Antigenic reactivity of ribosomal protein S6 and the calcium-binding ATPase inhibitor protein of mammalian mitochondria.
Phosphorylation of ribosomal protein S6 of mammals precedes activation of cell growth in numerous biological systems. We have cloned a cDNA for ribosomal protein S6 from T-47D human breast cancer cells by immunoscreening a lambda gt11 expression library with antibody raised against the mitochondrial Ca(2+)-binding ATPase inhibitor protein (CaBI) of bovine heart mitochondria (Yamada & Huzel: J Biol Chem 263: 11498-11503, 1988). Similar clones were obtained by the immunoscreening of a rat heart expression library. In agreement with others, the open reading frames of the cDNAs from the two species coded for the same amino acid sequence. No difference in S6 of the human neoplastic cells compared to that of non-neoplastic cells was found. However, common antigenic determinants in S6 and CaBI were indicated. Accordingly, S6 was purified from rat liver ribosomes and antiserum prepared. Immuno-dot blot and Western blot analyses showed high specific reactivity between S6, the cloned chimeric beta-galactosidase fusion protein from a cDNA clone, and CaBI with anti-S6 and anti-CaBI antibodies. The antibodies also showed a high degree of discrimination for S6 and CaBI. Neither interacted with the other ribosomal proteins nor with another ATPase inhibitor protein from bovine heart mitochondria. Neither interacted with the Ca(2+)-binding proteins, calmodulin, oncomodulin, Protein C, or Factor X. Prothrombin was weakly reactive with anti-CaBI but not with anti-S6. Thus, the results fulfill the specific criteria for the concept and operational definition of common protein epitopes in S6 and CaBI. However, neither prothrombin nor S6 fusion protein inhibited mitochondrial ATPase activity even at 20 times the concentrations at which CaBI gave 97% inhibition.
Penner
C G
CG
Department of Biochemistry and Molecular Biology, University of Manitoba, Winnipeg, Canada.
Murphy
L C
LC
Huzel
N J
NJ
Yamada
E W
EW
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology; D002147:Calmodulin / Q000737:chemistry / Q000276:immunology; D008929:Mitochondria, Heart / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000235:genetics / Q000276:immunology*; D014407:Tumor Cells, Cultured; D000097795:ATPase Inhibitory Protein
D000251:Adenosine Triphosphatases; D000906:Antibodies; D002118:Calcium; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D011506:Proteins; D011993:Recombinant Fusion Proteins; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D000097795:ATPase Inhibitory Protein; C034733:oncomodulin