Mechanisms Governing the Level of Susceptibility of Erythrocyte Membranes to Secretory Phospholipase A₂

¹ Brigham Young University
² Utah Valley State College

^* To whom correspondence should be addressed. E-mail: john_bell{at}byu.edu.

Submitted on November 20, 2004
Revised on December 28, 2004
Accepted on 21 January 2005

	Abstract

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A2 (sPLA2), they become susceptible during apoptosis or following cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of calcium ionophore to cause susceptibility depends on temperature, occurring best above about 35 °C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA2 on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA2 was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 °C. Calcium loading enhanced packing at temperatures in the low end of this range but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.

Key Words: NBD, enzyme binding, enzyme kinetics, lipid packing, membrane properties, merocyanine 540

This article has been cited by other articles:

				B. M. Stott, M. P. Vu, C. O. McLemore, M. S. Lund, E. Gibbons, T. J. Brueseke, H. A. Wilson-Ashworth, and J. D. Bell Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes J. Lipid Res., June 1, 2008; 49(6): 1202 - 1215. [Abstract] [Full Text] [PDF]

				A. L. Heiner, E. Gibbons, J. L. Fairbourn, L. J. Gonzalez, C. O. McLemore, T. J. Brueseke, A. M. Judd, and J. D. Bell Effects of Cholesterol on Physical Properties of Human Erythrocyte Membranes: Impact on Susceptibility to Hydrolysis by Secretory Phospholipase A2 Biophys. J., April 15, 2008; 94(8): 3084 - 3093. [Abstract] [Full Text] [PDF]

				R. W. Bailey, E. D. Olson, M. P. Vu, T. J. Brueseke, L. Robertson, R. E. Christensen, K. H. Parker, A. M. Judd, and J. D. Bell Relationship between Membrane Physical Properties and Secretory Phospholipase A2 Hydrolysis Kinetics in S49 Cells during Ionophore-Induced Apoptosis Biophys. J., October 1, 2007; 93(7): 2350 - 2362. [Abstract] [Full Text] [PDF]

				H. A. Wilson-Ashworth, Q. Bahm, J. Erickson, A. Shinkle, M. P. Vu, D. Woodbury, and J. D. Bell Differential Detection of Phospholipid Fluidity, Order, and Spacing by Fluorescence Spectroscopy of Bis-pyrene, Prodan, Nystatin, and Merocyanine 540 Biophys. J., December 1, 2006; 91(11): 4091 - 4101. [Abstract] [Full Text] [PDF]

				C. Leidy, L. Linderoth, T. L. Andresen, O. G. Mouritsen, K. Jorgensen, and G. H. Peters Domain-Induced Activation of Human Phospholipase A2 Type IIA: Local versus Global Lipid Composition Biophys. J., May 1, 2006; 90(9): 3165 - 3175. [Abstract] [Full Text] [PDF]