Glucose regulation of pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase.
The effect of glucose and 2-deoxy-D-glucose on pre-steady state kinetics of ATP hydrolysis by Na,K-ATPase has been investigated by following pH transients in a stopped-flow spectrophotometer. A typical pre-steady state signal showed an initial decrease then subsequent increase in acidity. Under optimal Na+ (120 mM) and K+ (30 mM) concentrations, magnitudes of both H+ release and H+ absorption were found to be approximately 1.0/ATPase molecule. The presence of 1 mM glucose significantly decreased H+ absorption at high Na+ concentrations, whereas it was ineffective at low Na+. H+ release was decreased significantly in the presence of 1 mM glucose at Na+ concentrations ranging from 30 mM to 120 mM. Similar to the control, K+ did not show any effect on either H+ release or H+ absorption at all tested combinations of Na+ and K+ concentrations. Pre-steady state H+ signal obtained in the presence of 2-deoxy-D-glucose did not vary significantly as compared with glucose. Delayed addition of K+ (by 30 ms) to the mixture (enzyme+120 mM Na++ATP+glucose) showed that only small fractions of population absorb H+ in the absence of K+. No H+ absorption was observed in the absence of Na+. Delayed mixing of Na+ or K+ did not have any effect on H+ release. Effect of 2-deoxy-D-glucose on H+ absorption and release was almost the same as that of glucose at all combinations of Na+ and K+ concentrations. Results obtained have been discussed in terms of an extended kinetic scheme which shows that, in the presence of either glucose or 2-deoxy-D-glucose, significantly fewer enzyme molecules reached the E-P(3Na+) stage and that K+ plays an important role in the conversion of E1.ADP.P(3Na+) to H+.E1~(3Na+) complex.
Haque
Mohammad Mahfuzul
MM
Enzyme Kinetics Laboratory, Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India.
Manzoor
Nikhat
N
Amin
Mohammad
M
Hussain
Mohammad Ejaz
ME
Khan
Luqman Ahmad
LA
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003847:Deoxyglucose / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology*; D006041:Goats; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods
D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D005947:Glucose