Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii |
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Authors: | Reiner Dolle Wilhelm Nultseh |
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Affiliation: | (1) Botanisches Institut, Fachbereich Biologie der Philipps-Universität Marburg, Karl-von-Frisch-Strasse, D-3550 Marburg 1, Federal Republic of Germany |
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Abstract: | Specific binding of the calcium antagonist [3H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K+-stimulated, Mg2+-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [3H]verapamil were found at 22°C, one with higher and a second with lower affinity to [3H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [3H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [3H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [3H]verapamil binding in both fractions.Abbreviations Bmax maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC50 concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid |
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Keywords: | Chlamydomonas reinhardtii Phototaxis Galvanotaxis Calcium Calcium channels Calcium channel blockers Radioligand binding Polymer twophase system |
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