PARALLEL DETECTION OF NA,K-ATPASE ALPHA-SUBUNIT ISOFORMS BY PAN-SPECIFIC MONOCLONAL MAB 9A7

While emphasis has been placed upon those proteins which either mediat e or respond to the rapid influx of calcium following depolarization, there has been little emphasis upon those proteins which aid in the re equilibration of the membrane potential, In an effort to identify pres ynaptic membrane proteins implicated in neurosecretion, monoclonal ant ibodies were screened against proteins which cosegregated with neurona l voltage-dependent calcium channels (VDCC) following immunoprecipitat ion. One monoclonal antibody (mAb 9A7) identified a 110-kDa protein, M icropeptide sequencing of (i) the mAb 9A7 immunoaffinity purified anti gen and (ii) the 110-kDa protein present in the neuronal (N-type) VDCC preparation (McEnery et al., 1991, Proc, Natl, Acad, Sci, 88, 11095-1 1099) indicated identity with the alpha subunit(s) of the Na,K-ATPase. Further characterization by Western blotting, immunochemical localiza tion, and immunoaffinity purification indicated that mAb 9A7 not only recognized the alpha3 isoform which is predominant in neuronal tissues but also identified the alpha1 and alpha2 isoforms, mAb 9A7 exhibited a wide cross-species reactivity and recognized human, rat, and mouse alpha subunit isoforms at an internal epitope, The pan-specificity of mAb 9A7 and the differential mobility of the alpha1 isoform relative t o the alpha2 and alpha3 permitted parallel detection of multiple alpha isoforms. Western blot analysis of undifferentiated rat pheochromocyt oma cell line (PC12) and human neuroblastoma (IMR32) cells indicated c oexpression of the alpha1 and alpha3 isozymes, Upon differentiation of IMR32 cells by dibutrylyl-cAMP, a substantial increase in the alpha3 relative to the alpha1 isoform was observed, While the enrichment of t otal Na,K-ATPase may reflect the increased demand for ATP-dependent io n transport as IMR32 cells become more excitable, the specific increas e in the alpha3 isoform suggests a unique role of this isoform during IMR32 cell differentiation. (C) 1997 Academic Press.