Formation of 1,3-cyclic glycerophosphate by the action of phospholipase C on phosphatidylglycerol.

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.