Purification of the (Na+ + K+)-adenosine triphosphatase from human renal tissue.
(Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was purified from human cadaver renal tissue and exhibited a linear reaction rate with time. 100 g of whole kidney would yield 1--3.5 mg protein with a specific activity of 50--200 mol - kg-1 - h-1 for (Na+ + K+)-ATPase. The preparation was completely inhibited by 100 micronM ouabain with a Ki of 1.8 micronM. K+-dependent phosphatase increased during purification of (Na+ + K+)-ATPase to 7.8 mol - kg-1 - h-1. There was no detectable Mg2+-ATPase in the final preparation. Sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis yielded three protein peaks of 117 000, 92 500, and 56 000 daltons. The peptide band corresponding to 92 500 daltons underwent an Na+-dependent phosphorylation with [gamma-32P]-ATP. The band at 56 000 daltons stained for glycoprotein. The Km for ATP was 0.38 mM and that for Mg2+ was 0.5 mM. The formation of ADP and inorganic phosphate from ATP was stoichiometric. The Km for Na+ in the presence of 20 mM K+ was 16 mM and the Km for K+ in the presence of 100 mM Na+ was 1.5 mM. The temperature optimum was 51degrees C and the pH optimum was 7.0. (Na+ + K+)-ATPase in whole homogenate, microsomes, and NaI-treated microsomes exhibited a slowing of reaction rate (non-linearity) with time such that the enzyme was inactive by 10--15 min of reaction. This non-linearity was eliminated during purification. The significance is discussed.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000328:Adult; D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology*; D012265:Ribonucleotides; D012964:Sodium / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013696:Temperature
D012265:Ribonucleotides; D010042:Ouabain; D012964:Sodium; D000251:Adenosine Triphosphatases; D008274:Magnesium; D011188:Potassium