Red Cell Hydrolases

Abstract

Human erythrocyte plasma membranes solubilized in 0.1% Triton X-100, with tritium-labeled 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid fetuin as substrate, were found to contain considerable neuraminidase activity. The enzyme was purified 470-fold, with 17% recovery, by centrifugation and gel chromatography on Sephadex G-100 and Sephadex G-150. The highest activity of the purified enzyme was at pH 4.2, but the neuraminidase was active over a broad pH range. No cofactors were necessary for neuraminidase activity ; the enzyme was inhibited by 0.05 m Ca acetate, 0.1 m CuSO(4), 0.01 m FeCl(3), 0.01 m FeCl(2), 0.01 m HgCl(2), and 0.005 m p-chloromercuribenzene sulfonate. The purified enzyme had maximal activity at 30-40 °C, and a linear relationship was demonstrated for activity versus time and for activity versus amount of enzyme. Using the Eadie-Hofstee plot and a fetuin molecular weight of 48,000, a V(max) of 4.5 X 10^4 cpm · h^-1 · mg^-1, and a Km of 9 x 10^-6 m were calculated. On whole-cell electrophoresis, it was demonstrated that purified human erythrocyte plasma membrane neuraminidase, like Clostridium perfringens neuraminidase, could cause release of terminal sialic acid residues from the external surfaces of intact human erythrocytes.