V M Berthoud1, A J Cook, E C Beyer. 1. Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract
PURPOSE: To characterize the chicken lens gap junction protein, connexin56 (Cx56). METHODS: The methods used were immunoblotting, immunofluorescence, alkaline phosphatase treatment, in vitro translation, and primary tissue culture. RESULTS: Connexin56 translated in vitro showed a single band with an electrophoretic mobility of approximately 66 kd. Multiple bands of 67 to 90 kd were detected in immunoblots of chicken lens homogenates using antibodies raised against a peptide from Cx56. Most, if not all, of these bands represented different phosphorylated forms of Cx56, because immunoreactive Cx56 was detected as a doublet of 65 to 67 kd after treatment of lens homogenates with alkaline phosphatase. Indirect immunofluorescence demonstrated that Cx56 was localized at membrane appositions between lens fibers and bow region cells. Levels of Cx56 increased from embryonic days 4 to 15; thereafter, levels remained fairly constant until hatching, after which they declined. Before embryonic day 9, the slowest migrating bands were not as abundant as they were at later ages. After embryonic day 20, less Cx56 was observed by immunofluorescence in the nucleus than in the cortex; however, both regions had similar levels of Cx56 as measured by immunoblotting. The pattern of bands differed between the two lens regions, suggesting differential protein modification. Immunoreactive Cx56 bands of 35 to 38 kd were detected unless homogenates were prepared in the presence of ethylenediaminetetraacetic acid (EDTA). Cx56 was also detected in lentoid-containing primary cultures derived from chicken lens. CONCLUSIONS: Cx56 is a phosphoprotein. Its appearance and modification by phosphorylation, as detected by immunoblotting, correlate with lens fiber differentiation.
PURPOSE: To characterize the chicken lens gap junction protein, connexin56 (Cx56). METHODS: The methods used were immunoblotting, immunofluorescence, alkaline phosphatase treatment, in vitro translation, and primary tissue culture. RESULTS:Connexin56 translated in vitro showed a single band with an electrophoretic mobility of approximately 66 kd. Multiple bands of 67 to 90 kd were detected in immunoblots of chicken lens homogenates using antibodies raised against a peptide from Cx56. Most, if not all, of these bands represented different phosphorylated forms of Cx56, because immunoreactive Cx56 was detected as a doublet of 65 to 67 kd after treatment of lens homogenates with alkaline phosphatase. Indirect immunofluorescence demonstrated that Cx56 was localized at membrane appositions between lens fibers and bow region cells. Levels of Cx56 increased from embryonic days 4 to 15; thereafter, levels remained fairly constant until hatching, after which they declined. Before embryonic day 9, the slowest migrating bands were not as abundant as they were at later ages. After embryonic day 20, less Cx56 was observed by immunofluorescence in the nucleus than in the cortex; however, both regions had similar levels of Cx56 as measured by immunoblotting. The pattern of bands differed between the two lens regions, suggesting differential protein modification. Immunoreactive Cx56 bands of 35 to 38 kd were detected unless homogenates were prepared in the presence of ethylenediaminetetraacetic acid (EDTA). Cx56 was also detected in lentoid-containing primary cultures derived from chicken lens. CONCLUSIONS:Cx56 is a phosphoprotein. Its appearance and modification by phosphorylation, as detected by immunoblotting, correlate with lens fiber differentiation.