Lister M D, Crawford-Redick C L, Loomis C R
Sphinx Pharmaceuticals Corporation, Biomolecular Research, Durham, NC 27717.
Biochim Biophys Acta. 1993 Jan 10;1165(3):314-20. doi: 10.1016/0005-2760(93)90142-v.
A neutral pH-optimum sphingomyelinase (N-SMase), solubilized from rat brain membranes, was characterized with respect to metal and membrane lipid effects. Chromatofocusing chromatography, which separates proteins according to pI, showed two N-SMase activities. One eluted at pH 4.7 and the other required 0.4 M NaCl before elution. Kinetically, the two preparations appeared similar. The N-SMase eluting at pH 4.7 was most extensively studied here. Of the phospholipids studied, only phosphatidylserine showed any influence on N-SMase and that was to increase its activity by as much as 50%. Neither serine nor phosphatidic acid had any effect. Of the cations tested, none was able to replace Mg2+ as a required activator. However, it was found that several metals were inhibitory, with Cu2+ being most effective (IC50 = 5 microM). Gangliosides, particularly the monosialoganglioside, GM3 (IC50 approximately 50 microM), inhibited N-SMase. Other glycolipids showed little effect on activity, even the immediate precursor to GM3 - lactosylceramide. The ganglioside sugar, N-acetylneuraminic acid, also had no effect on N-SMase activity. None of these inhibitors affected the acidic pH-optimum sphingomyelinase. Other sphingolipid compounds such as ceramide - the enzymatic product - and sphingosylphosphorylcholine (lysosphingomyelin) showed no capacity to inhibit N-Smase, implying that the enzyme may have a selective substrate-binding site.
从大鼠脑膜中溶解得到的一种最适pH为中性的鞘磷脂酶（N-SMase），就金属和膜脂的影响进行了特性研究。根据蛋白质等电点进行分离的色谱聚焦层析显示有两种N-SMase活性。一种在pH 4.7时洗脱，另一种在洗脱前需要0.4 M NaCl。从动力学上看，这两种制剂表现相似。这里对在pH 4.7时洗脱的N-SMase进行了最广泛的研究。在所研究的磷脂中，只有磷脂酰丝氨酸对N-SMase有任何影响，且这种影响是将其活性提高多达50%。丝氨酸和磷脂酸均无任何作用。在所测试的阳离子中，没有一种能够替代Mg2+作为必需的激活剂。然而，发现几种金属具有抑制作用，其中Cu2+最为有效（IC50 = 5 μM）。神经节苷脂，尤其是单唾液酸神经节苷脂GM3（IC50约为50 μM），抑制N-SMase。其他糖脂对活性几乎没有影响，甚至GM3的直接前体——乳糖基神经酰胺也是如此。神经节苷脂糖N-乙酰神经氨酸对N-SMase活性也没有影响。这些抑制剂均不影响最适pH为酸性的鞘磷脂酶。其他鞘脂类化合物，如酶促产物神经酰胺和鞘氨醇磷酸胆碱（溶血鞘磷脂），没有抑制N-Smase的能力，这意味着该酶可能有一个选择性底物结合位点。
