Phosphorylated intermediates of two Ca++-ATPases in membrane preparations from lens epithelial cells

By incubating preparations enriched in membranes from lens epithelial cells with [gamma 32P]-ATP and Ca++ at 0 degrees C for 15 seconds followed by SDS-PAGE analysis, it was possible to demonstrate a Ca++-dependent [32P]-phosphate incorporation in two polypeptides with Mr 105,000 and 140,000. Treatment of phosphorylated preparations with 0.06 N hydroxylamine at pH 5.4 and 25 degrees C removed the label from both polypeptides indicating that the phosphate was attached to the proteins by an anhydride linkage characteristic of the phosphorylated intermediates of the ATPases. Membrane preparations from sarcoplasmic reticulum and red blood cell studied under the same conditions showed a Ca++-dependent [32P]-phosphate incorporation into polypeptides with Mr 105,000 and 138,000, respectively, corresponding to the phosphorylated intermediates of the Ca++-ATPases present in these preparations. The results suggest the presence of two Ca++-ATPases in lens epithelial cells which, in terms of Mr, appear to be similar to those present in the sarcoplasmic reticulum and the red blood cell plasma membrane, respectively.