The quantitative dependence of glucose-6-phosphohydrolase upon phospholipids: Effects of phospholipase C at 50 degrees and 370 degrees.

Since the classical, pioneering studies of Beaufay and De Duve [ 11, it has been known that glucose-6phosphohydrolase (G-6-Pase) is a phospholipid-dependent enzyme. However, the specificity of this dependence is still unknown. Duttera et al. [2] found that Cl. welchii phospholipase C, which hydrolyses microsomal phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin, causes 80-90% inhibition of G-6-Pase. Addition of PE or lysolecithin regenerated G-6-Pase activity in phospholipase treated microsomes but PC did not. Our investigations of the quantitative dependence of G-6-Pase upon PC and PE show that in microsomes treated with phospholipase C at 37” there is a fairly close correlation between PC hydrolysis (90-100%) and loss of G-6-Pase activity (95-lOO%), but no apparent relationship with PE hydrolysis which plateaus at 50-70%. When, however, phospholipase C treatment was done at 5” the situation was reversed: G-6Pase plateaued at 40-60% of its initial activity like PE while 90-95% PC hydrolysis was still obtained as at 37’. In addition, unlike the situation after phospholipase treatment at 20” [2] , PC completely restored the G-6-Pase activity lost after 5” treatment. The different effects of phospholipase C treatment at 5”, 20”and 37” do not reflect difference in phospholipid hydrolysis, but are related to heat inactivation. Postincubation at.20’ or 37”) of 5’ phospholipase treated microsomes after the addition of EGTA, leads to loss of G-6-Pase approximately proportional