Catalytic subunit isoforms of mammalian lens Na,K-ATPase

To identify the Na,K-ATPase isoforms present in the mammalian lens, seven antisera were prepared to selected peptide sequences of the catalytic (alpha) subunit. Three antisera were prepared to peptide sequences at the N-terminus of the three sequenced rat alpha isoforms. There is < 53% sequence homology among the isoforms in this region. Three antisera were prepared to peptide sequences at the ouabain binding site in the extracellular loop between membrane spanning sequences 1 and 2 of the sequenced rat alpha isoforms; sequence homology among the isoforms in this region is < 69%. An antiserum was also prepared to the carboxyl terminal region of the alpha 2 rat isoform. The sequenced isoforms (rat and human) in this region are > 94% homologous. The results from stains of Western blots of SDS-PAGE separations of lens membranes are presented. Alpha 1 is the predominant isoform of the epithelium. It is not found in cells of the central epithelium but is present in cells located more toward the equator. Alpha 3 is the catalytic subunit of the central 43% of the epithelium. The lens fiber cell membranes have a catalytic subunit that is related to the alpha 2 isoform. In the fiber cell a 98-100 kDa band stains with the antiserum to the alpha 2 N-terminus and the antiserum to the alpha 2 ouabain site. The antiserum to the alpha 2 C-terminus does not stain the 98-100 kDa band. (Preliminary reports of these results were presented at the 1992 and 1993 meetings of the Association for Research in Vision and Ophthalmology).