Using polyclonal
antibodies raised against a rat liver nuclear envelope
protein,
lamin protein A, the
nuclear matrix proteins of a Walker 256 rat mammary
carcinoma wild-type (WS) and a selected cell line with acquired resistance to
nitrogen mustards (WR) were found to possess
antigenic determinants which were recognized by the
antibodies. In one-dimensional immunoblotting analysis, the
nuclear matrix protein fractions of both cell lines revealed a common band at Mr 75,000; however only the WS
nuclear matrix protein fraction contained a broad band at approximately Mr 70,000. Two-dimensional gel blotting studies of these
proteins showed that this Mr 70,000 WS
protein had a pI of approximately 7.5. Immunoprecipitation analysis revealed that the altered mobility of this
protein could be a function of phosphorylation. The
nuclear matrix proteins from both WS and WR cells were shown to bind 3':5'-cyclic
adenylic acid (cAMP), as judged by photoaffinity labeling and gel electrophoresis studies. The WS
nuclear matrix proteins showed a quantitatively greater level of cAMP binding compared to WR, with predominant binding to
proteins with molecular weights of 45,000, 55,000, and 70,000. In WR cells, there was no cAMP binding in the Mr 70,000 region. These data indicate that the Mr 70,000 nuclear matrix
lamin proteins are antigenically similar in WS and WR but differ in that the WR
protein is hypophosphorylated and does not bind cAMP.