D L Boyle1, L Takemoto. 1. Division of Biology, Ackert Hall, Kansas State University, Manhattan 66506, USA.
Abstract
PURPOSE: To integrate past biochemical findings with past morphological observations of area insoluble material isolated from cataract and aged normal lenses, by determining the spatial distribution of alpha-crystallins associated with the plasma membrane (PM) of nuclear cataractous and age matched normal human lenses. METHODS: Lenses were homogenized, pelleted and washed several times in 0.05M Tris-Cl (pH 7.2) containing 100mM KCl, 1 mM MgCl2 and 2mM beta-mercaptoethanol, followed by several washes in 8M urea. Urea insoluble pellets (UIP) were labeled before fixation and embedding with rabbit serum raised against alpha-crystallins, followed by goat anti-rabbit IgG conjugated to 5nm gold. Approximately 300 gold particles associated with the PM were counted, for each lens, on several electron microscopy (EM) micrographs. The number of gold particles/um of PM, number of individual vs clusters of gold particles were determined. RESULTS: Micrographs from both normal and cataractous human lenses clearly demonstrated the association of alpha-crystallins with the PM. Also apparent was the abundant labeling of the PM for cataractous lenses as compared to normal lenses. Quantification of the gold labeling revealed that not only was there an increase in the amount of labeling/um of PM in cataract lenses, but there was also an increased percentage of gold in clusters. These clusters were not only more numerous in cataractous lenses, but also contained a greater number of gold/cluster. CONCLUSIONS: These findings provide morphological evidence that the PM in nuclear cataract lenses is associated with large aggregates of alpha-crystallin.
PURPOSE: To integrate past biochemical findings with past morphological observations of area insoluble material isolated from cataract and aged normal lenses, by determining the spatial distribution of alpha-crystallins associated with the plasma membrane (PM) of nuclear cataractous and age matched normal human lenses. METHODS: Lenses were homogenized, pelleted and washed several times in 0.05M Tris-Cl (pH 7.2) containing 100mM KCl, 1 mM MgCl2 and 2mM beta-mercaptoethanol, followed by several washes in 8M urea. Urea insoluble pellets (UIP) were labeled before fixation and embedding with rabbit serum raised against alpha-crystallins, followed by goat anti-rabbit IgG conjugated to 5nm gold. Approximately 300 gold particles associated with the PM were counted, for each lens, on several electron microscopy (EM) micrographs. The number of gold particles/um of PM, number of individual vs clusters of gold particles were determined. RESULTS: Micrographs from both normal and cataractoushuman lenses clearly demonstrated the association of alpha-crystallins with the PM. Also apparent was the abundant labeling of the PM for cataractous lenses as compared to normal lenses. Quantification of the gold labeling revealed that not only was there an increase in the amount of labeling/um of PM in cataract lenses, but there was also an increased percentage of gold in clusters. These clusters were not only more numerous in cataractous lenses, but also contained a greater number of gold/cluster. CONCLUSIONS: These findings provide morphological evidence that the PM in nuclear cataract lenses is associated with large aggregates of alpha-crystallin.