Johansson F, Sommarin M, Larsson C
Rapid Purification of the Plasma-Membrane H+-Atpase in Its Nonactivated Form Using Fplc
Physiologia Plantarum: 1994 92:389-396
The plasma membrane H+-ATPase (EC 3.1.6.35) was solubilized from isolated spinach (Spinacia oleracea) leaf plasma membranes using the detergent dodecyl-beta-D-maltoside and subsequently purified to near homogeneity by ion exchange chromatography (FPLC). The enzyme purified in the presence of glycerol and ATP showed no loss in activity during 8 h on ice nor upon freezing at -80 degrees C and thawing, and the recovery was up to 75%. Addition of a phospholipid mixture only marginally increased the activity, whereas addition of lysophosphatidylcholine (lyse-PC) resulted in a 2-fold increase in activity and a decrease in K-m for ATP from ca 300 mu M to 100 mu M. The membrane-bound and the purified H+-ATPases showed very similar properties, also in their responses to lyse-PC, which is believed to activate the enzyme by displacement of its C-terminal inhibitory domain. Taken together, the data indicate that the H+-ATPase is purified in a non-activated form suitable for regulatory studies.
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