Isolation, purification and characterization of the ATPase complex from the thermophilic cyanobacterium Synechococcus 6716

Abstract

The ATPase complex is isolated and purified from membrane vesicles of the thermophilic cyanobacterium Synechococcus 6716 by octyl glucoside and cholic acid by a modification of the procedure for its extraction from spinach chloroplasts. The complex is purified by differential centrifugation and ammonium sulfate precipitation and by gel filtration on Sepharose 6B. The purified fraction, without any phycocyanin contamination, shows ATP hydrolysis activity and Pi/ATP exchange activity of 1564 and 350 nmol · min−1· mg protein−1, respectively. N, N′‐Dicyclohexylcarbodiimide inhibits the ATP hydrolysis activity of this purified fraction. On polyacrylamide gels most typical F1 ATPase polypeptides are identified, but the low‐molecular weight polypeptides visible cannot be ascribed to the Fo part of the complex with certainty; non‐identified bands around 30kDa are also present.

Solubilization and separation of ethacrynic acid(EA) highly sensitive and EA less sensitive Mg2+-ATPases in the rat brain.

“…The superiority of the detergent combination has been reported in the solubilization of ATPases from renal brush-border membranes (16) and thermophilic cyanobacteria (17). As previously described (10,12), Mg2+ ATPases in the microsomes were inhibited by EA biphasically with a deflecting point at 0.3-0.4 mM.…”

Reassembly of Synechocystis sp. PCC 6803 F1‐ATPase from its over‐expressed subunits

“…It has been shown previously that either biochemically prepared or recombinant nucleotide binding proteins, including F, subunits c~ [20] andfl [21], are still capable of binding Synechocystis thylakoid vesicles have been prepared according to [16]. Depletion of F I was achieved by washing the membranes in a buffer, containing 50 mM sucrose, 2 mM Tris, adjusted to pH 7.5 by addition of solid tricine.…”

“…Lubberding et al [16]. Measurement of ATP synthesis and hydrolysis were carried out according to published procedures [17].…”

Interaction of photosynthesis, respiration and nitrogen fixation in cyanobacteria

“…1), only one enzyme seems reasonable, which has been partially isolated and characterized (e.g. Lubberding et al 1983) without evidence for a second enzyme.…”