Effect of phospholipase digestion and lysophosphatidylcholine on dopamine receptor binding.
[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.
D000818:Animals; D002090:Butyrophenones / Q000378:metabolism*; D002421:Caudate Nucleus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D008861:Microsomes / Q000378:metabolism*; D009439:Neuraminidase / Q000494:pharmacology; D010740:Phospholipases / Q000494:pharmacology*; D010741:Phospholipases A / Q000494:pharmacology*; D054467:Phospholipases A2; D011954:Receptors, Dopamine / Q000378:metabolism*; D012756:Sheep; D013134:Spiperone / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D002090:Butyrophenones; D008081:Liposomes; D008244:Lysophosphatidylcholines; D011954:Receptors, Dopamine; D013134:Spiperone; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D009439:Neuraminidase; D014357:Trypsin