Several methods of purification of Na+, K+-adenosine triphosphatase (ATPase) have been previously described for a wide variety of tissues. In general, highest activity preparations have necessitated large amounts of tissue and many purification steps. This article describes a technique that allows partial purification of Na+,K+-ATPase from as few as 15 rat brains and should be of interest to investigators of the pharmacology of this particular enzyme system. In this modified version of the Jorgensen procedure (Biochim Biophys Acta 356:36-52,1974) we purified the Na+,K+-ATPase from 15-90 rat brains, and obtained enzyme preparations with a mean specific activity of 552 ± 37.6 μmol Pi/mg of protein/hr (95.5% ouabain sensitive). This "purified" enzyme had an activity ratio (Mg2+ + Na+ + K+)/(Mg2+ + Na+) of 47.4 ± 12.3 SEM, compared to 3.29 ± 0.17 SEM for the untreated microsomes. Ouabain inhibited the "purified" enzyme with an I50 of 6 × 10-9 M. Ouabain binding (644 pmol/mg of protein) yielded a turnover number of 13,700 min-1. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the enzyme revealed predominantly the α and β subunits with some minor contaminant bands. Previous methods of purification of rat brain Na+, K+-ATPase have employed sodium deoxycholate and high concentrations of Nal; the reported specific activity obtained was generally 150-350 μmol Pi/mg of protein/hr. We have employed higher SDS concentrations than in Jorgensen's technique for rabbit kidney but the procedure is simpler because sucrose gradients are not used. Final wash steps also include 10-20% glycerol in the media. These modifications have yielded Na+,K+-ATPase of significantly higher specific activity than previously reported for rat brain.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Pharmacological Methods|
|State||Published - Jun 1982|
Bibliographical noteFunding Information:
The authors thank Mr. Alan Davis for valuable assistance. This work was supported in part by the Medical Research Fund of the Veterans Administration and by grant No. HL-21457 from the National Institutes of Health, Department of Health and Human Services.
- Rat brain