Kinetic properties of the calcium pump in basolateralvesicles from crayfish liver, gill and kidney at 37C

The objective was to characterize ATP-dependent Ca uptake into inside out basolateral membrane vesicles prepared from three crayfish tissues that routinely transport Ca during stages of the crustacean molting cycle. The initial uptake rate of ⁴⁵Ca (free Ca 5 uM) into vesicles in the presence or absence of ATP (5 mM) was determined by rapid filtration and found to be linear for 10 mins. ATP independent accumulation was typically 20% of ATP dependent uptake. ATP dependent uptake was abolished by pretreatment with either vanadate or the ionophore A₂₃₁₈₇- Calcium efflux was observed when A₂₃₁₈₇ was added to actively loading vesicles. Ca affinity was equivalent in liver and gill (Km 0.27 uM) but was higher in kidney (0.11 uM). Maximal uptake was 10.13 pmol/mg/min in liver and was 5-6 fold higher in gill and kidney. An ATP titration curve indicated a Km of 0.01 mM in liver and gill and 0.024 mM in kidney. EGTA treatment of liver and kidney vesicles decreased Ca uptake by 50%. Uptake was restorable by calmodulin. However in gill Ca uptake was unaffected by EGTA treatment and calmodulin decreased uptake. Q₁₀ was 1.43 in liver, 2.06 in gill and 1-64 in kidney. Addition of Na (5 mM) caused a 30% increase in Ca uptake that was inhibitable by preincubation with ouabain indicating that the Na pump generates a Na gradient favorable for Ca accumulation via the Na/Ca exchanger. Kinetics have been related to the role of crayfish liver, gill and kidney in Ca homeostasis and compared with other known Ca transporting epithelia.