Becker G W, Lester R L
J Bacteriol. 1980 Jun;142(3):747-54. doi: 10.1128/jb.142.3.747-754.1980.
Incubation of membranes prepared from Saccharomyces cerevisiae with [32P]phosphatidyl[3H]inositol resulted in the transfer of both labels to two products which were characterized as two species of inositolphosphoceramide, differing in the ceramide portion of the molecule. The products were characterized on the basis of stability in mild alkali, mobility on silica gel-impregnated paper, chromatography on silicic acid columns, and release of inositol phosphate upon base hydrolysis. The reaction did not require the addition of metals, nor was it inhibited by ethylenediaminetetraacetic acid. The detergents Triton X-100 and Tween 20 provided little, if any, stimulation. At relatively high concentrations of phosphatidylinositol (1 to 4 mM), the in vitro rate was about 20% of the in vivo rate. Although ceramide was a logical substrate, the reaction could not be greatly stimulated by the addition of ceramides containing mono- and dihydroxy fatty acids. In addition, incubation of yeast membranes with [32P]phosphatidylinositol gave rise to a product that was chromatographically indistinguishable from the major yeast phosphosphingolipid, mannose-(inositol-P)2 ceramide.
用[32P]磷脂酰[3H]肌醇孵育酿酒酵母制备的膜，导致两种标记物都转移到两种产物上，这两种产物被鉴定为两种肌醇磷酸神经酰胺，它们在分子的神经酰胺部分有所不同。这些产物根据在弱碱中的稳定性、在硅胶浸渍纸上的迁移率、在硅酸柱上的色谱分析以及碱水解后肌醇磷酸的释放来进行鉴定。该反应不需要添加金属，也不受乙二胺四乙酸的抑制。去污剂Triton X - 100和吐温20即使有刺激作用也很小。在相对高浓度的磷脂酰肌醇（1至4 mM）下，体外反应速率约为体内反应速率的20%。虽然神经酰胺是合理的底物，但添加含有单羟基和二羟基脂肪酸的神经酰胺并不能极大地刺激该反应。此外，用[32P]磷脂酰肌醇孵育酵母膜会产生一种在色谱上与主要的酵母磷酸鞘脂甘露糖 - （肌醇 - P）2神经酰胺无法区分的产物。
