Freeze-stable sialidase activity in human leucocytes: 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 freeze-labile enzyme. In order to discriminate between the sialidase activities detected with the synthetic fluorimetric substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (MU-Neu5Ac), different tritiated sialoglycoconjugate substrates were prepared. Using this sensitive radioactive assay system, leucocyte sialidase activity towards glycoproteins was shown to be labile to repeated freeze-thawing, but a Triton-stimulated activity towards gangliosides was entirely freeze-stable. Assay conditions were optimized for this freeze-stable ganglioside sialidase activity. Subcellular fractionation of mononuclear leucocytes (MNLs) on Percoll-density gradients showed that this ganglioside sialidase activity was entirely associated with the plasma membrane. Study of the detergent requirements showed that MNLs also demonstrated ganglioside sialidase activity when sodium cholate was present in place of Triton. Cholate-stimulated ganglioside sialidase activity was found to be entirely freeze-stable and localized at the plasma membrane. Studies on whole homogenates of MNLs demonstrated that the Triton-stimulated and cholate-stimulated activities showed similar acidic pH optima at < or = 3.9 and were both strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and Cu2+, but not by free N-acetylneuraminic acid, N-(4-nitrophenyl)oxamic acid or heparan sulphate. These results suggest that human MNLs contain, in addition to the lysosomal freeze-labile sialidase, a single sialidase activity which is freeze-stable, ganglioside-specific, plasma membrane-associated and stimulated both by Triton and by cholate.
Waters
P J
PJ
Department of Biochemistry, Bath University, U.K.
Corfield
A P
AP
Eisenthal
R
R
Pennock
C A
CA
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D004795:Enzyme Stability; D005615:Freezing; D005732:Gangliosides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007962:Leukocytes / Q000201:enzymology* / Q000648:ultrastructure; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000097:blood*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011205:Povidone / Q000494:pharmacology; D012822:Silicon Dioxide / Q000494:pharmacology; D013379:Substrate Specificity
D002793:Cholic Acids; D003902:Detergents; D005732:Gangliosides; D011092:Polyethylene Glycols; C016039:Percoll; D012822:Silicon Dioxide; D009439:Neuraminidase; D011205:Povidone