An azide‐insensitive low‐affinity ATPase stimulated by Ca²⁺ or Mg²⁺ in basal‐lateral and brush border membranes of kidney cortex

Abstract

Basal‐lateral and brush border membranes from pig kidney cortex were prepared by differential centrifugation followed by free‐flow electrophoresis. In each type of membrane, azide‐insensitive, low‐affinity Ca2+‐ATPase and Mg2+‐ATPase activities are demonstrated. A comparative study for both membranes further reveals the following analogies between these ATPase: (a) they show maximal activity between pH 8 and 8.5; (b) they exhibit Km values for Ca‐ATP or Mg‐ATP in the millimolar range and have a comparable low substrate specificity; (c) they are insensitive to 10 μM of vanadate, N,N′‐dicyclohexylcarbodiimide, diethylstillbestrol, quercetin, harmaline and amiloride. The partial inhibition by 1 mM of the various compounds is rather aspecific. In view of these similarities it is concluded that only one enzyme entity is responsible for the activity which is measured in both membrane types. The HCO3−‐stimulated Mg2+‐ATPase activity in pig kidney cortex was also studied. This enzyme, however, is clearly of mitochondrial origin since the HCO3−‐stimulation coincides with the distribution profile of succinate dehydrogenase, a mitochondrial marker; and since it is inhibited by azide.

Autoradiography-based cytochemical detection of ecto-ATPase, ecto-ADPase, 5?-nucleotidase, and extracellular adenosine production, employing 141Ce3+ as a capturing agent

“…Azide is an inhibitor of mitochondrial H+-ATPase, but has no influence on the ecto-ATPase activity (Ilsbroux et al, 1985). As expected, sodium azide was unable to cause changes in the intensity of the observed signal (Fig.…”

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

“…An activation by Ca 2+ [5,20,31,32,44,45] and by other nucleotides besides ATP [13,31,44,45] has also been observed in earlier experiments on ATPases in cortex homogenates, isolated tubules, and membrane vesicles, suggesting that many of the previous investigations including partial purification [44] have been performed on ecto-ATPases. Our data showing the presence of Mge+-ATPases in both brush-border and basolateral membranes are in agreement with some earlier investigations [30,53] and disagree with others, which have demonstrated a Ca 2+ or Mg2+-activated ATPase exclusively in the luminal [20] or contraluminal [34] membrane of proximal tubule ceils.…”

“…The "Mg2+-ATPase '' measured in cortex homogenates [45], microdissected nephrons [32], and isolated brush-border membranes [5,20,31,44] is stimulated by Ca ~-+ and other nucleotides besides ATP [13,31,44,45], This pattern is typical for ecto-ATPases found in other cells [14,18,28,29,40,42] rather than for an ATPase involved in H + transport. The "bicarbonate-stimulated ATPase" in a rat kidney brush-border membrane preparation was related to ATP-driven H+se -cretion [35,36,41,43], but was later shown to be of mitochondrial origin [13,31,38]. Another candidate for H + secretion, the N,N'-dicyclohexylcarbodiimide (DCCD) and N-ethylmaleimide (NEM) sensitive ATPase was measured in microdissected and permeabilized proximal tubules [l], but a localization to the brush-border membrane was not demonstrated.…”