ATP dependence of Na(+)‐K+ pump of cold‐sensitive and cold‐tolerant mammalian red blood cells.

Abstract

SUMMARY1. The ATP concentration of intact, cold-tolerant (ground squirrel) red cells and cold-sensitive (guinea-pig and human) red cells was monitored by use of the firefly tail, luciferin-luciferase assay. ATP kinetics of the pump in intact red blood cells was investigated by altering cell [ATP] by progressive depletion of ATP in the presence of 2-deoxy-D-glucose and then by measurement of ouabain-sensitive K+ influx at each level of [ATP] at various temperatures between 37 and 5 'C. Na+-K+-ATPase activity of broken membranes was also determined in parallel experiments using ouabain-sensitive release of 32p from [y-32P]ATP as a measure of activity.2. Without depletion, there is no immediate decrease in [ATP] of intact coldsensitive cells at low temperature (5 'C) at times when there are marked differences in the activities of the Na+-K+ pump of cold-tolerant and cold-sensitive cells.3. At 37 'C Na+-K+-ATPase of all three species exhibited two components of ATP dependence at 37 0C, one with high velocity, low affinity, the other with low velocity, high affinity. Affinities of both components rose with cooling. 4. A similar, two component pattern was observed in intact guinea-pig and human red cells at 37 'C, except that the segment corresponding to the high affinity component had an apparent Km (Michaelis-Menten constant) 3-to 4-fold higher than that of the broken membrane preparation.5. Cooling intact guinea-pig and human red cells decreased the apparent affinity of the high velocity, low affinity component for ATP, so that at 20 'C the value of Km approached or exceeded the levels of physiological ATP concentration. Below 20 'C only one component with values corresponding to that of the low velocity, high affinity component could be observed.6. In intact ground squirrel cells only the low affinity, high velocity component was apparent between 37 and 5 'C. Its affinity for ATP rose with cooling between 37 and 5 'C.

Physiological adaptation of an Antarctic Na+/K+-ATPase to the cold

“…extracellular K + or intracellular Na + and ATP). However, we consider this unlikely because substrate concentrations were at saturating levels at room temperature, and previous studies on the Na + /K + -ATPase have shown that the cold increases the apparent affinity for extracellular K + and intracellular Na + (Ellory and Willis, 1982), and for ATP (Marjanovic and Willis, 1992). Furthermore, both the ATPbinding pocket and cation-binding sites of the Antarctic and temperate orthologs are conserved.…”

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates

“…-K ? -ATPase activity is just as temperaturesensitive as that of the guinea pig [15]. The capability of ground squirrel cells to maintain low levels of ion leakage during cold exposure appears to be primarily based on the better preservation of intracellular Ca 2?…”

“…-K ? -ATPase activity is relatively smaller [15] and rates of passive ion leakage are generally lower in the squirrel cells [16], which together enable preservation of ion gradients at significantly lower metabolic cost. It is worth noting that the capability of maintaining higher Na ?…”

Release of adenosine triphosphate by adenosine diphosphate in whole blood and in erythrocyte suspensions

“…Because ADP is released in a higher proportion than ATP, a passive leakage was excluded by these authors. Erythrocytes contain large amounts of ATP [28]; the content of ADP is much lower. In each volume of normal blood, the ATP content in the erythrocytes exceeded the releasable platelet ATP by about 50-fold.…”