Effects of phospholipases C from bacteria on binding of enkephalin to rat brain membranes

Abstract The effects of phospholipases C on the equilibrium constants and maximal binding capacities of tritiated [ d -Ala 2 ,- d -Leu 5 ] enkephalin to rat brain membranes were investigated using phosphatidylcholine-hydrolyzing phospholipase C and sphingomyelinase C of Bacillus cereus and, phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis . When 72% of the phosphatidylinositol in the rat brain membranes was hydrolyzed by phosphatidylinositol-specific phospholipase C, the affinity of opiate receptor for [ d -Ala 2 ,- d -Leu 5 ] enkephalin was almost doubled and maximal binding of [ d -Ala 2 ,- d -Leu 5 ] enkephalin was decreased to 87% of control. Although specific [ d -Ala 2 ,- d -Leu 5 ] enkephalin binding was decreased with phosphatidylinositol hydrolysis when measured at higher concentration (30 nM) of [ d -Ala 2 ,- d -Leu 5 ] enkephalin, the specific binding was increased with the hydrolysis of phosphatidylinositol when measured at lower concentration (6 nM) of the ligand. On treatment of membranes with phosphatidylcholine-hydrolyzing phospholipase C, specific [ d -Ala 2 ,- d -Leu 5 ] enkephalin binding was drastically decreased with the progressive hydrolysis of phosphatidylcholine in the rat brain membranes, and specific binding was completely lost after 81% hydrolysis of phosphatidylcholine. However, the affinity of opiate receptor for [ d -Ala 2 ,- d -Leu 5 ] enkephalin was not influenced, and maximal binding was decreased to 32% of the control when 61% of phosphatidylcholine was hydrolyzed. Treatment with sphingomyelinase C did not cause any appreciable reduction of specific [ d -Ala 2 ,- d -Leu 5 ] enkephalin binding. From these results, it is concluded that the binding of [ d -Ala 2 ,- d -Leu 5 ] enkephalin to opiate receptor is influenced by changes in the phospholipid environment of the rat brain membranes, and that phosphatidylinositol may be a modulator for the function of the receptor.