PURIFICATION OF BOVINE LENS CELL-TO-CELL CHANNELS COMPOSED OF CONNEXIN44 AND CONNEXIN50

Cell-to-cell channels composed of connexin44 and connexin50 were purif ied from plasma membranes of calf and fetal bovine lenses, The channel s were treated with the nonionic detergents octyl-beta-D-glucopyranosi de and decyl-beta-D-maltopyranoside, and the channel/detergent complex es purified by ion and gel filtration column chromatography. In negati ve staining, the channels appeared as annuli 11 +/- 0.6 nm (s.d,, n=10 5) in diameter and as 16 +/- 0.8 nm (s.d,, n=96) long particles which corresponded to top and side views of 'complete' cell-to-cell channels , The purified cell-to-cell channels were composed principally of a pr otein, called MP70, that appeared as a diffuse 55-75 kDa band in SDS-P AGE, Dephosphorylation with alkaline phosphatase transformed the diffu se 55-75 kDa band into two distinct bands of almost equal intensity, I mmunoblotting showed the bands to be connexin44 and connexin50, respec tively, The antibodies also recognized weaker bands composed of the un phosphorylated form of both connexins, The connexins appear to be proc essed independently 'in vivo', The unphosphorylated form of connexin50 was present in channels and membranes from fetal, calf and adult bovi ne lenses, while unphosphorylated connexin44 only in channels purified from fetal lenses, Therefore, lens cell-to-cell channels are composed principally of equal amounts of phosphorylated connexins 44 and 50 th at appear to be assembled in the same channel ('hybrid).