomas showed qualitative as well as quantitative differences. In Morris hepatomas 7800 and 5123C, two polypeptides with molecular weights of 160,000 and 145,000 were phosphorylated, while Morris hepatoma 7777 phosphorylated only the M.W. 160,000 polypeptide. With the exception of Morris hepatoma 7777, the phosphoproteins existed in the native membrane primarily as homo- and/or heterodimers joined by disulfide bridges. Using partial enzymic digestion on sodium dodecyl sulfate gels, a close sequence homology between the M.W. 145,000 and 130,000 polypeptides was demonstrated; however, no such homology was detected between the M.W. 160,000 and 145,000 polypeptides.

Time course studies showed that the reaction was biphasic for control microsomes, but, in membranes derived from 8-day-old rats and the hepatomas, the second burst of phosphorylation was absent, and overall ³²P incorporation into these polypeptide(s) was one-third to one-fifth that of the control. This study demonstrates a subtle phenotypic heterogeneity among the tumors and livers derived from 8-day-old rats and control animals. The M.W. 160,000 phosphoprotein, shared by all three tumors but not by the livers of control or of the 8-day-old rats, may be an important component of cancer.

¹ This work was supported by Grants CA-23076, CA-17300, and CA-07175 from the National Cancer Institute, NIH, United States Department of Health and Human Services.

² Present address: Stanford University School of Medicine, Palo Alto, Calif. 94304.

Received 8/18/80. Accepted 11/24/80.