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Cellular Compartment Model for Exploring the Effect of the Lipidic Membrane on the Kinetics of Encapsulated Biochemical Reactions Takeshi Sunami,† Kazufumi Hosoda,‡ Hiroaki Suzuki,‡ Tomoaki Matsuura,‡ and Tetsuya Yomo*,†,‡,§ †
Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan, ‡Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan, and § Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan Received December 3, 2009. Revised Manuscript Received January 11, 2010
One of the important characteristics of the cellular system is that interactions between the plasma membrane and water-soluble molecules in the cytoplasm are enhanced by the confinement of the molecules to the small volume of the intracellular space. Studying this effect in a model cell system, we measured the time evolution of an enzymatic hydrolysis reaction and a cell-free protein synthesis reaction taking place in giant liposomes having various size and phospholipid compositions by a flow cytometry. This single vesicle-based assay of a large number of liposomes enabled us to examine the volume dependence of enclosed reactions in detail, revealing that the presence of specific lipid affected the specific kinetic parameters of encapsulated reactions.
Introduction In cellular systems, cytosolic biochemical reactions are confined in a vesicle of plasma membrane. Because the confinement of biochemical reactions to such small volumes should substantially enhance physical interactions between the membrane and the enclosed water-soluble molecules, constituents of the membrane should affect the dynamics of chemical reactions in cells. However, intact cells are not appropriate for studying this effect because both the cellular membranes and the compartments they bound contain numerous kinds of components that are difficult to identify, and these components cannot be changed as the experimental parameters. Liposomes have been extensively used to investigate the fundamental interactions such as biomolecule encapsulation1-4 and enzyme reactions5-12 within membrane compartments, mainly to engineer the vesicles for gene/enzyme replacement therapies and food processing. Kinetics of enzyme reactions taking place in liposomes have been examined by absorbance and fluorescence measurements after removing the external enzymes by centrifugation8,9 or chromatography.12 In addition *To whom correspondence should be addressed: Tel þ81-6-6879-4171; Fax þ81-6-6879-7433; e-mail
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8544 DOI: 10.1021/la904569m
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Published on Web 02/04/2010
Langmuir 2010, 26(11), 8544–8551
Sunami et al.
Article
within liposomes, which cannot be obtained by bulk measurement methods. Previously, we examined concentrations of the final product of cell-free protein synthesis and substrate hydrolysis reaction and found that they distributed about an order of magnitude even in a population of liposomes with identical volume. This is presumably because in-liposome reactions should involve two inherent sources of fluctuations, i.e., fluctuations in liposome structure and reaction rates. Liposomes are known to have large heterogeneity in their size and structure, which should affect the extent of the interaction between the membrane and enclosed reactive species. Moreover, as the above-mentioned complex reactions involve many different components at various concentrations, encapsulation of the species at low concentrations (
