Purification and Some Properties of Na, K-transport ATPase

Abstract

To purify Na,K-ATPase [EC 3.6.1.3], its solubilization with Lubrol was examined. Pretreatment of the enzyme with a high concentration of Nal was also examined in detail. Ouabain-insensitive ATPare was almost completely removed by the latter procedure. Fiske and SubbaRow's method was slightly modified for the determination of phosphate in the presence of Lubrol. The inhibition of the ATPase by Lubrol and the time course of loss of activity in Lubrol extracts were studied. Pretreatment of Lubrol with a mixed resin increased its effectiveness for extraction and reduced its inhibitory effect on activity. The inhibitory effect of ouabain was apparently decreased by Lubrol, but restored by addition of heat-treated microsomes. The effective factor in the microsomes may be a lipid fraction. Conditions for efficient extraction of the enzyme with Lubrol were examined. Extraction was best in the absence of salt and ATP, using resintreated Lubrol. The supernantant fraction obtained by centrifugation of the Lubrol extract at 160,000 × g for 60 min showed higher specific activity than that reported previously for soluble enzymes. It also showed higher sensitivity to ouabain (2×10−5 M ouabain caused complete inhibition) and slightly higher stability. The extract was eluted as a single, symmetrical peak from a Sepharose 6B column, with an apparent molecular weight of 500,000. From the elution profile the preparation seemed to be homogeneous, but the time course of the reaction suggested the existence of at least two active components.