Ca2+ Uptake by Endoplasmic Reticulum from Zucchini Hypocotyls 1: The Use of Chlorotetracycline as a Probe for Ca2+ Uptake

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Ca2+ uptake into microsomal vesicles was measured using the fluorescent probe chlorotetracycline. The Ca2+ uptake was ATP-dependent and did not occur in the presence of the calcium ionophore A23187. There was a linear relationship between the rate of ATP-dependent fluorescence increase using chlorotetracycline and ATP-dependent 45Ca2+ uptake, indicating that chlorotetracycline can be used as a quantitative probe for Ca2+ uptake. The fluorescent probe allows measurements to be made in real time, and avoids the use of radioisotopes. Ca2+ transport was associated with endoplasmic reticulum on linear gradients when the endoplasmic reticulum was in either rough or smooth form. The Ca2+ uptake had a pH optimum of 7.5, a Km for ATP of 0.1 millimolar, a Km for Ca2+ of about 70 nanomolar, and was stimulated 2-fold by calmodulin. Vanadate inhibited uptake completely at a concentration of 50 micromolar, half-maximally at 5 micromolar. Carbonyl cyanide 4-(trifluoromethoxy)-phenyl-hydrazone, oligomycin, azide, and nitrate caused only slight inhibition. Dicyclohexylcarbodiimide (DCCD) stimulated slightly at concentrations as high as 400 micromolar. The hormones gibberellic acid, indoleacetic acid, and abscisic acid at 10 micromolar had no significant effect. Myo-inositol 1,4,5-trisphosphate did not cause release of Ca2+ after uptake. The properties of the enzyme suggest that it has a functional role in regulating cytosolic Ca2+ levels. Based on the lack of an effect by hormones, it may not act as a mediator of second messenger roles of Ca2+. The inhibition by vanadate and slight stimulation by DCCD may be useful as a `signature' for this endoplasmic reticulum Ca2+ uptake system.

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