Antiserum against Proteins of the Na+, K+,2CI– Cotransporter in Ehrlich Ascites Tumor Cells Inhibits Volume Regulation and Bumetanide-Sensitive K+ Influx

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Abstract
Solubilized membranes of Ehrlich cells separated on a furosemide-Sepharose CL-4B affinity matrix has major bands at 45 and 100 kD. The 45-kD protein was by immunodetection and protein sequencing found to be actin (1/5 of the protein was sequenced showing 100% homology). Antiserum against the purified proteins immunodetected the 100-kD protein in a Western blot. A protein with the same molecular weight was immunodetected on crude membrane preparations from several other cells and tissues. The antiserum against the purified proteins inhibits the Na+, K+,2C1- cotransport measured by two independent methods: (1) The regulatory volume increase after a hypertonic challenge was inhibited by the antibodies. (2) The antiserum inhibited the bumetanide-sensitive K+ influx mediated by the Na+, K+,2Cl- cotransport during regulatory volume increase. The specific inhibition strongly suggests that the 100-kD protein either is a constituent of the Na+, K+,2C1- cotransporter or a protein that effects the co-transporter’s regulation. The 100-kD protein isolated in the present investigation is clearly different from the 80-kD protein which was previously purified on a bumetanide-Sepharose column.

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