title: Characterisation of the major intrinsic protein (MIP) from bovine lens fibre membranes by electron microscopy and hydrodynamics.
abstract: The major intrinsic protein (MIP) from bovine lens fibre membranes has been purified from unstripped membranes using a single ion-exchange chromatography step (MonoS) in the non-ionic detergent octyl-beta-D-glucopyranoside (OG). SDS-PAGE has confirmed the purity of the preparation and thin-layer chromatographic analysis has shown that the protein is virtually lipid-free. To establish a stable and monodisperse protein sample, we exchanged OG with decyl-beta-D-maltopyranoside (DeM), another non-ionic detergent, by gel-filtration column chromatography. We conclude that the resulting protein/detergent complex is composed of four copies of MIP (a tetramer) and a detergent micelle. This conclusion is based on: (1) measurement of the weight-average molecular mass (Mw,app) of the protein moiety in the protein/detergent complex by sedimentation equilibrium; (2) measurement of the apparent molecular mass of the complexes formed by MIP in OG, in DeM, in dodecyl-beta-D-maltopyranoside (DoM) and in sodium dodecylsulphate (SDS) by gel filtration; (3) measurement of the apparent molecular mass of pure detergent micelles; (4) measurement of the predicted change in the molecular 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 tetragonal arrangement of MIP observed in both plasma membranes and junctional membranes in lens fibre cells is maintained in solution with non-ionic detergents.
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: N
authors.initials: N
authors.lastname: König
authors.forename: G A
authors.initials: GA
authors.lastname: Zampighi
authors.forename: P J
authors.initials: PJ
authors.lastname: Butler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D003902:Detergents; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007365:Intercellular Junctions / Q000737:chemistry; D007908:Lens, Crystalline / Q000737:chemistry*; D008055:Lipids / Q000302:isolation & purification; D008562:Membrane Glycoproteins; D008823:Micelles; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation
chemical_list: D020346:Aquaporins; D003902:Detergents; D005136:Eye Proteins; D008055:Lipids; D008562:Membrane Glycoproteins; D008823:Micelles; C030328:aquaporin 0