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Synthesis of affinity derivatives of ouabain and their interaction with the sodium pump

University of British Columbia

Affinity derivatives of the cardiac glycoside, ouabain, were synthesized and characterized with respect to their interactions with Na,K ATPase. The rhamnose sugar moiety of ouabain was subjected to periodate oxidation and the resulting aldehyde groups derivatized through Schiff base formation with nucleophilic amino reagents. Subsequent reduction with borohydride yielded chemically stable conjugates which could be isolated in pure form. Ouabain was covalently linked to copolymer methacrylate microspheres via a heptane-1,7-diamine spacer arm. The ouabain-microsphere conjugates were inhibitors of beef brain Na,K ATPase activity with an apparent K[sub i] of 3.0 X10⁻⁶M (for 200 Å diameter microspheres), with respect to the immobilized glycoside concentration. Fluorescently tagged ouabain-microsphere conjugates could not be demonstrated to bind to Hela cells, either by fluorescence microscopy or radioimmunoassay. Ouabain was also derivatized with the fluorochrome DANS-lysine and the resulting conjugate displayed a K[sub i] for the inhibition of beef brain Na,K ATPase of 7.5 X 10⁻⁷M. Native ouabain was shown to have a Ki of 6.5 X 10⁻⁸M for the inhibition of beef brain Na,K ATPase. Half-maximal protection against the binding of (³H)-ouabain to beef brain microsomal membranes could be provided by either 3.0 X10⁻⁷M DANS-lys-ouabain or 3.0 x 10⁻⁸M untreated ouabain. Studies on the binding of (³H)-ouabain to Hela cells indicated that these cells possess about 2.5 x 10⁵ ouabain binding sites with a single dissociation constant of 5.7 x 10⁻⁸M. The binding of DANS-lys-ouabain to Hela cells could not be directly demonstrated under the fluorescence microscope and a radioimmunoassay utilizing (¹²⁵I)-anti-DANS IgG also gave negative results in this regard. A hapten sandwich labelling technique using anti-DANS antibodies and DANS derivatized iron microspheres was developed to demonstrate the distribution of DANS derivatized cell surface probes at the level of the scanning electron microscope, and the use of X-ray microprobe analysis with respect to cell surface labelling was investigated. As an example of the hapten sandwich labelling technique, mouse fibroblast cells were initially labelled with DANS-ricin. Cells were then labelled sequentially with anti-DANS IgG followed by DANS derivatized iron microspheres. Labelled cells were then visualized in the SEM using the secondary electron mode or by analysis of energy dispersed X-rays.

Text

2010-03-23

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http://hdl.handle.net/2429/22331

Biochemistry and Molecular Biology

University of British Columbia

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