Waters P J, Corfield A P, Eisenthal R, Pennock C A
Department of Biochemistry, Bath University, U.K.
Biochem J. 1994 Aug 1;301 ( Pt 3)(Pt 3):777-84. doi: 10.1042/bj3010777.
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.
除了特性更为明确的溶酶体冻融不稳定酶外，人类白细胞还含有一种冻融稳定的唾液酸酶（神经氨酸酶；EC 3.2.1.18）活性。为了区分用合成荧光底物4-甲基伞形酮基-α-D-N-乙酰神经氨酸（MU-Neu5Ac）检测到的唾液酸酶活性，制备了不同的氚标记唾液酸糖缀合物底物。使用这种灵敏的放射性检测系统，发现白细胞对糖蛋白的唾液酸酶活性对反复冻融不稳定，但经 Triton 刺激的对神经节苷脂的活性完全冻融稳定。针对这种冻融稳定的神经节苷脂唾液酸酶活性对检测条件进行了优化。在 Percoll 密度梯度上对单核白细胞（MNLs）进行亚细胞分级分离表明，这种神经节苷脂唾液酸酶活性完全与质膜相关。对去污剂需求的研究表明，当用胆酸钠代替 Triton 时，MNLs 也表现出神经节苷脂唾液酸酶活性。发现胆酸盐刺激的神经节苷脂唾液酸酶活性完全冻融稳定且定位于质膜。对 MNLs 全匀浆的研究表明，Triton 刺激的活性和胆酸盐刺激的活性在≤3.9 时显示出相似的酸性 pH 最佳值，并且都受到 2-脱氧-2,3-二脱氢-N-乙酰神经氨酸和 Cu2+的强烈抑制，但不受游离 N-乙酰神经氨酸、N-(4-硝基苯基)草氨酸或硫酸乙酰肝素的抑制。这些结果表明，除了溶酶体冻融不稳定的唾液酸酶外，人类 MNLs还含有一种单一的唾液酸酶活性，该活性冻融稳定、对神经节苷脂具有特异性、与质膜相关且受 Triton 和胆酸盐刺激。
