Real-time observation for the enzymatic reaction of phospholipid membrane: application of the time-resolved quasi-elastic laser scattering method.
An analytical technique to measure reactions in biological membranes was developed and applied to monitoring the hydrolysis reaction of phospholipids (dipalmitoylphosphatidylcholine, DPPC) by phospholipase A(2). The technique uses the time-resolved quasi-elastic laser scattering (TR-QELS) method to measure an oil/phospholipid monolayer/water membrane system by monitoring the change of interfacial tension under a noncontact condition and in real time. When the TR-QELS method is used with the newly developed oil/phospholipid monolayer/water membrane system, measurement of the hydrolysis reaction of phospholipids with long alkyl chains (C >or=16), which are the major components in biological membranes, becomes possible. The reaction progress is monitored by the increase of interfacial tension at the oil/water interface caused by the decrease of surface-active DPPC molecules due to the reaction. The characteristic phases, namely, lag, burst, and equilibrium, are observed. The relationship between the duration of the lag phase (the rate-limiting step of the reaction) and the concentration of calcium ion (an essential cofactor of the reaction) is also investigated. Increase of calcium ion concentration in the subphase is found to shorten the duration of the lag phase. In addition, the real-time measurement simplifies the estimation process for the reaction activation energy.
Morisaku
Toshinori
T
Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Yui
Hiroharu
H
Iwazumi
Masanori
M
Ikezoe
Yasuhiro
Y
Fujinami
Masanori
M
Sawada
Tsuguo
T
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D000473:Alkanes / Q000737:chemistry; D001514:Bee Venoms / Q000201:enzymology; D002462:Cell Membrane / Q000378:metabolism*; D006868:Hydrolysis; D007834:Lasers; D008567:Membranes, Artificial; D008954:Models, Biological; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism*; D012542:Scattering, Radiation; D013499:Surface Properties; D013997:Time Factors; D014867:Water / Q000737:chemistry
D000473:Alkanes; D001514:Bee Venoms; D008567:Membranes, Artificial; D010743:Phospholipids; C024713:n-tetradecane; D014867:Water; D015060:1,2-Dipalmitoylphosphatidylcholine; D010741:Phospholipases A