Cytidine 5′-Monophosphosialic Acid Hydrolase: SUBCELLULAR LOCATION AND PROPERTIES

Abstract

CMP-sialic acid hydrolase catalyses the reaction: CMP-sialic acid → sialic acid + (CMP) Enzymatic activity was measured by following both the disappearance of substrate, CMP-[14C]sialic acid, and the appearance of product, [14C]sialic acid, after separation by ion exchange column chromatography. The stoichiometry of this relationship was 1:1. The activity of CMP-sialic acid hydrolase in crude homogenates of rat liver (in 0.25 m sucrose) was 53 units of enzyme per g of liver. The specific activity was 0.29 unit per mg of protein. It was demonstrated that CMP-sialic acid hydrolase is an enzyme of the plasma membrane. The yield of the hydrolase and its increase in specific activity in plasma membranes was similar to that of marker enzymes for this subcellular site. About 25% recovery of the hydrolase was obtained in purified plasma membranes, accompanied by 13- to 18-fold increases in specific activity. The presence of the hydrolase in nuclear and microsomal fractions could be accounted for by the presence of plasma membranes in these fractions. Liver and kidney were the most active of 13 tissues that were examined. The apparent Km (CMP-sialic acid) was 0.40 m m . In Tris-HCl buffers, the pH optimum was about 9.0. The action of the hydrolase was not reversible. No metal requirement could be demonstrated. The hydrolase was inhibited by nucleosides, nucleotides, sugar nucleotides, EDTA, PPi, and compounds that contain free sulfhydryl groups. UDP-galactose and UDP-N-acetylglucosamine acted as competitive inhibitors, having inhibition constants of about 0.3 m m . The inhibition constant for glutathione was about 5 m m . The enzyme was unaffected by —SH-reactive reagents, free sialic acid, sugars, sugar 1-phosphates, and Pi.