Isolation and characterization of BLMV from freshwater crayfish (Procambarus clarku) gills, antennal gland and hepatopancreas

Crustacean calcium transporting epithelia include the gills hepatopancreas and antennal gland The models for crustacean epithelial calcium transport have been derived exclusively form vesicle studies in intermolt marine species. To study calcum transport. basolateral membrane vesicles (BLMV) know to be the location of both the calcium pump and exchange were isolated and characterized from the antennal gland, hepatopancreas and gills of the freshwater erayfish. The tissues were homogenized, hepatopancreas BLMV were further isolated EDTA. The antenual gland and hepatopancreas BLMV were further isolated by a process differentiol centifigation The gill BLMV were isolated using a discontmuous surose gradient (65% ± 40%). The BLMV were fully charecterized and vesicle orientation/resealing determined. The antennal gland BLMV were 9.5±1.5 fold enriched while there was no enrichment of mitochondia or apical membrances and reseeling was found to be 21.4%±4.2 inside-out vesicles (IOV) 38.5%±4.5 rightside-out vesicles (ROV) and 40%±4.5 leaky/sheets. The gill BLMV were 18.0%±6.1 fold enriched of apical membrance and were (1.58±0.2 fold) enrichment of mitochondria and no enrichment of apichal membrance and were 17%±4.2 IOV 26.9%±4.6 ROV and 60%±6.3 leaky/sheets. The hepatopancreas BLMV were found to be 9.2±1.2 fold enriched with no enriched of the mitochondria or apical membrance and the reseaing was found to be 14.2%±3.8 IOV 25.6%±3.9 ROV and 60.2%±3.8 leak/sheets. The BLMV have been used to study radiotracer calcium uptake as de scribed in Wheatly et al. abstract.