Detection of Mg2+-K+ dependent plasma membrane ATPase activity through purification of cucumber (Cucumis sativus L.) root cells
Ha, S.H.; Chung, G.C.
Journal of the Korean Society for Horticultural Science 35(4): 294-300
ISSN/ISBN: 0253-6498 Accession: 002591243
Plasma membrane was isolated from fresh cucumber roots (from plants grown in nutrient film technique culture) and callus (induced in vitro from root tissue) and the purity of separation was investigated by measuring ATPase activity and protein content. The ATPase activity of callus supernatant, obtained at 1500 x g, was not increased by the addition of Mg2+ and K+. However, the pellet from further centrifugation at 10 000 x g did respond to the addition of these metal ions. Supernatant separated at 10 000 x g was further centrifuged at 80 000 x g and the resulting pellet showed metal ion-dependent ATPase activity. ATPase activity was decreased by up to 50% by the addition of vanadate. Interface collected from a 30/42.9% sucrose density gradient and centrifuged at 80 00 x g for 2 h showed increased metal ion-dependent activity.