Nonenzymatic glycation of Na,K-ATPase. Effects on ATP hydrolysis and K+ occlusion.
Glycation of the Na,K-ATPase in vitro (formation of Schiff base with glucose followed by reduction with NaCNBH3) shows the presence of three classes of reactive amino groups that differentially affect catalysis and cation binding. Reaction in the absence of ATP results in irreversible inhibition of enzyme activity with a t1/2 of 53 min. This is due to modification of one class of amino groups that affect the catalytic domain of the enzyme. In the presence of ATP, glycation first results in a shift in the steady state kinetics of ATP hydrolysis from substrate activation to Michaelis-Menten kinetics accompanied by an increase in the apparent affinity for K+ in the p-nitrophenylphosphatase reaction. This change in kinetic properties occurs with a t1/2 of 9 min and results in the complete loss of K+ occlusion. Incorporation of glucose is into the catalytic subunit, remote from the N-terminal end. Apparent total inhibition of K+ occlusion occurs with a stoichiometry 0.8 mol of glucose incorporated per mol of enzyme. Therefore, there is a rapidly reacting amino group that affects the cation binding domain of the Na,K-ATPase. More slowly, with a t1/2 of 9 h, the ATP hydrolysis kinetics change from Michaelis-Menten to substrate inhibition without recovery of K+ occlusion, showing that, in the E1 conformation, there is a third, slower reacting class of amino groups in the Na,K-ATPase that affects low affinity nucleotide interaction with the catalytic subunit.
Garner
M H
MH
Department of Ophthalmology, California College of Medicine, University of California, Irvine 92717.
Bahador
A
A
Sachs
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008954:Models, Biological; D011188:Potassium / Q000378:metabolism*; D011817:Rabbits; D012545:Schiff Bases; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D046911:Macromolecular Substances; D012545:Schiff Bases; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D005947:Glucose; D011188:Potassium