Properties of a synthetic plasma membrane marker: Fluorescent-Mercury-Dextran

Summary

Aminoethyl-Dextran T 10 (mol wt approx. 10,000) was conjugated withp-Chloromercuribenzoic acid (pCMB) and labeled with fluorescein isothiocyanate (FITC). This coupling procedure does not affect the mercurial function ofpCMB moiety of Fluorescent Mercury Dextran T 10 (FMD) since on the basis of mercury content itsK i-value for the (Na⁺−K⁺)-ATPase from rat kidney plasma membranes is identical with theK _i -value of unconjugatedpCMB (3×10⁻⁶ m). FMD binds to plasma membranes if applied in vivo, which could be shown in experiments in which rat kidneys were perfused with FMD and the plasma membranes isolated after the perfusion. The membrane-FMD complex is stable during common isolation steps such as differential centrifugation, sucrose density gradient centrifugation and free-flow electrophoresis. This was shown by in vitro binding studies of FMD with isolated plasma membranes from rat kidney cortex. FMD may be removed from the plasma membranes by the addition of 1×10⁻⁴ m dithiotreitol. Since F-aminoethyl-Dextran T 10 (withoutpCMB) does not interact with the plasma membranes, it is suggested that the binding of FMD to plasma membranes may involve a Hg-SH reaction. FMD does not penetrate into rat kidney cells in contrast topCMB, but can cross capillaries. Thus, FMD seems to be suitable (a) to label luminal and contraluminal surfaces of plasma membranes of epithelial structures, and (b) to be used as a fluorescent marker for plasma membranes during succeeding isolation procedures.