Properties of phospholipase A2 activity from bovine retina rod outer segments

CASTAGNET, P.I.; GIUSTO, N.M.

EXPERIMENTAL EYE RESEARCH

ACADEMIC PRESS LTD-ELSEVIER SCIENCE LTD

Año: 1993 vol. 56 p. 709 - 719

0014-4835

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]araehidonoyl-sn-glycero-3-phosphocholine as substrate, the apparent Km value was 36·5 μM and the Vmax 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 [3 H]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.