Abstract
A technique has been devised for isolation of lumenal plasma membranes from transitional epithelial cells lining the urinary bladder in rabbits and for subsequent separation of particle-bearing plaque regions from particle-free areas of the membranes. The success of the procedures employed and their effects on the isolates were assessed by electron microscopy of conventional plastic sections, negatively stained preparations, and freeze-etch replicas. When bladders are distended with a solution of 0.01 M thioglycolic acid, which reduces sulfhydryl bridges, cytoplasmic filaments are disrupted, and large segments of the lumenal membranes rupture and float free into the lumen. A centrifugation procedure was developed for isolating a fraction enriched with the large fragments. A comparison of membranes isolated in the presence of thioglycolate with those isolated from epithelial cells homogenized in sucrose medium indicates that thioglycolate has little effect on their fine structure except for the removal of filaments which are normally associated with their cytoplasmic surface. The curved plaques of hexagonally arrayed particles and the particle-free interplaque regions, both characteristic of membranes before exposure to thioglycolate, are well preserved. Subsequent treatment of thioglycolate-isolated lumenal membranes with 1% sodium desoxycholate (DOC) severs many of the interplaque regions, releasing individual plaques in which the particles are more clearly visible than before exposure to desoxycholate. Presumably, DOC acts by disrupting the hydrophobic bonds within the membrane; therefore, this type of cohesive force probably is a major factor maintaining the structural integrity of interplaque regions. This conclusion is consistent with the observation that interplaque regions undergo freeze-cleaving like simple bilayers with a plane of hydrophobic bonding.
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Selected References
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