Examination of a lens 'native' plasma membrane fraction and its associated crystallins.
A bovine lens "native" plasma membrane fraction containing its full compliment of extrinsic proteins was prepared by sucrose density centrifugation of the water insoluble fraction. The major membrane fraction was found at the 25/45% sucrose interface. This fraction contained 73% of the total water insoluble phospholipid, 74% of the total water insoluble cholesterol and 58% of the total urea-insoluble protein. Only 9% of the total urea-soluble protein was membrane associated (extrinsic protein), most (75%) was recovered from the pellet. The major intrinsic protein (8 M-urea-insoluble) of the membrane fraction was MIP28, with lesser amounts of MP17. Extrinsic proteins (8 M-urea-soluble) were examined by SDS-PAGE, isoelectric focusing, immunoblotting and amino acid composition analysis. Approximately 70% of the total extrinsic protein appeared to be alpha A-crystallins and modified alpha A-crystallins. About 20% of the extrinsic protein was apparently beta- and gamma-crystallins. The remainder contained presumed cytoskeletal proteins and perhaps other unidentified polypeptides. The native plasma membrane was found distributed throughout the lens with only minor differences in the quantitative composition of the membrane fraction. We have concluded that the native membrane fraction represents the lens plasma membrane with its extrinsic proteins which exist in vivo. These extrinsic proteins appeared to be primarily acidic alpha-crystallin polypeptides with minor amounts of beta- and gamma-crystallins, and presumed cytoskeletal elements. We speculate that these extrinsic proteins may serve as a nucleation site for the association of other water insoluble protein through protein-protein interactions such as those found in the non-membrane associated urea-soluble protein. Together, these interactions may form a structured cytoplasmic matrix important for the maintenance of lens transparency.
Fleschner
C R
CR
Department of Biochemistry, Kirksville College of Osteophathic Medicine, MO 63501-1497.
Cenedella
R J
RJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000032:analysis; D003459:Crystallins / Q000032:analysis*; D003598:Cytoskeletal Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007526:Isoelectric Point; D007908:Lens, Crystalline / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis; D012995:Solubility
D000596:Amino Acids; D003459:Crystallins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D010743:Phospholipids; D002784:Cholesterol