CHARACTERIZATION OF THE MAJOR INTRINSIC PROTEIN (MIP) FROM BOVINE LENS FIBER MEMBRANES BY ELECTRON-MICROSCOPY AND HYDRODYNAMICS

The major intrinsic protein (MIP) from bovine lens fibre membranes has been purified from unstripped membranes using a single ion-exchange c hromatography step (MonoS) in the non-ionic detergent octyl-beta-D-glu copyranoside (OG). SDS-PAGE has confirmed the purity of the preparatio n and thin-layer chromatographic analysis has shown that the protein i s virtually lipid-free. To establish a stable and monodisperse protein sample, we exchanged OG with decyl-beta-D-maltopyranoside (DeM), anot her non-ionic detergent, by gel-filtration column chromatography. We c onclude that the resulting protein/detergent complex is composed of fo ur copies of MIP (a tetramer) and a detergent micelle. This conclusion is based on : (1) measurement of the weight-average molecular mass (( M) over bar(w,app)) of the protein moiety in the protein/detergent com plex by sedimentation equilibrium; (2) measurement of the apparent mol ecular mass of the complexes formed by MIP in OG, in DeM, in dodecyl-b eta-D-maltopyranoside (DoM) and in sodium dodecylsulphate (SDS) by gel filtration; (3) measurement of the apparent molecular mass of pure de tergent micelles; (4) measurement of the predicted change in the molec ular mass of the MIP/DeM complex after partial enzymatic proteolysis; and (5) measurement of the size and shape of the MIP/detergent complex by electron microscopy and single-particle analysis. Therefore, the t etragonal arrangement of MIP observed in both plasma membranes and jun ctional membranes in lens fibre cells is maintained in solution with n on-ionic detergents. (C) 1997 Academic Press Limited.