Effects of Cholesterol on Physical Properties of Human Erythrocyte Membranes: Impact on Susceptibility to Hydrolysis by Secretory Phospholipase A₂

¹ Brigham Young University

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

Submitted on July 26, 2007
Revised on September 5, 2007
Accepted on 6 December 2007

	Abstract

The ability of secretory phospholipase A₂ (sPLA₂) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA₂ activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA₂. To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl--cyclodextrin (MBCD). Treatment of cells with MBCD increased the hydrolysis rate and total product hydrolyzed by sPLA₂. In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA₂ activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the hydrolysis rate and altered the relationship with membrane order. These data demonstrated that interactions with sPLA₂ observed in artificial bilayers apply to biological membranes. It is also proposed that the high level of cholesterol in erythrocyte membranes is a protective mechanism to guard against hydrolytic enzymes.

Key Words: ionomycin, kinetics, laurdan, methyl-beta-cyclodextrin, temperature, two-photon microscopy