We have recently shown that an endogenous phospholipase A2 from bovine erythrocytes does not hydrolyse NAPEs (N-acyl l-α-phosphatidylethanolamines), which accumulate remarkably in this system [Florin-Christensen, Suarez, Florin-Christensen, Wainszelbaum, Brown, McElwain and Palmer (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 7736–7741]. Here we investigate the causes underlying this resistance. N-acylation of PE (l-α-phosphatidylethanolamine) results in alteration of charge, head-group volume and conformation, the last two features depending on the N-acyl chain length. To evaluate each effect separately, we synthesized NAPEs with selected N-acyl chain length. We found that phospholipase A2 has considerable activity against N-acetyl PE, but is poorly active against N-butanoyl PE and only marginally active against N-hexanoyl PE, whereas the activity is completely lost when N-hexadecanoyl PE is presented as a substrate. On the other hand, N-hexanoyl PE does not inhibit phospholipase A2 activity, suggesting that this substrate fails to enter the hydrophobic channel. Phospholipase C presents a similar, but less sharp pattern. Molecular dynamics simulations of the polar head group of selected NAPEs reveal a substantially increased conformational variability as the N-acyl chain grows. This larger conformational space represents an increased impairment limiting the access of these molecules to the active site. Our data indicate that, whereas a change in charge contributes to diminished activity, the most relevant effects come from steric hindrance related to the growth of the N-acyl chain.
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August 2003
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Research Article|
August 15 2003
Phospholipase activity on N-acyl phosphatidylethanolamines is critically dependent on the N-acyl chain length
Julio J. CARAMELO;
Julio J. CARAMELO
∗Institute of Biophysics and Biochemistry (IQUIFIB, UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, RA-1113, Buenos Aires, Argentina
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Jorge FLORIN-CHRISTENSEN;
Jorge FLORIN-CHRISTENSEN
†Department of Microbiology, School of Medicine, University of Buenos Aires, Junín 956, RA-1113, Buenos Aires, Argentina
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José M. DELFINO
José M. DELFINO
1
∗Institute of Biophysics and Biochemistry (IQUIFIB, UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, RA-1113, Buenos Aires, Argentina
1To whom correspondence should be addressed (e-mail delfino@qb.ffyb.uba.ar).
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Publisher: Portland Press Ltd
Received:
November 26 2002
Revision Received:
May 02 2003
Accepted:
May 23 2003
Accepted Manuscript online:
May 23 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 374 (1): 109–115.
Article history
Received:
November 26 2002
Revision Received:
May 02 2003
Accepted:
May 23 2003
Accepted Manuscript online:
May 23 2003
Citation
Julio J. CARAMELO, Jorge FLORIN-CHRISTENSEN, José M. DELFINO; Phospholipase activity on N-acyl phosphatidylethanolamines is critically dependent on the N-acyl chain length. Biochem J 15 August 2003; 374 (1): 109–115. doi: https://doi.org/10.1042/bj20021840
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