Adenosine Triphosphate-dependent Calcium Pump in the Plasma Membrane

function as a second messenger in neutrophils. Since the plasma membrane seems to be a major regulator of in-tracellular Ca in many cells, we characterized an energy-dependent Ca transport system in plasma membrane-enriched fractions ("podosomes") from phorbol myristate acetate-stimulated guinea pig and human neutrophils. The active Ca transport system in guinea pig podosomes exhibited a high affinity for Ca (Michaelis constant [Km]Ca 280±120 nM) and a maximum velocity of 0.83 nmol Ca/mg protein per min. Uptake showed an absolute re-quirement for Mg ATP (Km ATP 67,M), whereas other trinucleotides were inactive. Ca uptake was optimal at pH 7, was azide insensitive and temperature dependent. Vanadium, an inhibitor of the Ca/Mg ATPase of heart sarcolemma, inhibited Ca pump activity by 50 % at 1 1M. Ca transport was not affected in a NaCl-containing me-dium, an observation arguing against the presence of a Na/Ca exchange system. Calmodulin at 0.5-10 gg/ml stimulated the Ca pumping activity ofEGTA-washed po-dosomes. Calmodulin depletion decreased the affinity of the Ca pump for Ca (Km Ca 2.07 MM) and its readdition restored it (Km Ca 0.55 MM). ATP-dependent Ca transport by podosomes an