Enhanced Adsorption of Ca-ATPase Containing Vesicles on a Negatively Charged Solid-Supported-Membrane for the Investigation of Membrane Transporters (original) (raw)
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Bioelectrochemistry, 2004
Sarcoplasmic reticulum (SR) native vesicles incorporating Ca-ATPase are adsorbed on a solid-supported lipid membrane (SSM). Upon adsorption, the ion pumps are chemically activated by concentration jumps of ATP and the capacitive current transients generated by SR Ca-ATPase are measured under potentiostatic conditions. The Michaelis -Menten constant, K M , for ATP is evaluated by varying the concentration of ATP in the activating solution. This preliminary result shows that ion transport by SR Ca-ATPase can be suitably investigated by a technique based on concentration jumps on an SSM. D
Biophysical Journal, 2004
Sarcoplasmic reticulum vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps both in the absence and in the presence of K 1 ions and at different pH values. Ca 21 concentration jumps in the absence of ATP were also carried out. The resulting capacitive current transients were analyzed together with the charge under the transients. The relaxation time constants of the current transients were interpreted on the basis of an equivalent circuit. The current transient after ATP concentration jumps and the charge after Ca 21 concentration jumps in the absence of ATP exhibit almost the same dependence upon the Ca 21 concentration, with a half-saturating value of ;1.5 mM. The pH dependence of the charge after Ca 21 translocation demonstrates the occurrence of one H 1 per one Ca 21 countertransport at pH 7 by direct charge-transfer measurements. The presence of K 1 decreases the magnitude of the current transients without altering their shape; this decrease is explained by K 1 binding to the cytoplasmic side of the pump in the E 1 conformation and being released to the same side during the E 1 -E 2 transition.
Biosensors based on membrane transport proteins
Biosensors and Bioelectronics, 1991
We propose a novel class of biosensors based on membrane bound receptors or transport proteins as the sensing element The protein is incorporated in a planar lipid bilayer which covers the transducer. The transducer may detect an electric current, a voltage, or a change in fluorescence. A prototype lactose sensor is presented which consists of a quartz slide covered by a lipid membrane containing the protein lactose permease from Escherichia coli. This protein is a lactose/H+ cotransporter, hence lactose in the external medium initiates lactose/H+ cotransport across the lipid membrane. This leads to a rise in proton concentration in the small volume between the lipid membrane and the quartz surface which can be detected by a pH-sensitive fluorescence dye.
Bulletin of the American …, 2010
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Journal of Biological Chemistry, 2015
Membrane proteins require phospholipids for being biologically active. Results: An increase of phosphatidylcholine/detergent molar ratio leads to a biphasic behavior of the PMCA Ca 2+ -ATPase activity whose maximum depends on phosphatidylcholine characteristics. Conclusion: The optimum hydrophobic thickness for PMCA structure and Ca 2+ -ATPase activity is about 24 Å. Significance: Differential modulation by neutral phospholipids could be a general mechanism for regulating membrane protein function.
Biochimica et Biophysica …, 2010
The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and educational use, including for instruction at the author's institution, sharing with colleagues and providing to institution administration.
Optical study of active ion transport in lipid vescicles containig reconstituted NaK-ATPase
A fluorescence method is described for the measuremeni of ATP-driven ion fluxes in lipid vesicles containing purified Na.K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made seleciively perméable to K" so thaï the membrane voltage approaches the Nernst potential for K". Wiih constant e\ternal K* concentration, ihe lime course of internai K" concentration can be continuousiy measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time résolution than tracer-flux experimenls and ailows an accurate détermination of inilial flux rates. From the lemperaiure dependence of active K" transport ils activation energy was determined to be 115 kj/mol. ATP-stimulaied elecirogenic pumping can be measured as a fast fluorescence change when the membrane conduclance is low (i.e.. at low or zéro valinomycin concentration). In accordance with expeclalion. the amplitude of the fast signal change increases with decreasing passive ion permeabiliiy of ihe vesicle membrane. The resolution of the charge movemenl is so high that a few pump turnovers can be easily detected.
Charge transfer in P-type ATPases investigated on planar membranes
Archives of Biochemistry and Biophysics, 2008
Planar lipid bilayers, e.g., black lipid membranes (BLM) and solid supported membranes (SSM), have been employed to investigate charge movements during the reaction cycle of P-type ATPases. The BLM/SSM method allows a direct measurement of the electrical currents generated by the cation transporter following chemical activation by a substrate concentration jump. The electrical current transients provides information about the reaction mechanism of the enzyme. In particular, the BLM/SSM technique allows identification of electrogenic steps which in turn may be used to localize ion translocation during the reaction cycle of the pump. In addition, using the high time resolution of the technique, especially when rapid activation via caged ATP is employed, rate constants of electrogenic and electroneutral steps can be determined. In the present review, we will discuss the main results obtained by the BLM and SSM methods and how they have contributed to unravel the transport mechanism of P-type ATPases.
The Journal of Membrane Biology, 1977
The kinetics of the transport of the 1-anilino-8-naphthalenesulfonate (ANS-, an anionic fluorescent probe of the membrane surface) across phospholipid vesicle membranes have been studied using a stopped-flow rapid kinetic technique. The method has been used to gain detailed information about the mechanism of transport of this probe and to study ionophore-mediated cation transport across the membrane. The technique has also been exploited to study differences between the inside and outside surfaces of vesicles containing pbosphatidyl choline (PC).