A QUANTITATIVE BIOCHEMICAL AND HISTOCHEMICAL STUDY OF THE LEAD METHOD FOR LOCALIZATION OF ADENOSINE TRIPHOSPHATE-HYDROLYZING ENZYMES

Abstract

The purpose was to study the nature of the adenosine triphosphatase (ATPase) localized to plasma membranes by the lead method and the significance of the lead-catalyzed hydrolysis of adenosine triphosphate (ATP) for the staining of fixed kidney. A modified Wachstein and Meisel's medium was used. The staining of sections of microsomal sediments from rat kidney was compared with the in vitro activity of (Na⁺ + K⁺)–, Mg⁺⁺– and Ca⁺⁺– ATPase in the presence of lead. The histochemical relevance of these observations was tested on sections of rat kidney. Localization of (Na⁺ + K⁺)–ATPase was not possible at concentrations of lead below the inactivating level (0.5 mM). Mg⁺⁺–ATPase and Ca⁺⁺– ATPase were only partially inhibited by 3.6 mM lead and were localized to plasma membranes in glutaraldehyde-fixed kidney. The staining followed a course of activation by Ca⁺⁺ and Mg⁺⁺ characteristic for enzymatic hydrolysis and different from the activation of nonenzymatic hydrolysis of ATP. Staining of fixed tissue was maximal at 2-3 mM lead and decreased at higher concentrations. In Mg⁺⁺–deficient media membrane staining was weak or absent at 5.2-6.8 mM lead, where the rate of nonenzymatic hydrolysis of ATP was high. Thus, in the medium used the lead-catalyzed hydrolysis of ATP contributed little to the staining of fixed kidney.