PROPERTIES OF PHOSPHOLIPASE-A(2) ACTIVITY FROM BOVINE RETINAL ROD OUTER SEGMENTS

In the present paper the properties of a phospholipase A2 (PLA2) activity associated with rod outer segments (ROS) have been studied. Under adequate experimental conditions, ROS PLA2 activity presented a maximum at pH 9.0. When using 1-palmitoyl-2[1-14C]arachidonoyl-sn-glycero-3-phosphocholine as substrate, the apparent K(m) value was 36.5 μM and the V(max) value was 0.612 nmol h-1 (mg protein)-1. The enzyme was fully activated at free calcium concentrations in the range 100-300 μM. Concentrations of CaCl2 above 1 mM inhibited its activity as a function of the ion concentration. The presence of EGTA or EDTA caused a 73% inhibition of PLA2 activity in ROS relative to the activity observed when no calcium was added. Treatment of the membranes with different kinds of detergents (Triton X-100, sodium deoxycholate and CHAPS) at concentrations below and above their critical micelle concentration resulted in an inhibition of PLA2 activity. However, when Triton X-100 was present at a concentration of 0.05 mM, no significant change in enzymatic activity could be observed. Maximum inhibition was observed in the presence of CHAPS 25 mM (87%). Seventy-five percent of PLA2 activity was recovered in ROS membranes after extraction of soluble and peripheral proteins. When retina phospholipids labelled with [3H]oleic acid and [3H]arachidonic acid were used as substrates,(diradyl)-ethanolamine glycerophospholipids (EtnGpl) were hydrolysed more efficiently than phosphatidylcholine (PtdCho). Moreover, hydrolysis of both phospholipids was stimulated when the substrates presented a higher degree of unsaturation in their fatty acyl components. © 1993 Academic Press, Inc.