Peptide mapping by limited proteolysis in sodium dodecyl sulfate of the main intrinsic polypeptides isolated from human and bovine lens plasma membranes

Abstract

The major intrinsic lens plasma membrane protein isolated from calves, young cows and young humans, is an approx. 26 000 dalton protein. In the adult human lens, a second major intrinsic protein ( M r 22 000 ) is found. The relationships between the bovine 26 000 dalton protein and the human 26 000 dalton protein, as well as between the human 26 000 dalton protein and the human 22 000 dalton protein, were examined using peptide mapping by limited proteolysis is sodium dodecyl sulfate. Purified membrane proteins were obtained by preparative sodium dodecyl sulfate gel electrophoresis. At concentrations of 1 mg/ml or more, the purified 26 000 dalton and 22 000 dalton proteins tend to aggregate in sodium dodecyl sulfate solution and form dimers, trimers and tetramers, as well as higher-order multimers. These aggregates cannot be dissociated by β-mercaptoethanol. Staphylococcus aureus protease exhibits differential proteolytic activity toward the intrinsic major plasma membrane proteins and the soluble lens proteins which are difficult to remove from membrane preparations. While the soluble lens proteins are very effectively cleaved by S. aureus into fragments of less than 12 000 daltons, both the human and bovine 26 000 dalton major intrinsic membrane proteins lost only an approx. 4000 dalton peptide. Under the same conditions, this protease did not proteolyse the human native approx. 22 000 dalton protein. The resistance to further proteolysis of the approx. 22 000 dalton proteins which were produced by the action of the protease on the human and bovine 26 000 dalton proteins, as well as the resistance to further proteolysis under the same conditions of the native 22 000 dalton human protein, suggest that all these proteins possess similar conformation and composition. Additional proof that the human 26 000 dalton and 22 000 dalton membrane proteins as well as the bovine 26 000 dalton protein are all closely related comes from the α-chymotrypsin peptide maps obtained from the purified proteins. These results suggest that the human 22 000 dalton membrane protein is a natural cleavage product of the 26 000 dalton main intrinsic plasma membrane protein.