Substrate sites of the (Na+ + K+)-dependent ATPase

Robinson, Joseph D.

doi:10.1016/0005-2744(76)90345-4

Cited by 141 publications

(42 citation statements)

References 41 publications

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“…The Km of the ATPase for N a + is about 80 mM (Logan, 1980). For K + , it is about 2-3 mM (Logan, 1980) although it may be higher for the glial enzyme, which seems to saturate at 18-20 mM K + (Grisar et al, 1979), and for ATP it is approximately 0.5 mM (Logan, 1980;Robinson, 1976). Thus, under physi ological conditions, the activity of the neuronal en zyme is controlled predominantly by the concentra tion of intracellular N a +.…”

Section: How Is the Utilization Of Atp Distributed Among The Various mentioning

confidence: 98%

“…Firstly, ATP is a substrate for the respective reaction cycles and a decrease in its concentration can limit the overall process kinetically. This is, however, unlikely to occur under normal in vivo conditions, because the Km values for ATP (Robinson, 1976;Gill et aI., 198 1) are much lower than its cytosolic concentra tion. On the other hand, when the rate of energy generation declines, as for example in pathological conditions of ischemia and hypoglycemia, a lack of ATP to maintain the pumps is one of the factors responsible for massive movements of ions down their concentration gradients (Hansen, 1985).…”

Section: What Are the Relationships Between The Energy Level And Ion mentioning

confidence: 99%

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ATP and Brain Function

Erecińska

Silver

1989

J Cereb Blood Flow Metab

745

400

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The importance of A TP as the main source of chemical energy in living matter and its involve ment in cellular processes has long been recog nized. The primary mechanism whereby higher or ganisms, including humans, generate ATP is through mitochondrial oxidative phosphorylation. For the majority of organs, the main metabolic fuel is glucose, which in the presence of oxygen under goes complete combustion to CO 2 and H 2 0:The free energy (.:lG) liberated in this exergonic re action is partially trapped as ATP in two consecu tive processes: glycolysis (cytosol) and oxidative phosphorylation (mitochondria). The first produces 2 mol of ATP per mol of glucose, and the second 36 mol of ATP per mol of glucose. In the latter case, 6 mol of ATP are contributed from the oxidation of 2 mol of NADH generated in the cytosol during gly colysis and transferred into the mitochondria indi rectly through various "shuttle" systems. (In the a-glycerophosphate shuttle, the yield of ATP per NADH is reduced from 3 to 2 because the relevant mitochondrial dehydrogenase is a flavoprotein linked enzyme). Thus, oxidative phosphorylation yields 17-18 times as much useful energy in the form of ATP as can be obtained from the same amount of glucose by glycolysis alone. It is there fore not surprising that limitation of O 2 supply pro duces very damaging effects on cellular function. The brain is one of the organs that is particularly sensitive to lack of oxygen and in humans at rest is responsible for 20% of total O 2 consumption al though it accounts for only 2% of the body weight. in the function of the CNS? It will become evident from our discussion that only partial an swers to these questions are currently available. However, the search for these solutions involves some of the most exciting areas of contemporary neurochemistry. CONCENTRA nONS OF HIGH ENERGY PHOSPHATE COMPOUNDS IN MAMMALIAN BRAINBrain, like all other organs in the body, contains phosphorylated nucleotides that yield energy upon hydrolysis of their phosphate bond(s); the most im portant of these is the adenine nucleotide ATP. In addition, the CNS, in common with other excitable tissues, possesses another high energy reservoir, the creatine phosphate/creatine (PCr/Cr) system,

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Section: How Is the Utilization Of Atp Distributed Among The Various mentioning

confidence: 98%

Section: What Are the Relationships Between The Energy Level And Ion mentioning

confidence: 99%

ATP and Brain Function

Erecińska

Silver

1989

J Cereb Blood Flow Metab

745

400

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“…Results using microsomal fractions of several vertebrate tissues showed that the stimulation of Na,K-ATPase activity by ATP occurs through two-phase curves, one with K 0.5 from 0.1 to 1 µM and the other with K 0.5 from 0.01 to 0.4 mM, which suggests the existence of two sites of substrate hydrolysis (21,39). Dual effects were also found on ATP hydrolysis by C 12 E 8 -solubilized and purified Na,K-ATPase with K 0.5 values of about 0.1 and 4 µM for the high-affinity site and of about 0.1 and 0.5 mM for the lowaffinity site, both with negative cooperativity, suggesting that the enzyme environment or oligomer formation is very important for substrate hydrolysis (21)(22)(23)40).…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

“…Dual effects were also found on ATP hydrolysis by C 12 E 8 -solubilized and purified Na,K-ATPase with K 0.5 values of about 0.1 and 4 µM for the high-affinity site and of about 0.1 and 0.5 mM for the lowaffinity site, both with negative cooperativity, suggesting that the enzyme environment or oligomer formation is very important for substrate hydrolysis (21)(22)(23)40). It should be emphasized that the contribution of the highaffinity site to the ATPase specific activity of the enzyme generally corresponds to values about 1 to 10% of the total activity, which hinders its kinetic characterization (21)(22)(23)39). It is very difficult to determine whether these arose from the presence of more than one ATP site per (aß) protomer or from a single site whose function and affinity change during the catalytic cycle (21)(22)(23)(24)40), and the functional relationship between the two hydrolysis sites and their behavior during the reaction cycle remains to be explored.…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Santos

Lamas

Ciancaglini

2002

Braz J Med Biol Res

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SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,KATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8 -solubilized and purified Na,KATPase by 99, 81 and 98.5%, respectively. We have shown that Na,KATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.

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“…at saturating levels of K is between 100 and 800 /M (e.g. Dunham & Glynn, 1961;Robinson, 1967Robinson, , 1974Robinson, , 1976Whittam & Wiley, 1967;Garrahan & Glynn, 1967b;Karlish & Glynn, 1974). Under normal conditions, therefore, cell ATP may be a rate limiting factor in Na pump turnover (Post, Hegyvary & Kume, 1972;Karlish, Yates & Glynn, 1978 An additional point to note is that at 10 min after A23187 addition in cells incubated with both A23187 concentrations, there appears to be a minimum level to which cell ATP falls (about 200 ,umole/l.…”

Section: Calculationsmentioning

confidence: 99%

The effect of intracellular calcium on the sodium pump of human red cells.

Brown¹,

Lew²

1983

The Journal of Physiology

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SUMMARY1. The inhibitory effect of cytoplasmic Ca on Na-pump-mediated Na-K exchange was investigated in intact red cells under conditions of constant cell volume, membrane potential and inorganic ion composition. The ionized cytoplasmic Ca concentration ([Ca2+]1) was controlled using the ionophore A23187.2. In normal cells, ouabain-sensitive 24Na efflux was inhibited with an apparent affinity for [Ca2+], which depended on the concentration of A23187; 50 % inhibition required 20-40 fM and 160-300 LM-cytoplasmic Ca2+ with 10 /tM and 0-63 SM-A23187 respectively. Cytoplasmic Ca also affected cell ATP content which fell rapidly on addition of A23187 and subsequently increased, steadied or continued to fall more slowly depending on the Ca and A23187 concentrations. Half-maximal fall required 5-15 AM and 110-170 /tM-cytoplasmic Ca2+ at 10/M and 0-63 /M-A23187 respectively.Removal of Ca from the cells failed to reverse either the Na pump inhibition or the fall in cell ATP.3. In ATP-enriched cells cytoplasmic Ca caused inhibition of ouabain-sensitive 24Na efflux in an A23187-dependent manner with apparent affinities for [Ca2+], similar to those observed in the normal cells. Inhibition was complete at high [Ca2+],. As in the normal cells, the ATP content of the cells fell in the presence of cytoplasmic Ca, but always remained above 1-2 m-mole/l. cells. This was higher than the ATP content of Ca-free normal intact cells. 4. A23187 had no effect on the inhibition by Ca of ouabain-sensitive ATPase activity in isolated red cell membrane preparations. Both under conditions near optimal for Na-K-ATPase activity and under conditions resembling those in the cytoplasm, inhibition was half-maximal at about 25 um-Ca2+ and in the latter case complete at below 400 /tM-Ca2 .5. The apparent ATP-dependence of ouabain-sensitive Na efflux in the presence of cytoplasmic Ca was distinctly different in the normal and ATP-enriched cells but in both groups of cells it was similar for data obtained with high and low concentrations of A23187.6. The data for Na pump inhibition by cytoplasmic Ca in the intact cells were well fitted by several kinetic models involving either [Ca2+], or CaATP as the inhibitory species and a low affinity dependence of pump activity on MgATP or total ATP. A. M. BROWN AND V. L. LEWHowever, for any model, the apparent affinities for CaATP or for Ca2+ required to fit the ATPase data were 25-10 times higher than those required to fit the data for Na efflux. 7. From the kinetic models fitted to the Na efflux data, it is predicted that physiological (i.e. 1-10 #m) fluctuations in cell [Ca2+], will inhibit the Na pump by less than 35 %.

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Substrate sites of the (Na+ + K+)-dependent ATPase

Cited by 141 publications

References 41 publications

ATP and Brain Function

ATP and Brain Function

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

The effect of intracellular calcium on the sodium pump of human red cells.

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