FREEZE-STABLE SIALIDASE ACTIVITY IN HUMAN-LEUKOCYTES - SUBSTRATE-SPECIFICITY, INHIBITOR SUSCEPTIBILITY, DETERGENT REQUIREMENTS AND SUBCELLULAR-LOCALIZATION

Human leucocytes contain a freeze-stable sialidase (neuraminidase; EC 3.2.1.18) activity in addition to the better-characterized lysosomal f reeze-labile enzyme. In order to discriminate between the sialidase ac tivities detected with the synthetic fluorimetric substrate 4-methylum belliferyl-alpha-D-N-acetylneuraminic acid (MU-Neu5Ac), different trit iated sialoglycoconjugate substrates were prepared. Using this sensiti ve radioactive assay system, leucocyte sialidase activity towards glyc oproteins was shown to be labile to repeated freeze-thawing, but a Tri ton-stimulated activity towards gangliosides was entirely freeze-stabl e. Assay conditions were optimized for this freeze-stable ganglioside sialidase activity. Subcellular fractionation of mononuclear leucocyte s (MNLs) on Percoll-density gradients showed that this ganglioside sia lidase activity was entirely associated with the plasma membrane. Stud y of the detergent requirements showed that MNLs also demonstrated gan glioside sialidase activity when sodium cholate was present in place o f Triton. Cholate-stimulated ganglioside sialidase activity was found to be entirely freeze-stable and localized at the plasma membrane. Stu dies on whole homogenates of MNLs demonstrated that the Triton-stimula ted and cholate-stimulated activities showed similar acidic pH optima at less than or equal to 3.9 and were both strongly inhibited by 2-deo xy-2,3-didehydro-N-acetylneuraminic acid and Cu2+, but not by fret N-a cetylneuraminic acid, N-(4-nitrophenyl)oxamic acid or heparan sulphate . These results suggest that human MNLs contain. in addition to the ly sosomal freeze-labile sialidase, a single sialidase activity which is freeze-stable, ganglioside-specific, plasma membrane-associated and st imulated both by Triton and by cholate.