Biochemistry of Bovine Lens Proteins: IV. SYNTHESIS AND AGGREGATION OF α-CRYSTALLIN SUBUNITS IN DIFFERENTIATING LENS CELLS

Abstract

Abstract The vertebrate lens is composed of two distinct cell types, the epithelial cells and the fiber cells. The absolute rate of synthesis of total protein and of purified α-crystallin, a structural polymeric protein found in both cell types, has been calculated using the kinetics of [3H]leucine incorporation into organ-cultured lenses. The data show that in adult epithelial cells about 2% of α-crystallin is renewed per day, as opposed to 0.3% in cortex fiber cells. The rate of α-crystallin synthesis is about 7 times higher in epithelial cells than in cortex fiber cells; for non-α-crystallin proteins, the corresponding ratio is approximately 24. Both epithelial cells and cortex fiber cells are shown to be highly specialized for the synthesis of α-crystallin; 60% of newly synthesized protein in adult epithelial cells is α-crystallin, whereas in cortex fiber cells the proportion is 75%. The polymeric α-crystallin has been dissociated into its component subunits and the resulting mixture of polypeptide chains has been analyzed by acrylamide gel electrophoresis. Four discrete bands are resolved by this technique, corresponding to two acidic subunits (αA1 and αA2) and two basic subunits (αB1 and αB2). The kinetics of aggregation of these subunits to form the polymeric α-crystallin has been investigated in adult epithelial cells. Two major points emerge from this study. (a) About 60% of the leucine incorporated into α-crystallin is accounted for by subunit αA2 and about 30% by subunit αB2. Since the relative concentrations of αA2 and αB2 within the aggregate are 60% and 30%, respectively, the incorporation data indicate that in adult epithelial cells the composition of newly formed α-crystallin is identical to that of the previously aggregated molecule. (b) No significant uptake of radioactive leucine in the polymeric molecule via subunit αA1 is detectable. This is consistent with our previous report that this subunit is not a product of direct genetic translation.