Kinetic studies of membrane (NA+-K+-Mg2+)-ATPase

(Na' + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(II1) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na' + K')-ATPase activity was accompanied by a parallel decrease of the associated K +-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na '-dependently, a phosphointermediate from [y-32P]ATP.The kinetics of inactivation and of phosphorylation with [ I > -~~P ] C~A T P and [ u -~~P I C~A T P are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme : ADP E + CrATP e E . C r A T P e E c r f E c r t E + C?' + P,. The dissociation constant of the CrATP complex of the pig kidney enzytne at 37 ' C was 43 pM. The inactivation rate constant (k+Z = 0.033 min-') was in the range of the dissociation rate constant k dof ADP from the enzyme of 0.011 min-'. The phosphoenzyme was unreactive towards ADP as well as to K + . N o hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na' reactivated it slowly. 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na f-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (drssociation constant ofthe enzyme ATP complex = 2.5 pM) as well as low concentrations of K'. CrATP was a competitive inhibitor of (Na' + K+)-ATPase.It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na' + K + ) -ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent protein kinase reaction as the phosphate acceptor site. magnetic resonance studies indicate the presence of one divalent metal ion bound at the active site [lo], where the divalent cation seems to maintain the phosphointermediate in the reactive state [ll]. There is evidence that the phosphointermediate formed in the presence of Na+ is converted from an ADPsensitive state into a K t -sensitive state [l, 3,11,12]. The mechanism for this interconversion is unclear, however; the earlier assumption that free Mg2 + might be necessary [1,3,12] has been doubted [8,13,14] since the proposed cyclical changes in the affinity of Mg2' for binding to and release from the enzyme need affinity changes of at least four orders of magni-

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“…MgATP with a Ki value of 4.8 mM has been reported earlier [8,55]. This value is two-to-three orders of magnitude higher than that found here.…”

Section: A Weak Competitive Inhibition Of Free Atp Towardssupporting

confidence: 54%

Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

1980

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“…Robinson (1967) working with brain Na+-K+ ATPase found that increasing K lowered the ATP affinity, although in this study it was impossible to tell on which side ofthe membrane K acted to lower the ATP affinity. This result has been confirmed by Hexum, Samson & Himes (1970) and Skou (1974). On the other hand, Peter & Wolf (1972) reported that increasing K raised the ATP affinity with no effect on the rate at saturating ATP concentrations.…”

Section: Ko+ep -E2k-e +Kimentioning

confidence: 53%

The interaction of potassium ions and ATP on the sodium pump of resealed red cell ghosts.

Eisner¹,

Richards²

1981

The Journal of Physiology

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SUMMARY1. Ouabain-sensitive K or Rb influx was measured into ghosts resealed to contain ATP concentrations of 1 ,uM-3 mm and no K.2. Increasing ATP from 1 to 100#sM, at saturating external K, increased K influx about twentyfold while having no effect on the ratio of ouabain-sensitive K influx to ouabain-sensitive ATPase activity.3. Increasing external K decreased the apparent affinity for ATP. Similarly increasing ATP decreased the apparent affinity for external K.4. The K influx can be empirically described as: influx = VmaxK2/(K + Kapp)2. Increasing ATP increased Vmax and (Kapp)2 by the same amount.5. These results are consistent with a consecutive model for the Na pump in which an ATP-dependent reaction follows a K-activated dephosphorylation.

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“…On the other hand, small changes in the ATP/ADP ratio were shown to inhibit K-sensitive Na+ efflux markedly. Hexum et al (1970) noted that the inhibitor constant Ki for Pi inhibition of Na-K-ATPase at the MgATP site is two orders of magnitude greater than the K, for MgADP inhibition. Consistent with these observations is the sensitivity of the garfish enzyme to ATP/ADP (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Caldwell, Hodgkin, Keynes & Shaw (1960) and IDeWeer (1970) suggested that the ATP/ADP ratio in squid axon is a critical factor in regulating pump activity. MgADP has been reported to be a competitive inhibitor of MgATP in rat brain Na-K-ATPase (Hexum, Samson & Himes, 1970). It was of interest to study the effect of ATP/ADP ratios on the Na-K-ATPase and to correlate it with ATP/ADP ratios found in stimulated nerve.…”

mentioning

confidence: 99%

Post‐tetanic hyperpolarization, sodium‐potassium‐activated adenosine triphosphatase and high energy phosphate levels in garfish olfactory nerve.

McDougal¹,

Osborn²

1976

The Journal of Physiology

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SUMMARY1. While much is now known about the Na-K-ATPase and the posttetanic hyperpolarization of nervous tissue, they have yet to be studied together in the same preparation.2. The post-tetanic hyperpolarization was studied in desheathed garfish olfactory nerve. The rate constant of decay of the post-tetanic hyperpolarization was determined by monitoring difference potentials after stimulation at 1/sec for 2-3 mmn.3. In membrane fractions prepared from these nerves, the ouabainsensitive ATPase activity (Na-K-ATPase) was determined by spectrophotometric measurements.4. Both the post-tetanic hyperpolarization. and the Na-K-ATPase showed a similar sigmoidal dependence on K+ concentration. The sequence of cation specificities measured at the K-site of the enzyme was the same as that determined by post-tetanic hyperpolarization measurements in whole nerve.5. The rate constants of the enzyme showed a dependence on Na+ concentration that paralleled the way in which the post-tetanic hyperpolarization rate constants varied as a function of the number of impulses. When Na+ was completely replaced by Li+, neither enzyme activity nor posttetanic hyperpolarization could be measured.6. The pH optimum for enzyme activity was between pH 7-0 and 7-8, while the optimal pH for post-tetanic hyperpolarization was above pH 8-0. 7. Metabolite levels in preparations of this nerve studied in vitro correspond to levels found in vivo.8 10. The accumulation of ATPase products was commensurate with the approach of post-tetanic hyperpolarization rate constants to their maximum level. This provides direct evidence for an ATPase functioning in active Na+ transport in nerve.11. The garfish Na-K-ATPase is sensitive to the ATP/ADP ratio of the incubating medium, but is relatively insensitive to orthophosphate, Pi.The fall in post-tetanic hyperpolarization rate constants observed with continued nerve stimulation may have been partially due to the falling ATP/ADP ratio measured in nerve under similar conditions.

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Kinetic studies of membrane (NA+-K+-Mg2+)-ATPase

Cited by 109 publications

References 23 publications

Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

Inactivation of (Na+ + K+)-ATPase by Chromium(III) Complexes of Nucleotide Triphosphates

The interaction of potassium ions and ATP on the sodium pump of resealed red cell ghosts.

Post‐tetanic hyperpolarization, sodium‐potassium‐activated adenosine triphosphatase and high energy phosphate levels in garfish olfactory nerve.

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