Stability of shark and human lens fiber cell membranes

1. Scanning electron microscopy of minced dogfish, shark, and human lens fiber cell preparations showed that neither 8 M urea or 1% SDS was capable of totally disrupting the fiber cells, but that their membranes were totally disrupted by 1% SDS containing 50 mM dithiothreitol. 2. Fiber cells were purified by sucrose centrifugation and extracted with Tris-buffer, 8 M urea, 1% SDS and 50 mM dithiothreitol plus 1% SDS. 3. A residual 10% of the protein was capable of maintaining membrane integrity. 4. DTT + SDS totally dissolved the membranes, implying that disulfide bonds are involved in maintaining structure. 5. Non-tryptophan fluorescence was nearly totally extracted prior to the SDS-DTT step, indicating that the fluorescence associated with the membrane protein was not serving as crosslinks between extrinsic and intrinsic proteins. 6. Polyacrylamide slab gel electrophoresis revealed that after exhaustive extractions of lens fiber cells with Tris-buffer, 8 M urea and 1% SDS, the addition of DTT released major heat stable 27,000 and 24,000 dalton peptides. 7. The data support the concept that fiber cell membranes contain high levels of extrinsic peptides and intrinsic peptides whose stability depends strongly on oxidized crosslinks, probably disulfide bonds.