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Abstract
Purpose. To examine the intermediate filament cytoskeletal proteins associated with native membrane fractions isolated from bovine lenses.
Methods. Decapsulated bovine lenses were divided into cortex and nucleus. The lens regions were homogenized and separated into water-soluble and water-insoluble fractions by centrifugation. Sedimenting membrane fractions were isolated from the water-insoluble fraction by discontinuous sucrose-density-gradient centrifugation and the non-sedimenting membrane fractions were isolated from the Kbr high-density water-soluble fractions by flotation, during overnight centrifugation. The intermediate filament peptides of the membrane fractions were examined by Western blot analysis, using monoclonal antibodies to filensin, cytoskeletal protein 49 (CP49) and vimentin.
Results. Filensin immunoreactive peptides were found in all membrane fractions of both cortex and nucleus. The parent 115 kDa filensin was found almost exclusively in the urea-soluble protein of cortical membrane fractions, and was the predominant filensin immunoreactive peptide only in the urea-soluble protein of the cortical sedimenting membrane fraction isolated from the 25%/45% sucrose density interface. The predominant filensin immunoreactive peptide of all other samples migrated with a Mr of 53 kDa. CP49 immunoreactive peptides were found almost exclusively in the urea-soluble protein of all membrane fractions from both the cortex and nucleus. The cortical non-sedimenting membrane fraction and the nuclear membrane fraction of the 25%/45% sucrose density interface were notably deficient in CP49. Vimentin immunoreactive peptides were found in both urea-soluble and urea-insoluble proteins of membrane fractions from the cortex only. Vimentin was particularly enriched in the cortical non-sedimenting membrane fraction. The urea-insoluble filensin immunoreactive peptides were only partially removed by alkali extraction, indicating a very avid association with the membrane. Two dimensional electrophoresis revealed that the urea-soluble protein of the major cortical membrane fraction contained two different filensin-derived 53 kDa fragments.
Conclusions. The non-sedimenting membrane fraction, which may reflect a distinct domain of the lens plasma membrane, possesses a membrane-associated cytoskeletal composition different from that of the major sedimenting membrane fractions. Curr. Eye Res. 17: 409–418, 1998.