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

Abstract

Some isoforms of secretory phospholipase A 2 (sPLA 2 ) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA 2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6 -48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA 2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform.During programmed cell death, or apoptosis, a variety of changes occur in the plasma membrane of the cell. These include morphological alterations that emerge late in the process such as blebbing and increased permeability of the membrane. Earlier in the process, several more subtle membrane changes occur. The best studied is a loss of the normal asymmetrical transmembrane distribution of phospholipid species. Consequently, anionic lipids like phosphatidylserine, which are typically confined to the inner leaflet of the membrane, become exposed on the outer surface (1). In addition, studies with fluorescent membrane probes have revealed possible increases in fluidity and/or the spacing between lipid molecules that may precede or coincide with the loss of membrane asymmetry, depending on the cell type and mode of apoptosis (2-9). Recently, a latent increase in the order of membrane lipids has also been reported (9).A potential consequence of these events during apoptosis is enzymatic attack of the cell membrane by secretory phospholipase A 2 (sPLA 2 ).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). Prelimina...

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A2 using cells deficient in the expression of scramblase

“…This source of enzyme was selected because of its previous use in biophysical studies of cell death by calcium ionophore and because of its strong similarity to the human group X sPLA 2 , the isoform that responds best to ionophore-stimulated cells [10,16,17]. We verified these assertions for human lymphocytes using samples of human groups IIa, V, and X sPLA 2 generously provided by Michael Gelb (University of Washington) and Wonhwa Cho (University of Illinois, Chicago).…”

“…This sensitivity is important because it prevents damage to the membranes of normal healthy mammalian cells while allowing efficient catalysis of bacterial membranes and those of damaged or dying cells [2–6]. In particular, the membranes of several types of cultured leukocytes have been reported to convert from being resistant to becoming susceptible to the action of the enzyme during apoptosis and other forms of biochemical cell death [4,5,7–10]. …”

“…Nevertheless, important clues have been identified from biophysical studies of both artificial bilayers and living cells [6–8,10–18]. Two prominent candidates have emerged: 1) a reduction in the strength of interactions among adjacent phospholipids resulting in increased lipid mobility and greater interlipid spacing and 2) the presence of anionic lipids on the membrane surface [6,10,14,19].…”

“…Nevertheless, important clues have been identified from biophysical studies of both artificial bilayers and living cells [6–8,10–18]. Two prominent candidates have emerged: 1) a reduction in the strength of interactions among adjacent phospholipids resulting in increased lipid mobility and greater interlipid spacing and 2) the presence of anionic lipids on the membrane surface [6,10,14,19]. The hypothesis is that reduced interactions among neighboring phospholipids allow substrates to migrate into the active site of adsorbed sPLA 2 , while negative charge facilitates initial adsorption of the enzyme to the membrane surface [11,18,20–22].…”

“…However, during apoptosis and other forms of cell death, PS is translocated to the outer leaflet [23–25]. Consequently, the exposed PS functions as a marker for processes that eliminate the dying cell such as phagocytosis [24,26–28] and, perhaps, hydrolysis by sPLA 2 [10,18,19]. Arguments for the importance of lipid-neighbor interactions and of surface PS are accumulating, although the existing evidence from cells depends on temporal correlations rather than direct experimental manipulations [6–8,10,14].…”