Characterisation and immunological identification of a calmodulin-stimulated Ca²⁺ATPase from maize shoots.

Microsomal membranes from shoots of Zed mays L. contain a Ca2+-ATPase which is stimulated over 4-fold by calmodulin. Colocalisation of calmodulin-stimulated Ca2+ transport with membrane-marker enzymes and with proteins containing HDEL or KDEL C-terminal sequences for retention in endoplasmic reticulum (ER), shows that at least part of the calmodulin-stimulated Ca2+-ATPase activity is in the ER. In an ER-enriched membrane fraction, vanadate inhibited, while carbonyl cyanide p-(trifluomethoxy) phenyl-hydrazine had no effect on, the calmodulin and non-calmodulin stimulated Ca2+-ATPase. The enzyme can thus be classified as a member of the P-type ATPase family. The calmodulin and non-calmodulin Ca2+-ATPase components showed a differential nucleotide triphosphate specificity, the activity in the absence of calmodulin being more specific for ATP. Ca2+-ATPase activity was inhibited by erythrosin B and by the specific inhibitor of animal ER/SR-type Ca2+-ATPases, 2,5-di-tert-butylhydroquinone. Antibodies raised against peptides which are conserved between the animal SR Ca2+-ATPase isoform 1 and two deduced sequences of putative plant Ca2+-ATPases, identified two bands of Mr = 102 kDa and 91 kDa. The 102 kDa band colocalised on linear sucrose gradients with the ER while the 91 kDa band colocalised with the Golgi apparatus. The 102 kDa band bound calmodulin and this binding was dependent on the presence of Ca2+. It is concluded that the ER is the site of a calmodulin stimulated Ca2+-ATPase of 102 kDa.</p