Isolation and Characterization of Acylneuraminate Cytidylyltransferase from Frog Liver

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Abstract
Frog liver (R. esculenta) is a rich source of acylneuraminate cytidylyltransferase. The soluble enzyme was purified 250-fold almost to purity with 25% yield and a specific activity of 9 mkat[milliKatals]/kg protein (0.54 U[units]/mg protein) using DEAE Sephadex and Sepharose 6B chromatography, followed by preparative polyacrylamide gel electrophoresis. The MW of the cytidylyltransferase was determined to be 163,000 with the aid of Sepharose 6B chromatography and gel electrophoresis, with or without dodecyl sulfate or urea. No subunits were found. The isoelectric point of the enzyme is at pH 6. Optimum reaction rate was observed at pH 9, 37.degree. C, 50 mM Mg2+ or Ca2+ and 1 mM mercaptoethanol. The Km values for N-acetylneuraminic acid, N-glycoloylneuraminic acid and CTP are 1.6 mM, 2.3 mM and 0.6 mM, respectively. O-Acetylated sialic acids are inactive with the cytidylytransferase from frog liver. Enzyme activity can be inhibited by SH reaggents and CMP (Ki = 0.5 mM).