Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.
Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D007774:Lactation; D008321:Mammary Glands, Animal / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium