Phosphorylation of red cell membranes at ambient temperatures with micromolar [32P]ATP in the presence of Na ions produced phosphoenzyme that was dephosphorylated rapidly upon the addition of ADP or K ions. However, as first observed by Blostein (1968, J. Biol. Chetn., 243:1957), the phosphoenzyme formed at 0°C under otherwise identical conditions was insensitive to the addition of K ions but was dephosphorylated rapidly by ADP. This suggested thatjthe conformational transition from ADP-sensitive, K-insensitive Na pump phosphoenzyme (E1-P) to K-sensitive, ADP-insensitive phosphoenzyme (E2P) is blocked at 0°C. Since the ATP: ADP exchange reaction is a partial reaction of the overall enzyme cycle dependent upon the steady state level of E1-P that is regulated by [Na], we examined the effects of temperature on the curve relating [Na] to ouabain-sensitive ATP:ADP exchange. The characteristic triphasic curve seen at higher temperatures when [Na] was between 0.5 and 100 mM was not obtained at 0°C. Simple saturation was observed instead with a K0.5 for Na of - 1 mM. The effect of increasing temperature on the ATP: ADP exchange at fixed (150 mM) Na was compared with the effect of increasing temperature on (Na + K)-ATPase activity of the same membrane preparation. It was observed that (a) at 0°C, there was significant ouabain-sensitive ATP:ADP exchange activity, (b) at 0°C, ouabain-sensitive (Na + K)-ATPase activity was virtually absent, and (c) in the temperature range 5-37 °C, there was an ~300-fold increase in (Na + K)-ATPase activity with only a 9-fold increase in the ATP:ADP exchange. These observations are in keeping with the suggestion that the Ei-P → E2P transition of the Na pump in human red cell membranes is blocked at 0°C. Previous work has shown that the inhibitory effect of Na ions and the low-affinity stimulation by Na of the rate of ATP: ADP exchange occur at the extracellular surface of the Na pump. The absence of both of these effects at 0°C, where Ei-P is maximal, supports the idea that external Na acts through sites on the E2P form of the phosphoenzyme. © 1985, Rockefeller University Press., All rights reserved.
CITATION STYLE
Kaplan, J. H., & Kenney, L. J. (1985). Temperature effects on sodium pump phosphoenzyme distribution in human red blood cells. Journal of General Physiology, 85(1), 123–136. https://doi.org/10.1085/jgp.85.1.123
Mendeley helps you to discover research relevant for your work.