Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.
Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.
Munzer
J S
JS
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Silvius
J R
JR
Blostein
R
R
D000818:Animals; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004285:Dogs; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008561:Membrane Fusion; D008861:Microsomes / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology; D012413:Rubidium / Q000097:blood; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012756:Sheep; D012964:Sodium / Q000097:blood; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013045:Species Specificity
D010042:Ouabain; D012964:Sodium; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D002118:Calcium