The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells.
Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for 1 min stimulates the bumetanide-inhibitable K+ influx approximately fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The KI for bumetanide was 0.19 +/- 0.06 microM for the cytoplasts as compared to 0.67 +/- 0.11 microM for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at approximately 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the approximately 80 kD protein.
Hoffmann
E K
EK
Institute for Biological Chemistry, August Krogh Institute, University of Copenhagen, Denmark.
Jessen
F
F
Dunham
P B
PB
D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D051379:Mice; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D002712:Chlorides; D007140:Immunoglobulin Fab Fragments; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D010042:Ouabain; D012964:Sodium; D011188:Potassium