Identification of the calmodulin‐binding components in bovine lens plasma membranes

Lens membranes, purified from calf lenses, have been labeled by covalent cross‐linking to membrane‐bound 125 I‐calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated two major 125 I‐containing products of M r = 49000 and 36000. That the formation of these two components was specifically inhibited by unlabeled calmodulin, or calmodulin antagonists, would indicate that the formation of these components was calmodulin‐specific. The size of these two 125 I‐labeled components was unchanged over a range of 125 I‐calmodulin or dithiobis(succinimidyl propionate) concentrations indicating that they represent 1:1 complexes between 125 I‐calmodulin ( M r = 17000) and M r ‐32000 and M r ‐19000 lens membrane components respectively. Although formation of both cross‐linked components exhibited an absolute dependence on Mg 2+ , the autoradiographic intensity of these components was enhanced when Ca 2+ was included with Mg 2+ during the cross‐linking reaction. Labeling was maximal in 10 mM MgCl 2 and approximately 1 μM Ca 2+ . Treatment of lens membranes with chymotrypsin resulted in the cleavage of MP26 (the major lens membrane protein), with the appearance of a major proteoloytic fragment of M r = 22000. This proteolysis was not associated with any significant change in either the size or amount of the 125 I‐calmodulin‐labeled membrane components. These results suggest that calmodulin interacts with two membrane proteins, but not significantly with MP26, in the intact lens cell membrane. Our results indicate the need to maintain caution in interpreting direct calcium plus calmodulin effects on MP26 and lens cell junctions.