Cytochemical demonstration of sodium, potassium-adenosine triphosphatase by a hemepeptide derivative of ouabain

Abstract

A cytochemical probe for the ultrastructural localization of the NaKATPase was devised, which utilizes the biological affinity of the noncompetitive inhibitor, ouabain (ouab) to which was coupled a hemepeptide (H11P) which possesses peroxidatic activity. The conjugate, ouab-H11P, had an apparent K(i) of ~ 8 x 10-7 M. When reacted with fixed tissue from the salt gland of osmotically stressed ducklings, the NaKATPase was localized to the basal and lateral infoldings of the plasma membranes of secretory epithelial cells. Reaction product consisted of fine textured deposits distributed in focal patches on the outer aspects of the membrane. Apical membranes were negative, as were intracellular membrane components. Preincubation of tissue with unlabeled ouabain or binding of ouab-H11P in the presence of 10 mM K+, no ATP and no Mg++, resulted in the absence or diminution of reaction product.