During apoptosis, changes occur in T-lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A2 (sPLA2). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was first applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: initial hydrolysis rate was elevated, total reaction product was increased four-fold, 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 substrate availability rather than enzyme adsorption. Fluorescence experiments identified three membrane alterations that might affect substrate access to the sPLA2 active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting increased lipid spacing. Second, laurdan detected increased solvation of the lower head group 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 was proposed that the apoptotic membranes become susceptible to sPLA2 through a reduction in lipid-neighbor interactions which facilitates migration of phospholipids into the enzyme active site. This proposal was then examined in T-lymphocytes treated with glucocorticoid, a more physiologically relevant apoptotic stimulant, using similar techniques. The following observations corresponded to induction of membrane susceptibility: increased merocyanine 540 intercalation; phosphatidylserine flip-flop, detected by annexin binding; and alterations in laurdan fluorescence properties. These observations implied a relationship among sPLA2 susceptibility, lipid spacing, and phosphatidylserine exposure. To clarify this relationship, additional assays were also performed using dibutyryl-cAMP to induce apoptosis, a drug reported to induce apoptosis in S49 cells without the typical translocation of phosphatidylserine. Our results indicated that in cells treated with dibutyryl-cAMP, the merocyanine 540 response and its correlation with sPLA2 susceptibility was similar to that observed with dex-treated samples. This suggests that the underlying mechanisms which promote sPLA2 hydrolysis lead to alterations that may be facilitated by but do not require phosphatidylserine exposure. Taken together, all of the results suggest that direct regulation of the biophysical microenvironment of the membrane is the mode of control of membrane susceptibility to the hydrolytic activity of sPLA2.



College and Department

Life Sciences; Physiology and Developmental Biology



Date Submitted


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apoptosis, cell membrane, hydrolysis, spectroscopy, flow cytometry, S49 cell, membrane biophysics