SDS purification of porcine H,K-ATPase from gastric mucosa.
A highly purified membrane fraction of H,K-ATPase was isolated from hog gastric mucosa by using differential centrifugation, sodium dodecyl sulfate (SDS:0.125%) treatment and density-gradient centrifugation. The final fraction showed a major band at 97 kD by SDS-gel electrophoresis. This purified H,K-ATPase sedimented at the interface of a 28-35% sucrose step gradient and displayed a specific activity of 140-170 mumol Pi/h/mg protein and a ratio of K-stimulated ATPase activity to Mg-stimulated ATPase activity of 6.5-8.7. The apparent Km for ATP was 0.154 mM and the Km for K+ was o.6 mM. The enzymatic activity recovered from this purification procedure was K(+)-ionophore-independent. SDS treatment in the presence of 2.5 mM ATP did not change the kinetic properties of the isolated enzyme. Exclusion of ATP during SDS solubilization diminished the enzymatic activity by 90%, indicating that ATP protection is essential for the full recovery of enzymatic activity. In summary, mild SDS solubilization can be used to purify relatively large quantities of active H,K-ATPase to near homogeneity without altering the enzyme's kinetic properties.
Yeh
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LA
Department of Metabolic Diseases, Pfizer Central Research, Groton, CT 06385.
Cosgrove
P
P
Holt
W F
WF
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000818:Animals; D002499:Centrifugation, Density Gradient; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009550:Nigericin / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification; D013552:Swine
D012967:Sodium Dodecyl Sulfate; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D009550:Nigericin