ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 335 (2004) 334–337 www.elsevier.com/locate/yabio
Notes & Tips
A continuous Xuorescent method for measuring Na+ transport Christoph von Ballmoos, Peter Dimroth¤ Institut für Mikrobiologie der Eidgenössischen Technischen Hochschule, ETH Zentrum, CH-8092 Zürich, Switzerland Received 6 July 2004 Available online 15 September 2004
In past decades, various primary sodium pumps that play important roles in the metabolism of diVerent organisms have been found . Examples are the ubiquitous Na+/K+ ATPases of eukaryotic cells and the bacterial Na+-translocating decarboxylases, ATP synthases, and NADH:ubiquinone oxidoreductase of the Nqr and complex I types [2,3]. The measurement of Na+ transport across a biological membrane is an important experimental step in the investigation of speciWc properties of these catalysts (as an example, see ). Unlike H+ transport, which can be followed by pH change in a particular compartment, the measurement of Na+ transport into proteoliposomes is more diYcult and laborious. Methods used previously have a common drawback. Since only the Na+ content entrapped in the interior compartment should be measured, this has to be previously separated from external Na+ ions. This separation is usually performed by passing the liposome suspension through a column of the strong cation exchanger DOWEX50, K+, which adsorbs all Na+ ions except those entrapped in the inner lumen of the liposomes. The incorporated sodium content is then easily measured by means of radioactive 22 Na+ or by atomic absorption spectroscopy. Both methods, however, have the disadvantage of being discontinuous, since for every data point the separation procedure described above has to be performed. Moreover, the usage of the strong radioactive 22Na+ ( radiation) is inconvenient. Although a number of Na+ Xuorophores are commercially available, their application has so far been restricted to the detection of sodium ions within whole cells (as an example, see ). Here we present a facile method to qualitatively measure sodium ion uptake into
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proteoliposomes by the Na+-speciWc Xuorophore sodium green, which is commercially available.
Materials and methods Reagents The two sodium-speciWc Xuorophores, SBFI (tetraammonium salt; S-1262) and sodium green (tetra(tetramethyl)ammonium salt; S-6900) were from Molecular Probes (Leiden, NL). Phosphatidylcholine from soy bean (Type II S) was from Sigma. Other reagents were from Fluka (Buchs, Switzerland). The Xuorophores were dissolved in dimethyl sulfoxide (20 mg/ml) and stored at ¡20 °C in the dark. Reconstitution of F1F0 ATP synthase into lipid vesicles Method A. The reconstitution of F-type ATP synthase from Ilyobacter tartaricus into liposomes was performed with minor modiWcations as described . Liposomes were formed by the vortexing of a suspension of 30 mg phosphatidylcholine and 20 g of a Na+-speciWc Xuorophore in 0.9 ml 50 mM potassium phosphate, pH 7.0, 1 mM dithioerythritol, 1 mM NaCl to homogeneity (5 min) and the subsequent sonication for 2 £ 30 s in a tip sonicator (MSE Soniprep 150 homogenizer; Sanyo). The puriWed F1F0 ATP synthase from I. tartaricus (0.3 mg protein) in 0.1 ml 5 mM potassium phosphate, pH 8.0, 2.5 mM MgCl2 was added to the preformed liposomes and the suspension was incubated at room temperature for 10 min with occasional shaking. The mixture was frozen in liquid nitrogen, kept there for 10 min, and subsequently thawed in a water/ice mixture which lasted about 1 h. The proteoliposomes were sonicated for 3 £ 5 s with the tip sonicator. To remove the external Xuorophore, the proteoliposomes were subjected to gel Wltration on a NAP-10 column (Amersham Biosciences) in 50 mM potassium phosphate, 2.5 mM MgCl2.
Notes & Tips / Analytical Biochemistry 335 (2004) 334–337
The turbid fractions were collected and directly used for transport measurement. Method B. Liposomes were formed after a modiWed protocol as described . Soy bean phosphatidylcholine (30 mg), dissolved in CHCl3:MeOH (2:1, v:v) in a roundbottomed Xask, was Xushed with nitrogen. The solvent was removed under reduced pressure Wrst in a rotary evaporator and then in high vacuum for 4 h. The lipids and 20 g of the Xuorophore were resuspended in 0.9 ml buVer (50 mM potassium phosphate, pH 7.0, 1 mM dithioerythritol, 1 mM NaCl) and subjected to seven freeze– thaw cycles in liquid N2 and at 37 °C with short vigorous shaking after each cycle. To obtain unilamellar vesicles of deWned size, the suspension was passed 15 times through a membrane (100-, 200-, or 400-nm) using a liposome extruder (LiposoFast Pneumatic; Avestin, Canada). The preformed liposomes were further utilized as described under Method A. Determination of Na+ transport into proteoliposomes All measurements were performed on a RF-5001PC spectroXuorometer (Shimadzu) in a 300-l quartz cuvette. Typically, 50 l of the proteoliposomes were mixed with 250 l of the assay mixture (50 mM potassium phosphate, pH 7.0, 2.5 mM MgCl2, 6 mM phosphoenolpyruvate, 10 U pyruvate kinase). Subsequently, 1 M of valinomycin was added and the Xuorescence emission was recorded (sodium green, 540 nm/488 nm excitation; SBFI, 505 nm/333 nm excitation). Na+ uptake into proteoliposomes was initiated with the addition of 1 mM Na2ATP. The dicyclohexylcarbodiimide (DCCD)1 treated sample was incubated with 50 M of the inhibitor at pH 6.5 30 min prior to measurement. Tributyltin chloride (50 M) was added directly into the Wnal solution without prior incubation.
the Na+ ion and can detect small changes (