Rapid purification of the plasma membrane H+‐ATPase in its non‐activated form using FPLC

The plasma membrane H‐ATPase (EC 3.1.6.35) was solubilized from isolated spinach (Spinacia oleracea) leaf plasma membranes using the detergent dodecyl‐β‐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°C and thawing, and the recovery was up to 75%. Addition of a phospholipid mixture only marginally increased the activity, whereas addition of lysophosphalidylcholine (lyso‐PC) resulted in a 2‐fold increase in activity and a decrease in Km lor ATP from ca 300 μM to 100 μM. The membrane‐bound and the purified H‐ATPases showed very similar properties, also in their responses to lyso‐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.