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KMID : 0369819990290010013
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Jorunal of Korean Pharmaceutical Sciences
1999 Volume.29 No. 1 p.13 ~ p.19
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Comparative Study on the Transport Characteristics of Canalicular Liver Plasma Membrane Vesicles Prepared by Two Different Methods
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송임숙/Song IS
정석재/심창구/Chung SJ/Shim CK
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Abstract
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Canalicular liver plasma membrane vesicles (cLPM) were prepared according to two different methods (Inoue method and Meier method), and were evaluated for their protein yield, enzyme activity and transport characteristics. No difference was found between the methods in the protein yield (i.e., 0.14±0.031 and 0.15±0.050 mglg liver for Inoue method and Meier method, respectively). The activity of alkaline phosphatase, a marker enzyme of canalicular membrane, was significantly (P<0.05) higher in the vesicles of Meier method (3.52±0.91mmol/mg/hr)than in the vesicles of Inoue method (2.28±0.94 mmol/mg/hr) indicating that more purified cLPM were obtained from Meier method compared with Inoue method. ATP-dependent vesicular uptake of taurocholate and tributylmethylammonium (TBuMA) was observed for vesicles of both methods, and the kinetic parameters responsible for the transport were similar between the vesicles of both methods (for example, Vmax: 9.72 nmol/mg protein/30sec and Km: 0.63 mM for Inoue method; Vmax: 10.1 nmol/mg protein/30sec and Km: 0.70 mM for Meier method). A pH gradient dependent counter transport of TBuMA was also observed for both vesicles with similar kinetic characteristics. Either the uptake of taurocholate in the absence of ATP or that of TBuMA in the absence of pH gradient, which may represent passive diffusion of respective compound into the vesicles, was more rapid for the vesicles of Meier method than for the vesicles of Inoue method. For example, passive diffusion rate constants (Kd) for TBuMA uptake into the vesicles were 0.00030 and 0.00052\;{\mu}l/mg$ protein/min for the vesicles of Inoue method and Meier method, respectively. It may indicate that more leaky vesicles are obtained form the Meier method compared with the Inoue method. These aspects together with the time necessary to prepare the vesicles (i.e., 8 hr for Inoue method and 23 hr for Meier method) should be considered before selecting an appropriate method for the preparation of cLPM.
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KEYWORD
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Canalicular liver plasma membrane vesicles (cLPM), Inoue method, Meier method, Uptake, Taurocholate, TBuMA, ATP, pH-gradient
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