Solubilization by lysolecithin and purification of the plasma membrane ATPase of the yeast Schizosaccharomyces pombe.

J.P Dufour,A Goffeau

doi:10.1016/s0021-9258(17)38024-9

Abstract

Purified plasma membranes of Schizosaccharomyces pombe were obtained by precipitation at pH 5.2 of a crude particulate fraction, followed by differential centrifugations and isopycnic centrifugation in a discontinuous sucrose gradient. The specific activity of the Mg2+-requiring plasma membrane ATPase activity (EC 3.6.1.3) was enriched from 0.3 mumol min-1 x mg-1 of protein in the homogenate to 26 in the purified membranes. The optimal conditions for solubilization of the ATPase activity by lysolecithin were found to be: 2 mg/ml of lysolecithin, a lysolecithin to protein ratio of 8 at pH 7.5, and 15 degrees C in the presence of 1 mM ATP and 1 mM ethylenediaminetetraacetic acid. A 6- to 7-fold purification of the solubilized ATPase activity was obtained by centrifugation of the lysolecithin extract in sucrose gradient. Part of the ATPase activity which was inactivated during the centrifugation in the sucrose gradient could be restored by addition of a micellar solution of 50 microgram of lysolecithin/ml during the assay. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the purified enzyme showed only one band of Mr = 105,000 stained with Coomassie blue. Another ATPase component of apparent molecular weight lower than 10,000 was stained by periodic Schiff reagent but not colored by Coomassie blue. The purified enzyme was 85% inhibited by 50 micrometer N,N'-dicyclohexylcarbodiimide and 94% inhibited by 53 microgram of Dio-9/ml.

Highlights

Purified plasma membranes of Schizosaccharomyces pombe were obtained by precipitation at pH 5.2 of a crude particulate fraction, followed by differential centrifugations and isopycnic centrifugation in a discontinuous sucrose gradient
ATPase activity were recovered by centrifugation at 100,000 x g for 1 h of the 3 ml of the 1.20 g x crne3 sucrose layer previously diluted in 25 ml of 1 mru ATP, 1 mM EDTA, 10 mru Tris adjusted at pH 7.5 with CH:&OOH
@-One criticai factor for successful solubilization of the plasma membrane ATPase is to use enriched plasma membrane fractions of high specific pH 6.0 ATPase activity. Such plasma membrane fractions of S. pombe with high ATPase activity were obtained by selective pH precipitation followed by centrifugation through a discontinuous sucrose gradient

Summary

AND METHODS

Chemicals-ATP, phosphoenolpyruvate, pyruvate kinase, and pgalactosidase were from Boehringer. ATPase activity were recovered by centrifugation at 100,000 x g for 1 h of the 3 ml of the 1.20 g x crne sucrose layer previously diluted in 25 ml of 1 mru ATP, 1 mM EDTA, 10 mru Tris adjusted at pH 7.5 with CH:&OOH. ATPase was to suspend 1 mg of purified plasma membrane in 350 fi of the following solution referred to in the text as “solubilization buffer”: 1 rnM EDTA, 1 mM ATP, 10 m&r Tris adjusted at pH 7.5 with. This suspension kept at 4’C was added to 4.65 ml of so&a&d solubilixation buffer containing 5 mg of lysolecithin and incubated for 10 min at 15°C. The pellets were washed several times with ether, resuspended in 100 d of 1% b-mercaptoethanol, 2% sodium dodecyl sulfate, 1% glycerol, 0.005% bromophenol blue, 27 rnM HzSOI, pH 6.14, adjusted with Tris and heated at 100°C for 6 min. Spectrophotometric scanning was carried out with the 580 to 650 nm filter of the Kipp and Zonen DD2 densitometer

RESULTS

50 M die clohexykrboknide

DISCUSSION