Activity and Positional Specificity of a Rat Plasma Phospholipase on Phosphatidylethanolamine

Abstract

Activity and positional specificity of a rat plasma phospholipase on phosphatidylethanolamine. Cm. J. Biochem. 51,855-862 ( 1973 ) .Lysophosphatidylethanolamine (LPE), rich in arachidonk acid, has been proposed by others as a physiologically important inhibitor of the renal enzyme renln that circulates in blood. Intravenously infused phosphatidylethanolamine (PE) has proposedly inhibited the acute pressor effect of injected renin in rats, suggesting a rapid conversion of PE to arachidmate-rich LPE by blood phospholipase activity. Therefore a definition of the activity was attersrpteb. Incubation at 37 "C, pH7.7-7.9, of a highly purified, aaP-labeled rat liver PE added to fresh rat plasma (heparin) in diethyl ether (0.2 ml/ml plasma) or sodium deoxycholate solution (5 mg/ml plasma) indicated that one phospholipase was dominant, and its activity greater when deoxycholate, rather than ether, was present. Lysophospholipase activity was negligible in either case. Two milliliters of plasma (with deoxycholate) deacylated 47% of 1.5 mg PE (approximately 2 pmol) during 24 min of incubation. Fresh serum had higher activity than corresponding plasma, suggesting activation associated with blood coagulation. C-2 specificity was demonstrated by selective removal of the label from 2-[1-C'4]arachidonoyl rat liver PE, without concurrent cholesterol esterification, and by virtually identical cornpositions (gas-liquid chromatography) of fatty acids removed from PE by plasma or by C-2 specific snake venom phospholipase (Crotalus atrox). Our data appear to rule out C-1 specific post-heparin phospholipase and also C-2 specific lecithin : cholesterol acyltransferase (LCAT) , and we present a plasma phospholipase Aa that is highly active against PE and creates an arachidonate-poor LPE. presumably not a renin inhibitor. OSMOND, D. H., HOLUB, B. J., et Ross, L. J. Activity and positional specificity of a rat plasma phospholipase on phosphatidylethanolamine. Can. 9. Biochern. 51, $55-862 (1973 ).D'apr&s certains auteurs, la lysophosphatidylCthanola~ne (LPE), riche en acide arachidonique, serait un important inhibiteur physiologique de Ia rCnine, enzyme rCnal en circulation dans le sang. L'infusion intraveineuse de phosphatidylCthanolamine (PE) inhiberait l'effet presseur aigu de la rCnine injectCe 5 des rats, ce qui suggZre une conversion rapide de la PE en LPE riche en arachidonate grAce A l'activitk ghospholipasique sanguine. Nous avons alors tent6 de dCfinir cette activitC. Une PE hautement purifiCe de foie de rat, marquCe au 8aP et ajoutCe 2 du plasma (hCparinC) frais de rat est incubBe 2 37 "C et A pH 7.7 A 7.9 dans 1'Cther dikthylique (0.2 ml/ml de plasma) ou dans une solution de dCsoxycholate de sodium (5 mg/ml plasma). On observe alors la prkdominance d'une phospholipase dont l9activitC est plus grande en prCsence du dCsoxycholate qu'en prCsence de l'Cther. L'activitC de la lysophospholipase est nCgligeable dans le deux cas. Deux ml de plasma (avec la dCsoxycholate) dhacyle 47% de 1.5 mg de PE (environ 2 pmol) en 24 min &incubatio...

Molecular Species of Ethanolamine Phosphatides of Dog and Pig Kidney

“…It might have a physiological activity different from that of the I -isomer containing largely saturated and nnonotansaturated fatty acids and released by an enzyme activity similar to that of pkospholipase A2 (30).…”

Some Characteristics of the Exchange of Lecithin between Rabbit Erythrocytes and Serum

“…Th ... post-hepar1n phospholipaàes have litt le of no (1 activity in normal plasma. By contrast, a simi1ar enzyme has tieen found in normal human plasma which hydrolyses lecithin (197) and in normal rat i 'plasma which hy4r01yse~ phosphatidylethanolamine (192). The reason for these appar~nt1y contradictory observations is unclear.…”

“…No es~erification or condehsation~ pathways analogous to those found in the erythrocyte have been observed~ysophospholipase activity appears to be negligible (191,192). In post-heparin plasma of man a phospholipase which o 0 sel~ctively hydrolyses phosph8tidy1ethanolamine has been described (193,194) and in heparinized rat plasma.a p~ospholipase,hydro1yses 1ecithin significant1y (195,196).…”

Plasma Phospholipase A2 Activity in Nephrectomized Rats and the Question of Renin Inhibition

“…Our incubation conditions were designed to provide an excess of sulbstrate ("preinhibitor") of which only a small proportion would be deacylated to form "inhibitor", in order to conform with requirements for linear kinetics (10). The underlying rationale was that plasma phospholipase activity would determine the proportions of "preinhibitor" and "inhibitor" existing at the end of the incubation period.…”

“…After twice washing with volumes of distilled water, the extract was concentrated in a flash evaporator under reduced pressure, quantitatively transferred to a thin layer silica gel chromatography (TLC) plate, and developed in chloroform : methanol: H20 (95:35:6, v/v/v) (4, 10, 11). After drying, the "preinhibitor" (PE) and "inhibitor" (LPE) spots were visualized with ninhydrin reagent, scraped off into counting vials, liqiiifluor added, and radioactivity determined (10,11). Baseline counts in the PE and LPE zones were derived from the control unincubated samples in each experiment; with these we compared the proportions of counts obtained after incubating labeled PE with plasma from sham-opera ted or nephrectomized rats, thus estimating relative phospholipase activities, Results.…”