Characterisation of the H+‐ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Abstract

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two‐phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P‐type ATPase. This was verified by using antibodies against P‐type ATPase from Arabidopsis, which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (Ki= 0.9 μM) but not affected by inhibitors specific for F‐ or V‐type ATPases. demonstrating the purity of the plasma membranes. Mg‐ATP was the substrate, and the rate of ATP‐hydrolysis followed simple Michaelis‐Menten kinetics giving a Km= 0.46 mM. Free Mg2+ stimulated the activity, K1/2= 0.68 mM. Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20‐fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right‐side‐out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H+‐pumping as shown by the optical pH probe acridine orange. H+‐pumping was dependent on the presence of valinomycin and K+ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside‐out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.