Abstract
The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.
Highlights
THEJOURNAOFLBIOLOGICAL CHEMISTRYVOl. 267, No, 18, Issue of June 25, pp. 12673-12681,1992 Printed in U.S.A. Role of Carnitine and Carnitine PalmitoyltransferaasseIntegral Components of the Pathway foMr embrane PhospholipidFatty Acid Turnover in IntacHt uman Erythrocytes*
The deacylationand reacylation procesosf phospho- zymes for palmitate and oleatew, hich support the imlipids is the major pathway of turnover and repair in portance of carnitine palmitoyltransferase inmodulaterythrocyte membranes
ATP depletion decreased incorporation of [l-’4C]pal- shown that suchenzymes use substrates from the inner hemmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, ileaflet of the erythrocyte membrane [11]and that the reabuttheincorporationinto phosphatidylcholine and cylating activity ismainly expressed at thesecond position of phosphatidylethanolamine was unaffected
Summary
VOl. 267, No, 18, Issue of June 25, pp. 12673-12681,1992 Printed in U.S.A. Role of Carnitine and Carnitine PalmitoyltransferaasseIntegral Components of the Pathway foMr embrane PhospholipidFatty Acid Turnover in IntacHt uman Erythrocytes*. Identification of ALC intheCourse of theReacylation Study-Erythrocyte membrane phospholipid fatty acid turnover can be examined by following the incorporation of the erythrocytes do not possess any metabolic activity toward radiolabeled fatty acid into phospholipid fractions of a red fatty acids, except for the ability to transfer the acyl moiety cell suspension previously exposed to BSA-radioactive fatty to different acceptor molecules, such as CoA, carnitine, and acid complex. Erythrocyte suspensions were incubated at 37 "C with [l-'4C]palmitic acid complexed to fatty acid-free BSA, and at fixed times aliquots were removed and processed as describedunder "Experimental Procedures." Results are given as picomoles of [l-'4C]palmitic acid/pg of lipid phosphorus present in each phospholipid fraction. This competition study offers additional clues on the potential modulatory activity of carnitine palmitoyltransferase on erythrocyte membrane phospholipid fatty acid turnover.Infact,the lower palmitate incorporationinto phospholipids fits well with the higher. Determinations of radiolabeled fatty acidincorporation into membrane PC, PE, andALC were carried out as described in Fig. 2, and the results arexpressed as percentage of control
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