Partial characterization of a high affinity [Ca2+ + Mg2+]-dependent adenosinetriphosphatase from bovine retina

Examination of retinal tissue homogenates indicated the presence of a [Ca2+ + Mg2+]-dependent adenosinetriphosphatase activity that exhibited high affinity for Ca2+ (K0.5 = 0.17 microM) and moderately high affinity for Mg2+ and ATP (K0.5 = 12.5 microM and Km = 22.8 microM, respectively). Maximum ATP hydrolysis occurred at pH 7.4. Under conditions of optimal substrate, cation and hydrogen ion concentrations, specific activity ranged from 15 to 18 nmol phosphate released min-1 mg-1 protein. Although the retinal [Ca2+ + Mg2+] adenosinetriphosphatase hydrolyzes both ATP and dATP, other nucleotides (CTP, GTP, ITP and UTP) were not hydrolyzed to any great extent. The monovalent cations, Li+, K+ and Na+, had no effect upon hydrolysis of ATP; whereas Cs+ and NH4+ ions were moderately (approximately 30%) inhibitory. All divalent cations tested were stimulatory. With the exception of rotenone which inhibited ATP hydrolysis approximately 25%; retinal adenosinetriphosphatase activity was insensitive to mitochondrial inhibitors (NaN3, KCN, ruthenium red and oligomycin). Adenosinetriphosphatase activity was observed to be very sensitive to low concentrations (I50 approximately 2 microM) of vanadate; whereas, lanthanum administration resulted in no inhibition. Removal of calmodulin (80%) resulted in reducing adenosinetriphosphatase activity 60% but addition of exogenous calmodulin back to calmodulin deficient membranes did not restore activity to starting levels. Calmodulin antagonists trifluoperazine and calmidazolium reduced significantly Ca2+ stimulated, Mg2+ dependent ATP hydrolysis. We conclude that the [Ca2+ + Mg2+]-dependent adenosinetriphosphatase of bovine retina is a non-mitochondrial protein exhibiting very high affinity for Ca2+ and appears to require calmodulin for maximum activity. Because of its high affinity for Ca2+, this protein may play an important role in reducing intracellular Ca2+ to nanomolar levels.