Properties of phospholipase A2 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 Km value was 36.5 microM and the Vmax value was 0.612 nanomol per hour per mg protein. The enzyme was fully activated at free calcium concentrations in the range 100- 300 microM. Concentrations of CaCl2 above 1 milliM 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 milliM, no significant change in enzymatic activity could be observed. Maximum inhibition was observed in the presence of CHAPS 25 milliM (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.
Castagnet
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Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sury CONICET, Bahía Blanca, Argentina.
Giusto
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D000818:Animals; D002417:Cattle; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D004795:Enzyme Stability; D005227:Fatty Acids / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008563:Membrane Lipids / Q000032:analysis; D010741:Phospholipases A / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D054467:Phospholipases A2; D010743:Phospholipids / Q000032:analysis; D012160:Retina / Q000201:enzymology; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013379:Substrate Specificity
D005227:Fatty Acids; D008563:Membrane Lipids; D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2