Relationship between calcium ion transport and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

Abstract Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca 2+ transport in this preparation were characterized and compared to those of (Ca 2+ + Mg 2+ )-ATPase activity. The time course of Ca 2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8–11.5 nmol Ca 2+ /mg protein after 3–4 min of incubation. The rate of Ca 2+ transport was increased by higher oxalate concentrations with a near linear rate of uptake at 20 mM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca 2+ /mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent K m values of 100 μM and 7 μM for ATP concentration ranges above and below 50 μM, respectively. The apparent K m values for Mg 2+ and Ca 2+ were 132 μM and 0.36–0.67 μM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal ( Ca 2+ + Mg 2+ )-ATPase. The presence of potassium was required for maximum Ca 2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca 2+ transport and ( Ca 2+ + Mg 2+ - ATPase activity was K + > Na + = NH 4 + > Li + . Ca 2+ transport and ( Ca 2+ + Mg 2+ )- ATPase activity were both inhibited 10–20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 μM p- chloromercuribenzene sulfonate. The results indicate that Ca 2+ transport in adipocyte endoplasmic reticulum is mediated by a ( Ca 2+ + Mg 2+ )-ATPase and suggest an important role for endoplasmic reticulum in control of intracellular Ca 2+ distribution.