CHARACTERIZATION OF THE GAP JUNCTION PROTEIN CONNEXIN56 IN THE CHICKEN LENS BY IMMUNOFLUORESCENCE AND IMMUNOBLOTTING

Purpose. To characterize the chicken lens gap junction protein, connex in56 (Cx56). Methods. The methods used were immunoblotting, immunofluo rescence, alkaline phosphatase treatment, in vitro translation, and pr imary tissue culture. Results Connexin56 translated in vitro showed a single band with an electrophoretic mobility of approximately 66 kd. M ultiple bands of 67 to 90 kd were detected in immunoblots of chicken l ens homogenates using antibodies raised against a peptide from Cx56. M ost, if not all, of these bands represented different phosphorylated f orms of Cx56, because immunoreactive Cx56 was detected as a doublet of 65 to 67 kd after treatment of lens homogenates with alkaline phospha tase. Indirect immunofluorescence demonstrated that Cx56 was localized at membrane appositions between lens fibers and bow region cells. Lev els of Cx56 increased from embryonic days 4 to 15; thereafter, levels remained fairly constant until hatching, after which they declined. Be fore embryonic day 9, the slowest migrating bands were not as abundant as they were at later ages. After embryonic day 20, less Cx56 was obs erved by immunofluorescence in the nucleus than in the cortex; however , both regions had similar levels of Cx56 as measured by immunoblottin g. The pattern of bands differed between the two lens regions, suggest ing differential protein modification. Immunoreactive Cx56 bands of 35 to 38 kd were detected unless homogenates were prepared in the presen ce of ethylenediaminetetraacetic acid (EDTA). Cx56 was also detected i n lentoid-containing primary cultures derived from chicken lens. Concl usions. Cx56 is a phosphoprotein. Its appearance and modification by p hosphorylation, as detected by immunoblotting, correlate with lens fib er differentiation.