Shinitzky M, Friedman P, Haimovitz R
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1993 Jul 5;268(19):14109-15.
The action of phospholipase C (PLC) from Bacillus cereus on phosphatidylglycerol (PG), derived from egg yolk phosphatidylcholine (PC), was examined in an ether-water mixture. The PLC cleavage of PG and PC followed a Michaelis-Menten kinetics with apparent Vmax values per 1 microgram enzyme of 0.26 and 0.91 mumol.min-1 and Km values of 10 and 12 mM, respectively. When the same enzymic reaction was carried out in minimally buffered aqueous solution of 1% Triton X-100, the decrease in pH with respect to phospholipid cleavage was as expected with PC but much less pronounced with PG. This could be accounted for by the formation of a cyclic glycerophosphate, rather than alpha-glycerophosphate, in the PLC hydrolysis of PG. Examination of the chemical nature of the water-soluble product of PG by phosphorus nuclear magnetic resonance (31P NMR) revealed a single band at 2.31 ppm, while the bands of alpha-glycerophosphate and beta-glycerophosphate appeared at 5.12 and 4.57 ppm, respectively. Basic hydrolysis of the phospholipase cleavage product of PG (0.1 M NaOH for 1 min at 80 degrees C) followed by neutralization shifted its 31P NMR band to 5.18 ppm, which practically coincided with that of alpha-glycerophosphate. Analogous experiments were carried out with PG labeled with 3H at the carbon 2 of the glycerol headgroup ([3H]PG). Autoradiography of thin layer chromatography (TLC) of the [3H]PG enzymic hydrolyzate displayed a single 3H-labeled compound, which could be converted to alpha-glycerophosphate by basic hydrolysis. These results strongly suggest that the phosphate headgroup of PG is cleaved off by PLC as 1,3-cyclic glycerophosphate. A series of PLC experiments with phosphatidyl dihydroxyacetone and phosphatidyl 1,3-propanediol as model substrates supported this assignment. Two-dimensional homonuclear 1H NMR correlated spectra as well as infrared spectra carried out on the isolated sodium salt of this product could further confirm such a structure. The unique structure and chemical nature of 1,3-cyclic glycerophosphate may bear a distinct physiological function.
研究了蜡样芽孢杆菌的磷脂酶C（PLC）在乙醚 - 水混合物中对源自蛋黄磷脂酰胆碱（PC）的磷脂酰甘油（PG）的作用。PG和PC的PLC裂解遵循米氏动力学，每1微克酶的表观Vmax值分别为0.26和0.91微摩尔·分钟⁻¹，Km值分别为10和12毫摩尔。当在含1% Triton X - 100的最低缓冲水溶液中进行相同的酶促反应时，相对于磷脂裂解的pH下降在PC中如预期的那样，但在PG中则不太明显。这可以通过在PG的PLC水解中形成环状甘油磷酸酯而不是α - 甘油磷酸酯来解释。通过磷核磁共振（³¹P NMR）检查PG的水溶性产物的化学性质，发现在2.31 ppm处有一个单峰，而α - 甘油磷酸酯和β - 甘油磷酸酯的峰分别出现在5.12和4.57 ppm处。PG的磷脂酶裂解产物（在80℃下用0.1 M NaOH处理1分钟）进行碱性水解然后中和后，其³¹P NMR峰移至5.18 ppm，这实际上与α - 甘油磷酸酯的峰重合。对甘油头部基团碳2位用³H标记的PG（[³H]PG）进行了类似的实验。[³H]PG酶促水解产物的薄层色谱（TLC）放射自显影显示有一个单一的³H标记化合物，该化合物可通过碱性水解转化为α - 甘油磷酸酯。这些结果强烈表明，PG的磷酸头部基团被PLC裂解为1,3 - 环状甘油磷酸酯。一系列以二羟基丙酮磷脂和1,3 - 丙二醇磷脂为模型底物的PLC实验支持了这一结论。对该产物的分离钠盐进行的二维同核¹H NMR相关光谱以及红外光谱可以进一步证实这种结构。1,3 - 环状甘油磷酸酯独特的结构和化学性质可能具有独特的生理功能。
