Abstract
The optimal kinetic parameters for the K-dependent, ouabain-sensitive hydrolysis of p-nitrophenyl phosphate by K-nitrophenyl phosphatase, under conditions closely approximating those employed for cytochemistry, were determined in the avian salt gland as a necessary prerequisite for the ultrastructural localization of the enzyme. The enzyme was characterized in 50-µ cryostat sections of paraformaldehyde-fixed tissue, incubated at room temperature in a medium containing 5 mM nitrophenyl phosphate, 10 mM MgCl2, 20 mM SrCl2 and 100 mM Tris-HCl buffer (pH 9.0), either with or without 10 mM KCl. For comparison, parallel assays were conducted in the absence of Sr, the heavy metal salt used to precipitate phosphate for cytochemistry. Enzymatic activity was determined by measuring spectrophotometrically the amount of nitrophenol hydrolyzed. In this system, Sr acts as a pure noncompetitive inhibitor of the enzyme, causing 50% inhibition at 3 mM and 87% at 20 mM. The Km for the enzyme is 4.5 mM. Sr (20 mM) causes an 8-fold reduction in the apparent affinity of the enzyme for Mg but has little effect on K affinity. The sensitivity of the enzyme to ouabain is decreased 50-fold in the presence of 20 mM Sr. The relationship of this enzymatic activity to Na-K-activated adenosine triphosphatase and the application of this defined medium to transport adenosine triphosphatase cytochemistry are discussed.