Characterisation of the major intrinsic protein (MIP) from bovine lens fibre 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 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.
König
N
N
MRC Laboratory of Molecular Biology, Cambridge, UK.
Zampighi
G A
GA
Butler
P J
PJ
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
D020346:Aquaporins; D003902:Detergents; D005136:Eye Proteins; D008055:Lipids; D008562:Membrane Glycoproteins; D008823:Micelles; C030328:aquaporin 0