Abstract
Purpose: :
A-crystallin (A) and B-crystallin (B) are historically considered to be the two subunits of a single protein, -crystallin. Both these small heat shock proteins are expressed in non-lenticular tissues, however they are not found together in the same tissue suggesting that they function as independent proteins. Outside of the lens, A is expressed predominantly in spleen and thymus, where no B is detected. B, on the other hand is highly expressed in the heart, where A is not found (J Biol. Chem. 267:23337-41, 1992). We have previously suggested that these two proteins may also have independent physiological functions inside the ocular lens (Eur. J. Biochem. 102: 775-81, 1991). In this investigation we have examined the status of A and B synthesis and localization in the developing rat lens.
Methods: :
Differential centrifugation and discontinuous density sucrose gradient fractionation were employed to study the association of A and B in different cellular compartments at various developmental stages. Free and bound polysomes were fractionated and the presence of A and B in each of these compartments was investigated. Immunoflourescence and confocal microscopy were used to localize the A and B in the lens epithelial cells and in the native ocular lens.
Results: :
Differential centrifugation shows that in postnatal day 3 (PND3) and PND10 lenses significantly more of B is found in the membrane fractions than A. Density gradient fractionation of post-nuclear supernatants reveals that A like B is associated with the Golgi membranes. Interestingly, examination of free and bound polyribosome fractions isolated from fetal and post-natal day 10 rat lenses reveals that A is associated with free polysomes and smooth membranes while B is predominantly found in the bound polysome fractions.
Keywords: crystallins • protein structure/function • gene/expression