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Antibodies against synthetic peptides corresponding to the carboxyl-terminal six amino acids, Lys-ArgSer-Arg-His-Phe (KF), and an internal region, Glu-Glu-GluGlu-Tyr-Met-Pro-Met-Glu (EE), of polyoma virus medium T antigen were used successively to purify medium T antigen by affinity chromatography. Medium T antigen from cell extracts was first bound to anti-KF antibodies and released from the immune complex with excess KF peptide; then it was bound to anti-EE antibodies and released with excess EE peptide. Two proteins, pp6OCSrC and a new protein of "61,000 Da (61-kDa protein), were copurified because they formed complexes with medium T antigen. The 61-kDa protein-medium T antigen complex was detected in extracts from wild-type-infected and transformed cells but not from cells infected with NG59 virus, which has a mutation in the medium T gene and is transformation defective. Instead, NG59 medium T antigen formed a complex with another cellular protein of =72,000 Da.The transforming protein of polyoma virus, medium T antigen, is a membrane protein (1-3) associated with a tyrosine-specific protein kinase activity (4-6). Biochemical studies have indicated that this activity is not intrinsic to medium T antigen (7,8) but instead resides in a minor fraction sedimenting with a size of 200,000 Da, whereas the bulk of medium T antigen is monomeric and kinase inactive (9). It was shown subsequently that the kinase activity is due to an association between medium T antigen and pp6O1(src, the cellular homolog of the transforming protein of Rous sarcoma virus, pp6OVSrC (10). The interaction between medium T antigen and pp60'-sr may be facilitated by the fact that both proteins are membrane associated and, therefore, come into close contact. In a similar fashion, medium T antigen might interact with other neighboring membrane proteins. We asked how one might identify such proteins without having a functional assay or a specific antiserum available. In the present paper, we demonstrate that in addition to pp60O-s>, medium T antigen also forms a complex with a cellular protein of -61,000 Da. This was achieved by affinity chromatography with two anti-peptide antibodies directed against different regions of medium T antigen.

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We have used antibodies against the synthetic peptide Lys-Arg-Ser-Arg-His-Phe, corresponding to the six COOHterminal amino acids of the polyoma virus medium tumor (T) antigen, to purify the medium T antigen by affinity chromatography. Release of the medium T antigen from the anti-peptide antibody was achieved under mild conditions by using a large excess of the peptide in an isotonic buffer at neutral pH containing mixed detergents. This procedure yielded a 2,500-fold purification of the medium T antigen in a single step. The protein kinase activity associated with the medium T antigen was also released and was studied in this active state in solution. Sedimentation analysis showed that the bulk of the purified medium T antigen was in a monomeric form (Mr about 42,000) not associated with protein kinase activity. A small fraction ofthe medium T antigen was found in a rapidly sedimenting form (Mr about 200,000) that possessed protein kinase activity.The early region of the polyoma virus genome encodes three proteins, the small, medium, and large tumor (T) antigens. The medium T antigen is of particular interest because it appears to be responsible for many of the changes in cell growth control that accompany transformation by polyoma virus (1-3). The medium T antigen is located in plasma membranes (4-6) and is associated with a protein kinase activity when isolated in immunoprecipitates (7-9). This protein kinase activity phosphorylates one or more tyrosine residues in the medium T antigen itself (9). Mutations in the medium T antigen that block cell transformation also affect the associated protein kinase activity (7)(8)(9)(10)(11). Whether the medium T antigen itselfis a protein kinase, or whether the medium T antigen is associated with a cellular protein kinase in immunoprecipitates is an important question, particularly in view of the fact that the transforming proteins of several retroviruses are tyrosine-specific protein kinases (refs. 11 and 12; for review see ref. 13).To examine the nature of the kinase activity associated with medium T antigen we have begun to purify the medium T antigen, expecting that an associated protein kinase might be removed during purification or that a homogeneous preparation of the medium T antigen might be enzymatically active. Purification by conventional procedures would be difficult because the medium T antigen represents only a small fraction (0.01-0.03%) of the total cellular protein in polyoma virus-infected cells. Consequently we have chosen the approach of affinity chromatography, using specific antibodies produced by immunization with a synthetic peptide. We previously described antibodies specific for the polyoma virus medium T antigen, produced by immunizing rabbits with the synthetic peptide Lys-Arg-Ser-Arg-His-Phe, corresponding to the unique COOH terminus of the medium T antigen (14). We now show that these antibodies can be used to purify the medium T antigen by affinity chromatography. The procedure described here should be applicable to many proteins...

The polyoma middle‐sized T antigen (MT antigen) is associated with a protein kinase activity which phosphorylates tyrosine residues in polyoma T antigens in vitro. We have studied the sites of tyrosine phosphorylation of MT antigens phosphorylated in immunoprecipitates or in soluble form after partial purification by immunoaffinity chromatography. By analyzing the amino acid sequences of tryptic peptides of MT antigen, and by analyzing deletion mutant MT antigens, we have identified two major sites of phosphorylation in MT antigen, tyrosines 250 and 315. Additional sites were phosphorylated under some conditions. A synthetic peptide (Glu.Glu.Glu.Glu.Tyr.Met.Pro.Met.Glu), corresponding to the sequence around tyrosine 315, was phosphorylated when added to immunoprecipitates containing MT antigen.