Use of Phospholipid Transfer Protein as a Probe to Study the Lipid Dynamics and Alkaline Phosphatase Activity in the Brush Border Membrane of Human Term Placenta by Sankar N. Sanyal*, Meenakshi Goyal, Shailender S. Kanwar, and Shivani Kaushal Department of Biophysics, Panjab University, Chandigarh, 160014, India (phone: þ 91 172 2534119; e-mail: sanyalpu@gmail.com) Incubation of placental brush border membrane (BBM) along with sonicated vesicles of exogenous lipids (egg yolk PC) in the presence of phospholipid-transfer protein (PL-TP) showed a decrease in the alkaline phosphatase activity due to the change in the membrane micro-environment, such as fluidity. Effect of substrate concentration was tested by Lineweaver–Burk plot, which showed decreased V max and K M . The effect of temperature was probed by the Arrhenius plot, which showed no change in transition temperature, but a decline in the energy of activation both below and above the transition temperature. The protein-catalyzed transfer of phospholipid from the donor unilamellar vesicles resulted in a substantial increase in the BBM phospholipid and a net decrease in cholesterol/phospholipid molar ratio. The change in membrane fluidity was assessed by translational as well as rotational diffusion of membrane extrinsic fluorescent probes, pyrene and diphenyl-hexatriene. An increased lateral mobility was recorded by the increased pyrene excimer formation. A decrease in fluorescent polarization of diphenyl-hexatriene was observed, which led to the decrease in fluorescence anisotropy and order parameter, and therefore, an increase in membrane fluidity (rotational diffusion). Mean anisotropy parameter was also decreasedin the presence of PL-TP. Thus, the placental BBM alkaline phosphatase activity showed a distinct lipid dependence which may have important physiological consequences. Introduction. – The plasma membrane of placental syncytial trophoblast cells contains two distinct regions, the microvillus or brush border membrane (BBM), and the basal cell membranes, which are distinguished morphologically, biochemically, and functionally [1 – 4] . The BBM forms the effective barrier separating the maternal blood from the fetal circulation and is important in the exchange of nutrients, hormones, waste products, and other molecules between the mother and the fetus [5]. The membrane is also richly endowed with alkaline phosphatase activity, which is believed to be intimately associated with the phosphate transport process [6]. Functional dependence of the membrane-bound enzymes on lipid micro-environment has been a subject of continued research interest in this laboratory [7 – 9]. The phospholipid- transfer proteins (PL-TPs) have been used as excellent membrane probes in modifying the physicochemical properties and functional dependence of the membrane protein [10 – 12]. In earlier studies, the lipid-transfer proteins have been effectively used in membrane modification and functional studies in myelin and brain microsomes [13 – 15], intestinal BBM [16] [17], and lung surfactant [18] [19]. In the present study, the molecular dynamics of the lipid bilayer and its interaction with alkaline phosphatase activity has been investigated in the BBM of human term placenta using the technique CHEMISTRY & BIODIVERSITY – Vol. 3 (2006) 527 # 2006 Verlag Helvetica Chimica Acta AG, Zürich