FORMATION OF 1,3-CYCLIC GLYCEROPHOSPHATE BY THE ACTION OF PHOSPHOLIPASE-C ON PHOSPHATIDYLGLYCEROL

The action of phospholipase C (PLC) from Bacillus cereus on phosphatid ylglycerol (PG), derived from egg yolk phosphatidylcholine (PC), was e xamined in an ether-water mixture. The PLC cleavage of PG and PC follo wed a Michaelis-Menten kinetics with apparent V(max) values per 1 mug enzyme of 0.26 and 0.91 mumol.min-1 and K(m) values of 10 and 12 mM, r espectively. When the same enzymic reaction was carried out in minimal ly buffered aqueous solution of 1% Triton X-100, the decrease in pH wi th respect to phospholipid cleavage was as expected with PC but much l ess pronounced with PG. This could be accounted for by the formation o f a cyclic glycerophosphate, rather than alpha-glycerophosphate, in th e PLC hydrolysis of PG. Examination of the chemical nature of the wate r-soluble product of PG by phosphorus nuclear magnetic resonance (P-31 NMR) revealed a single band at 2.31 ppm, while the bands of alpha-gly cerophosphate 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 neutralizatio n shifted its P-31 NMR band to 5.18 ppm, which practically coincided w ith that of alpha-glycerophosphate. Analogous experiments were carried out with PG labeled with H-3 at the carbon 2 of the glycerol headgrou p ([H-3]PG). Autoradiography of thin layer chromatography (TLC) of the [H-3]PG enzymic hydrolyzate displayed a single H-3-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 ex periments with phosphatidyl dihydroxyacetone and phosphatidyl 1,3-prop anediol as model substrates supported this assignment. Two-dimensional homonuclear H-1 NMR correlated spectra as well as infrared spectra ca rried out on the isolated sodium salt of this product could further co nfirm such a structure. The unique structure and chemical nature of 1, 3-cyclic glycerophosphate may bear a distinct physiological function.