MP26, a protein of intercellular junctions in the bovine lens: electrophoretic and chromatographic characterization.
We have characterized the membrane protein of apparent molecular weight 26 kD from bovine lenses (MP26 or MIP) with respect to six different electrophoretic and chromatographic procedures. These include one- and two-dimensional gel electrophoretic procedures, as well as SDS-hydroxylapatite chromatography. The two-dimensional gels include isoelectric focusing with both conventional ampholytes and buffer focusing methods. With buffer focusing, the membranes are solubilized without the use of SDS and the isoelectric focusing is performed in the absence of SDS. As specific probes for MP26, a monoclonal antibody and an anti-MP26 rabbit serum were used, the latter prepared against electrophoretically purified MP26. These separation techniques were adapted to MP26 in order to permit a more detailed characterization of this protein and to search for any heterogeneity in this size range, specifically other junctional proteins or protein fragments. We have found evidence for charge heterogeneity in MP26, but no evidence for multiple membrane proteins of Mr 26,000 in urea-treated membranes. The charge heterogeneity appears to be related to a phosphorylation of MP26. The results reported here aid the interpretation of a variety of data, especially findings on the reconstitution of MP26 in artificial membranes and results from work with polyclonal MP26 antibodies. These investigations are all designed to evaluate the proposed role of MP26 as a protein of cell-to-cell channels in the lens fiber cell.
Johnson
K R
KR
Department of Genetics and Cell Biology, University of Minnesota, St Paul 55108-1095.
Sas
D F
DF
Johnson
R G
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D000818:Animals; D020346:Aquaporins; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002845:Chromatography; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000302:isolation & purification*; D007908:Lens, Crystalline / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D008970:Molecular Weight
D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; C030328:aquaporin 0