FASTER HISTOCHEMICAL REACTION FOR ATPASE IN PRESENCE OF CHLORIDE SALTS, WITH STUDIES OF THE MECHANISM OF PRECIPITATION

Abstract

A very rapid localization of nucleoside triphosphate phosphohydrolase activity is given by incubation mixtures containing 2 M chlorides of monovalent cations. An optimal procedure is to fix overnight in cold formaldehyde-calcium, store in gum-sucrose, freeze-section and incubate 4 min or less at 30°C in a solution of 3 mM Na2 adenosine triphosphate (ATP), 2 M KCl, 3 mM MgCl2, and 1 mM Pb(NO3)2 in 24 mM Tris buffer adjusted to pH 7.2 with HCl. In rat intestine, for example, a strong, selective stain was developed in myenteric plexus, blood vessels, lamina propria mucosae and smooth muscle, while paired preparations without 2 M KCl were pale or unstained. The reaction was specific to nucleoside triphosphates and required Mg++. Acceleration by 2 M chloride was also found in the adenosine triphosphate phosphohydrolase reaction given by a single protein fraction (thin disc) in acrylamide gel electropherograms. The histochemical reaction for ATP phosphohydrolase produced residues of lead in formaldehyde-fixed sections, with two-thirds of lead captured as a precipitate of products of enzyme reaction and one-third bound directly to tissue. Only the enzyme-generated precipitate gave an anatomically and chemically selective and time-dependent pattern of staining, while direct binding gave a diffuse and time-independent background stain enhanced by omitting the substrate. Less than 1% of precipitate originated from nonenzymatic breakdown of ATP. Conditions of precipitation were studied. Reaction products were captured by lead as mixed crystals of nucleotides and phosphate. Addition of chloride salts decreased the concentration of lead required to saturate the reaction mixture, and this effect probably accounted for the enhanced histochemical reaction.