Solubilization and molecular weight determination of the (Na^+ + K^+)-ATPase from rectal glands of Squalus acanthias - Esmann, M.; Skou, J.C.; Christiansen, C. (Elsevier)
The membrane-bound (Na^+ +K^+)-activated ATPase (ATP Phosphohydrolase, EC 3.6.1.3) system was treated with the nonionic detergent octaethylene-glycoldodecyl ether, yielding a transparent supernatant after centrifugation. The supernatant was highly active with both ATPase and p-nitrophenylphosphatase, with initial specific activities of 2300 μmol P"i released . mg^-^1 protein . h^-^1 and 350 μmol p-nitrophenol released . mg^-^1 protein . h^-^1, respectively. The supernatant was purified to 95-100%, with respect to the 96 000 dalton and the 56 000 dalton peptides. The solubilized enzyme was gel filtered in Sepharose 4B-CL and displayed 2 peaks, both with catalytic activity. The low molecular weight particles eluted at K"a"v = 0.54, corresponding to a molecular weight of approximately 500 000 daltons and the particles had a specific activity of 2100 μmol P"i . mg^-^1 protein . h^-^1. Both peaks contained phospholipid with 60 mol phospholipid bound per 300 000 g protein. The low molecular weight particles had a molecular weight of 276 000 as determined by sedimentation equilibrium analysis.
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Keywords: W