Isolation, visualization, characterization, and osmotic reactivity of crayfish BLMV.
Procedures were developed to isolate basolateral membrane vesicles (BLMV) from gill, hepatopancreas, and antennal gland of intermolt freshwater crayfish, Procambarus clarkii. Individual procedures involved a discontinuous sucrose gradient (gill), a 65% sucrose cushion (hepatopancreas), or differential centrifugation (antennal gland). BLMV were visualized, characterized (37 degrees C), and tested for osmotic reactivity with a view to using them for Ca2+ uptake studies. Mean diameters of BLMV were 159 nm (gill), 363 nm (hepatopancreas), and 226 nm (antennal gland). Enrichments of basolateral membranes and mitochondria in BLMV were, respectively, 18- and 1.7-fold for gill, 9- and 0.4-fold for hepatopancreas, and 10- and 1-fold for antennal gland. Apical contamination was negligible in BLMV. Percentages of resealing of vesicles as inside out, right side out, or leaky/sheets were 17:27:56% (gill), 14:26:60% (hepatopancreas), and 21:39:40% (antennal gland). Vesicles exhibited osmotic reactivity, as indicated by a linear relationship between vesicular 45Ca2+ uptake and osmolality. Nonspecific 45Ca2+ binding was 20% in gill, 39% in hepatopancreas, and 31% in antennal gland. Data were compared with published values for marine crustaceans.
Wheatly
M G
MG
Department Of Biological Sciences, Wright State University, Dayton, Ohio 45435, USA.
Weil
J R
JR
Douglas
P B
PB
D000818:Animals; D003400:Astacoidea / Q000502:physiology*; D001693:Biological Transport, Active; D002118:Calcium / Q000502:physiology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000502:physiology; D016764:Cell Polarity; D004064:Digestive System / Q000378:metabolism* / Q000648:ultrastructure; D005880:Gills / Q000502:physiology* / Q000648:ultrastructure; D008722:Methods; D010042:Ouabain / Q000494:pharmacology; D012503:Saponins / Q000494:pharmacology; D012964:Sodium / Q000502:physiology; D013347:Subcellular Fractions / Q000201:enzymology; D014882:Water-Electrolyte Balance
D012503:Saponins; D010042:Ouabain; D012964:Sodium; D002118:Calcium