Colloid Osmotic Pressure of Steer and β-Crystallins: Possible Functional Roles for Lens Crystallin Distribution and Structural Diversity

Abstract

This study addresses the general mechanisms whereby the major cytoplasmic proteins from the adult bovine lens contribute both to transparency and maintenance of the refractive index gradient across the lens. Colloid osmotic properties and quaternary structure were measured for α- and β-crystallins isolated from the steer lens, including low-molecular-weight crystallins from the cortex (α Le and β L) and nucleus (α Ln) and high-molecular-weight crystallins from the nucleus (α H and β H). In electron microscopic images of rotary-shadowed preparations α Le appears as spherical particles 16 nm in diameter, α Ln appeared as individual spheres or small aggregates of spherical subunits, α H contained large irregular aggregates as large as 180 nm, and both β L and β H appeared as elliptical particles of 7-9 nm diameter. Secondary osmometry showed that for all these crystallins colloid osmotic pressure increased monotonically in a non-linear fashion with protein concentration. For the α-crystallins, osmotic pressure rose more steeply with concentration for α Le than for either α Ln or α H, so that at 0·3 g ml -1 at 0·1 M ionic strength, the colloid osmotic pressure of α Le, α Ln and α H were approximately 2·6 × 10 5 dyn cm -2, 1·6 × 10 5 dyn cm -2 and 1·0 × 10 5 dyn cm -2, respectively. In a similar manner, osmotic pressure rose more steeply with concentration of β L than for β H, so that at 0·3 g ml -1 at 0·1 M ionic strength the colloid osmotic pressures of β L and β H were 2·6 × 10 5 dyn cm -2 and 1·1 × 10 5 dyn cm -2, respectively. The osmotic pressure of α Le dropped as ionic strength was increased from 0·02 to 0·4 M. For β L and β H, osmotic pressure dropped as ionic strength was increased from 0·02 to 0·1 M but was nearly the same at 0·1 M and 0·4 M ionic strength. The data for steer α Ln and β H were similar to previous reports for calf cortical α L and β-crystallins, respectively. The osmotic pressure isotherms for α Le, β L and that previously reported for steer cortical extract were nearly identical, whereas the nuclear crystallins (α Ln, α H or β H) generated slightly higher pressures than those previously reported for steer nuclear crystallin extracts. In all cases, osmotic pressure rose more steeply with concentration for the cortical crystallins than for the nuclear crystallins. Calculations showed that the measured osmotic pressures generated by solutions of β L and β H were similar to those predicted to arise from excluded volume effects in solutions of uncharged spherical proteins of 60 and 200 kDa, respectively. In contrast, the osmotic pressures generated by each of the α-crystallins were substantially larger than those predicted for pure excluded volume effects between uncharged spherical proteins, pointing to a role for Donnan osmotic pressure and electrostatic repulsion in defining α-crystallin interactions. In particular, modeling studies indicated that the osmotic properties of α Le and α Ln are consistent with the behavior of charged 750-kDa spheres, where α H behaves as charged cylindrical particles. The comparison of the pressure isotherms of the isolated α- and β-crystallins with the nuclear and cortical extracts suggests that the osmotic properties of the lens cytoplasm depend importantly on both the α- and β-crystallins, and implies that the γ-crystallins also contribute to the osmotic properties of the nuclear cytoplasm. The unexpected similarities of the pressure isotherms of the crystallins within the cortex (α L and β L) or nucleus (α Ln, α H and β H) are interpreted as arising from compensatory variations in the size, shape and charge of the proteins and are hypothesized to promote both transparency and the miscibility of the crystallin species in lens fiber cells. The differences in the pressure-concentration isotherms of the cortical and nuclear α- and β-crystallins support the hypothesis that the spatial distribution of these proteins within the lens functions to maintain the radial gradient in refractive index that is essential to visual acuity.