Isolation and characterization of basolateral membrane vesicles (BLMV) from crayfish gills and kidney

Crayfish (Procambarus clarkii) are excellent models to study Ca transport mechanisms since they exhibit bidirectional Ca transfer associated with molting. Basolateral Ca transport in the gill and kidney is mediated by Ca ATPase and Na/Ca exchanger. To study these mechanisms, BLMV were isolated and characterized. Gills and kidney were homogenized in an isotonic solution (sucrose, EDTA, Imidazole and HEPES). A brief spin (20' @ 1000g) removed cellular debris. The preparation was then incubated following the addition of Mg-Gluconic acid. After two additional spins (3000g & 5000g), the upper pelleted layer was removed, resuspended and spun (30' @10,000g). The vesicles were resuspended and warmed to ensure resealing. The Na/K ATPase activity in BLMV for gill and kidney was 4.5±1.0 and 5.3±1.3 fold enriched, confirming basolateral membrane isolation. Vesicle orientation was determined from Na/K ATPase activity in the presence of ouabain. The orientation of gill BLMV was 32.8±6.6% rightside-out (ROV), 22.1±11.8% inside-out (IOV), 59.1±6.0% leaky sheets (LS). For kidney BLMV the orientation was 22.7±1.0% ROV, 22.1±11.8% IOV, 57.1±10.1% LS. BLMV Ca transport studies will employ radiotracer technology or fluorescent indicators. (Supported by NSF grants DCB 8916412 and IBN 9307290 to MGW.).