Ca²⁺-ATPase and Mg²⁺-ATPase activities distinct from alkaline phosphatase in rat jejunal brush-border membranes

Abstract

In rat jejunal brush-border membranes (BBM), ATP hydrolysis activity was specifically stimulated by CaCl₂ and by MgCl₂, allowing to identify Ca²⁺-ATPase and Mg²⁺-ATPase activities with a broad pH optimum near 8.0. Nonspecific ATPase activity (in the absence of cations) had a pH optimum above 9.5 as alkaline phosphatase. The effects of Ca²⁺ and Mg²⁺ concentrations on ATPase activity evidenced two apparent K_A for each cation. At high concentrations, a similar affinity for both cations was recorded (K_A : 0.35 mM). At low concentrations, the affinity for Mg²⁺ was greater than for Ca²⁺ (K_A : 0.02 mM and 0.07 mM respectively). In an attempt to differentially solubilize alkaline phosphatase and ATPase activities, eleven different detergents were assayed. They more or less successfully released Ca²⁺-ATPase and Mg²⁺-ATPase activities from BBM but the more membranes were solubilized by a detergent, the more activities were lost, suggesting a close dependence on integration in BBM. As to alkaline phosphatase and nonspecific ATPase, they almost cosolubilized with Ca²⁺-ATPase and Mg²⁺-ATPase but their total activity was little affected. After treatment of BBM with phosphatidylinositol-specific phospholipase C (E.C. 3.1.4.10), 58% of alkaline phosphatase activity and 45% of nonspecific ATPase activity were released in the supernatant while Ca²⁺-ATPase and Mg²⁺-ATPase activities remained totally incorporated in BBM pellets. These last results definitively demonstrate that Ca²⁺-ATPase and Mg²⁺-ATPase activities are not manifestations of alkaline phosphatase, as earlier suggested, but rather result from the existence of one or several intrinsic membrane enzymes.

Key Words:

• rat
• jejunum
• brush-border membranes
• alkaline phosphatase
• Ca²⁺-ATPase
• Mg²⁺-ATPase