Relationship between Membrane Physical Properties and Secretory Phospholipase A2 Hydrolysis Kinetics in S49 Cells during Ionophore-Induced Apoptosis

Abstract

During apoptosis, changes occur in lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A(2) (sPLA(2)). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: the initial rate of hydrolysis was elevated at all enzyme concentrations, the total amount of reaction product was increased fourfold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by availability of substrate rather than adsorption of enzyme. Fluorescence experiments identified three membrane alterations during apoptosis that might affect substrate access to the sPLA(2) active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting an increase in lipid spacing. Second, laurdan detected increased solvation of the lower headgroup region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it is proposed that the membranes of apoptotic cells become susceptible to sPLA(2) through a reduction in lipid-neighbor interactions that facilitates migration of phospholipids into the enzyme active site.

Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin

“…Previous studies suggested that membrane structure contributed to the level of membrane susceptibility to A. piscivorus piscivorus PLA 2 in addition to PS externalization (Bailey et al, 2007(Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. Nevertheless, the effect of membrane-interacted mode of PLA 2 enzymes and membrane compositions on PLA 2 activity does not explore in the studies of Bailey et al (2007Bailey et al ( , 2009 and Nelson et al (2011and Nelson et al ( , 2012. Nelson et al (2011) found that A. piscivorus piscivorus PLA 2 showed different activity on hydrolyzing the membrane of ionomycin-, dexamethasone-or thapsigargin-treated cells.…”

“…Previous research has reported that the membranes of cells undergoing apoptosis are susceptible to human secreted PLA 2 enzymatic activity (Atsumi et al, 1997). Bell and his colleagues suggest that PS externalization in concert with alteration of membrane structure increase the susceptibility of apoptotic cells to Agkistrodon piscivorus piscivorus PLA 2 and human secreted PLA 2 s (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. In their studies, ionomycin, dexamethasone or thapsigargin are used to induce apoptosis of cells, and thus the direct effect of these reagents on membrane structure and PLA 2 activity could not exclude out (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012.…”

“…Bell and his colleagues suggest that PS externalization in concert with alteration of membrane structure increase the susceptibility of apoptotic cells to Agkistrodon piscivorus piscivorus PLA 2 and human secreted PLA 2 s (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. In their studies, ionomycin, dexamethasone or thapsigargin are used to induce apoptosis of cells, and thus the direct effect of these reagents on membrane structure and PLA 2 activity could not exclude out (Harris et al, 2001;Bailey et al, 2007Bailey et al, , 2009Nelson et al, 2011Nelson et al, , 2012. Our previous studies revealed that Naja naja atra PLA 2 (NnaPLA 2 ) induced apoptosis of human leukemia K562 and U937 cells (Liu et al, 2009;Chen et al, 2010a), while Notechis scutatus scutatus notexin (presynaptic PLA 2 neurotoxin) induced apoptosis of human neuroblastoma SK-N-SH cells (Chen et al, 2010b).…”

Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains

“…Further results from our laboratory ( 34 ) have related the activity of phosphatidylinositol-specifi c PLC to the degree of molecular order in membranes. Also, Bell and co-workers ( 35,36 ) and Mouritsen and co-workers ( 37,38 ) have explored the relationship between PLA 2 activity and the physical state of the membrane. Holopainen et al ( 39 ) observed lipid microdomain formation that resulted from sphingomyelinase activity on fl uid membranes of PC/SM.…”

“…The present contribution is intended to provide a link between the bulk (in cuvette) studies of lipase activity on lipid bilayers in different physical states (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44) and the morphological data obtained largely with GUV of homogenous lipid compositions (45)(46)(47)(48)(49). This work is based on our previous studies of phase separation in GUVs ( 23 ) as well as our experience with PlcHR 2 ( 8,50 ).…”