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

Abstract

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees 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 sPLA(2) 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 sPLA(2) 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 degrees 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.

Kinetic Evaluation of Cell Membrane Hydrolysis during Apoptosis by Human Isoforms of Secretory Phospholipase A2

“…Instead, the increased v max in proportion to the number of cells engaged in cell death under these conditions (control Ͻ dexamethasone Ͻ ionomycin ϭ 100%) suggested that the second step in Scheme 1, substrate migration into the enzyme active site, was the one most facilitated by apoptosis. This conclusion substantiates interpretations reached previously for the AppD49 enzyme in model systems, human erythrocytes, and S49 cells treated with ionomycin (8,12,14,15,31).…”

“…3-5 can be described in terms of the proportions of two subpopulations of cells: the resistant healthy subpopulation and the susceptible subpopulation. The observed initial hydrolysis rate (dP/dt), then, is the sum of the rates pertaining to each subpopulation (Res and Sus) weighted by the fraction susceptible (f Sus ), Explicit generic descriptions of dP/dt that can be applied to each subpopulation are derived as described previously (8,12),…”

“…2 Ordinarily, healthy cells resist hydrolysis, but during apoptosis they become vulnerable to destruction by the enzyme (9 -11). Studies with snake venom phospholipase A 2 have identified possible ways by which this phenomenon relates to membrane physical properties (8,9,12). Preliminary investigations suggest that human groups IIa (hGIIa) and V (hGV) isoforms may also distinguish healthy and apoptotic cells, although the details of how they do so are uncertain (11,13,14).…”

Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

“…Indeed, the enzymatic activity of group X sPLA 2 has been shown to be influenced by the lipid composition of the plasma membrane, notably by the ratio between sphingolipids and phosphatidylcholine species (71). Furthermore, it has been shown that the enzymatic activity of several sPLA 2 s is dependent on lipid packing and phospholipid asymmetry (72). The ratio of phospholipids to sphingomyelin is changed during sperm maturation, and this is likely a way to control mGX activity on distinct sperm subpopulations.…”

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes

“…Human erythrocytes were obtained from blood samples remaining after physical exams at the Brigham Young University Student Health Center. For experiments, cells were washed and suspended in minimum basic salt solution [(MBSS) 134 mM NaCl, 6.2 mM KCL, 1.6 mM CaCl 2 , 1.2 mM MgCl 2 , 18 mM HEPES, 13.6 mM glucose, pH 7.4, at 37jC] at 3% of the original hematocrit (hct) as described previously (17).…”