CYTOPLASMIC SURFACE ULTRASTRUCTURES OF GAP-JUNCTIONS IN BOVINE LENS FIBERS

Purpose. To examine the cytoplasmic surface ultrastructures of lens fi ber gap junctions, where the cytoplasmic domains of connexons were exp ected to be exposed. Methods. Bovine lens fiber gap junctions, both in situ and in the form of isolated membranes, were examined with the de ep etching replica methods. Isolated membranes were also examined with the same methods after the treatment with endoproteinase glu-C, which is known to cleave off the cytoplasmic domain of a putative lens fibe r connexin MP70 to determine whether any structural changes should occ ur between proteolyzed and nonproteolyzed gap junctions. In addition, both proteolyzed and nonproteolyzed gap junctions were studied by sodi um dodecyl sulfate polyacrylamide gel electrophoresis and immunolabeli ng with the monoclonal antibody that recognized cytoplasmic domain of MP70 to clarify whether MP70 lost its cytoplasmic domain by the treatm ent with endoproteinase glu-C. Results. Gap junctions were shown to ha ve particulate substructures on their cytoplasmic surfaces; the distri butions of the particles were restricted within gap junctional plaques and the non-gap-junctional areas showed smooth cytoplasmic surfaces. Although the treatment with endoproteinase glu-C failed to remove the cytoplasmic particles of gap junctions in deep etching replica study, MP70 was shown to have lost its cytoplasmic domain in sodium dodecyl s ulfate polyacrylamide gel electrophoresis and immunolabeling studies. Conclusions. Each particle revealed on the cytoplasmic surfaces of len s fiber gap junctions corresponded to the cytoplasmic domain of a conn exon. The particles were not removed by the treatment with endoprotein ase glu-C, whereas MP70 was cleaved by the same treatment.