Characterization and localization of phospholipase A2 activity in sympathetic ganglia.
Superior cervical ganglion phospholipase A2 activity was characterized using 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme activity exhibited linearity with interval of incubation and tissue concentration; there appeared to be two pH optima of the enzyme, at pH 6.0 and 9.0. A Lineweaver-Burk plot of the reciprocal of activity versus substrate concentration yielded an apparent Km of 0.53 mM and a Vmax of 5.3 nmol/h/mg of protein. The enzyme exhibited a partial Ca2+ dependence; in the absence of Ca2+ and presence of EGTA, activity was reduced by 40%. The phospholipase A2 activity was heat sensitive and was completely inactivated after treatment at 100 degrees C for 30 min. For determination of whether the enzyme had a preference for hydrolysis of specific fatty acid substituents in the 2 position of phosphatidylcholine, several different substrates were tested. The order of preference for hydrolysis by the ganglionic enzyme was 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine = 1-palmitoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-2-[1-14C]palmitoyl-sn-glycero-3-phosphocholine. For determination of the localization of the phospholipase A2 enzyme in sympathetic ganglia, two approaches were used. Guanethidine, which results in destruction of adrenergic cell bodies in sympathetic ganglia, was administered to rats; an approximately 50% decline in phospholipase A2 activity was observed after this treatment. In other experiments, the preganglionic nerve to the ganglion was sectioned in rats; after 2 weeks of denervation, no significant change in ganglionic phospholipase A2 activity was seen.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003714:Denervation; D004285:Dogs; D005728:Ganglia, Sympathetic / Q000187:drug effects / Q000201:enzymology*; D006145:Guanethidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium; D006145:Guanethidine