ISOLATION, VISUALIZATION, CHARACTERIZATION, AND OSMOTIC REACTIVITY OFCRAYFISH BLMV

Procedures were developed to isolate basolateral membrane vesicles (BL MV) from gill, hepatopancreas, and antennal gland of intermolt freshwa ter crayfish, Procambarus clarkii. Individual procedures involved a di scontinuous sucrose gradient (gill), a 65% sucrose cushion (hepatopanc reas), or differential centrifugation (antennal gland). BLMV were visu alized, characterized (37 degrees C), and tested for osmotic reactivit y with a view to using them for Ca2+ uptake studies. Mean diameters of BLMV were 159 nm (gill), 363 nm (hepatopancreas), and 226 nm (antenna l gland). Enrichments of basolateral membranes and mitochondria in BLM V were, respectively, 18- and 1.7-fold for gill, 9- and 0.4-fold for h epatopancreas, and 10- and 1-fold for antennal gland. Apical contamina tion was negligible in BLMV. Percentages of resealing of vesicles as i nside out, right side out, or leaky/sheets were 17:27:56% (gill), 14:2 6:60% (hepatopancreas), and 21:39:40% (antennal gland). Vesicles exhib ited osmotic reactivity, as indicated by a linear relationship between vesicular Ca-45(2+) uptake and osmolality. Nonspecific Ca-45(2+) bind ing was 20% in gill, 39% in hepatopancreas, and 31% in antennal gland. Data were compared with published values for marine crustaceans.