Human lens fiber cell plasma membranes. I. Isolation, polypeptide composition and changes associated with ageing

Abstract

A detailed methodology is offered for the bulk isolation of human lens fiber cell plasma membranes. The human lens fiber plasma membrane fraction is isolable as the water-insoluble and urea-insoluble but detergent-soluble material, under reducing conditions. Ultrastructural characterization confirmed the homogeneity of this fraction. Biochemical, immunological and electrophoretic analyses were conducted upon the isolated fiber plasma membranes. Analyses were conducted of whole-lens, cortical and nuclear fiber plasma membranes from fetal, newborn, 30–50, 50–60, and 60–80-year-old normal human lenses. The fiber plasma membrane fraction remained a relatively constant 0·9% of the fresh (wet) weight of the human lens throughout lifespan; the protein-lipid ratio was determined as 1:1·2 with a slight increase in the lipid factorial of older lenses (50 years and older). Thirteen (13) polypeptides, ranging in molecular weight from 12–235 kilodaltons were resolved electrophoretically for the membranes. The phospholipid-containing 25 and 27 kilodalton polypeptides were found to constitute the main intrinsic protein of the human lens fiber membranes. α-Crystallin polypeptides (20 and 22·5 kilodaltons) were consistently recovered from the membranes, and their presence confirmed immunologically only following total detergent solubilization of the membranes. Reactions of an antiserum to the main intrinsic protein of chick lens fiber membranes and the human lens membranes were negative. The major age-dependent change in the protein composition of whole-lens human fiber membranes consisted of a gradual reversal in the preponderance of the 27 kilodalton polypeptide prenatally (and at birth) by the 25 kilodalton polypeptide as the main intrinsic polypeptide of the membranes postnatally. Additional changes comprised a gradual increase in the weight fraction of the 12 kilodalton polypeptide in the whole-lens fiber membranes throughout lifespan, and a gradual decrease of the α-crystallin content of cortical fiber membranes of lenses 50 years and older.