Phosphorylation is one of the most important post-translational modifications, and it is estimated that about one third of all eukaryotic proteins are phosphorylated. [1][2][3] Crystallins in lens are also phosphorylated, and the phosphorylation changes in their functions. a-Crystallin is a major protein of the mammalian lens, constituting as much as 50% of its dry weight. It exists as a large heterogeneous aggregate (ca. 800 kDa), which is composed of two types of subunits, aAcrystallin (173 amino acids) and aB-crystallin (175 amino acids).4) Amino acid sequences of the two subunits show 55% homology with each other. Both subunits also exist in non-lenticular tissues and are similar to a small heat-shock protein, hsp 27, in their amino acid sequence. In addition, acrystallin functions as a molecular chaperone.5) Previously we reported that truncation of the C-terminal region of acrystallin could lead to loss of its chaperone activity.6) On the other hand, Ito et al. reported that various agents and heat stress stimulated the phosphorylation of aB-crystallin, and by using antibodies they identified the sites of phosphorylation on aB-crystallin as three serine residues, 3) These observations suggested that the phosphorylation of aB-crystallin causes dissociation of large oligomers to smaller ones and reduces its chaperone-like activity, like in the case of HSP27.3) However, there are few reports on the phosphorylation of rodents aA-crystallin. 7,8) Bovine aAcrystallin is reported to be phosphorylated at In this research, the phosphorylation of crystalline lens proteins aB-crystallin and aA-crystallin was examined in cataractous and normal rats of different ages. ICR/f is a strain of hereditary cataractous rat in which opacity of the lens begins to develop about 75 d after birth. 10) Lenses of 22-d-old (before the opacity develops) and 85-d-old (after it develops) ICR/f rats were used as a cataract model, and the lenses of Wistar strain rats of the same ages were used as a normal model. Hereafter, these are abbreviated as follows: ICR22, lens of ICR/f rat of 22 d; ICR85, lens of ICR/f rat of 85 d; Wistar22, lens of Wistar rat of 22 d; Wistar85, lens of Wistar rat of 85 d. MATERIALS AND METHODS Determination of Protein Contents Protein contentswere determined by the method of Bradford using bovine serum albumin as a standard. 11)Two-Dimensional Electrophoresis Lenses of 22-d-old and 85-d-old rats were homogenized in 50-75 ml of water using a Potter-type homogenizer at 4°C. The homogenate was centrifuged at 15000ϫg for 50 min to remove the waterinsoluble material. Then the supernatant (lens water-soluble fraction) was submitted to two-dimensional electrophoresis.The supernatant (150 mg protein) was resuspended in the rehydration buffer (8 M urea, 2.2% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;), 2 mM tributyl-phosphine, Immobiline pH gradient (IPG) buffer, [pH 3-10 NL (non-linear)] (Bio-Rad)). The first-dimension electrophoresis was performed with the IPGphor system. Briefly, an 11-cm st...