F1‐Like ATPase from Anaerobic Bacterium Lactobacillus casei Contains Six Similar Subunits
Authors: BIKETOV Sergei F. , KASHO Vladimir N. , KOZLOV Igor A. , MILEYKOVSKAYA Yevgenia I. , OSTROVSKY Dmitrii N. , SKULACHEV Vladimir P. , TIKHONOVA Galina V. , TSUPRUN Vladimir L. 
Published: March 2005

Membrane‐linked ATPase of an anaerobic bacterium Lactobacillus casei has been studied.

It was found that L. casei membrane particles hydrolyzed 0.1–0.2 μmol of ATP × mg protein−1× min−1 in the presence of Mg2+ or Ca2+ ions at optimal pH 6.4. This activity was inhibited by N,N′‐dicyclohexylcarbodiimide. Oligomycin, ouabain, vanadate and hydroxylamine were ineffective.

The addition of valinomycin to the intact L. casei cells in the absence of extracellular K+ and glucose resulted in an ATP synthesis which was abolished by the addition of K Cl, N,N′‐dicyclohexylcarbodiimide or carbonylcyanide m‐chlorophenylhydrazone. In the presence of glucose, L. casei cells showed an energy‐dependent accumulation of tetraphenylphosphonium cations, which was sensitive to N,N′‐dicyclohexylcarbodiimide and carbonylcyanide m‐chlorophenylhydrazone.

A soluble ATPase was isolated from L. casei membranes by means of (a) washing with a low ionic strength buffer in the presence of EDTA, Mg2+ ions and a protease inhibitor ‐ diisopropylfluorophosphate or (b) a chloroform treatment. Crude ATPase extracts were purified using isotachophoresis and (or) Sepharose 6B gelfiltration. Preparations of purified ATPase had a specific activity 3.0–4.0 μmol ATP × mg protein−1× min−1 and were homogeneous when studied with analytical disc electrophoresis or analytical ultracentrifugation (s20,w= 12 ± 0.5 S). The molecular mass of the L. casei ATPase estimated with sieve chromatography on Sepharose 6B proved to be 270 kDa. Purified L. casei ATPase denaturated by 1% sodium dodecyl sulphate or 8 M urea and then studied with dodecyl sulphate or urea polyacrylamide gel electrophoresis showed a single 43‐kDa band. L. casei ATPase subunits could not be separated by isoelectrofocusing in 8 M urea/polyacrylamide gel (a pH gradient of 0.2–0.3 cm was used) and migrated as a single band of isoelectric point at pH 4.2.

An electron microscopic study of the purified L. casei ATPase negatively stained with uranyl acetate showed particles of very regular structure. The predominant type of image was found to be a hexagonal particle 9.0–10.0 nm in size with 6 n point symmetry. An analysis of five different projections indicated that six similar subunits are arranged in two layers (three subunits in each layer) at vertices of two triangles rotated at 180°. This structure has a 32 point group symmetry.

It is concluded that the detachable part of membrane N,N×‐dicyclohexylcarbodiimide‐sensitive ATPase of L. casei is composed of six similar subunits. Such a simplified version of factor F1 may be regarded as an evolutionary precursor of the catalytic part of the H+ ‐ATP synthase of aerobic and photosynthetic bacteria, mitochondria, and chloroplasts, containing two types of major and 2–3 types of minor subunits. The possibility is discussed that simplicity of L. casei ATPase is due to the fact that in the cell it operates as an ATP‐splitting, rather than a synthesizing, mechanism.