J. Biochem, 1974, Vol. 76, No. 6 1175-1182
© 1974 Japanese Biochemical Society
research-article |
Membrane-bound Adenosinetriphosphatase of Escherichia coli
II. Physicochemical Properties of the Enzyme*
Faculty of Pharmaceutical Sciences, University of Tokyo Hongo, Bunkyo-ku, Tokyo 113
Membrane-bound adenosinetriphosphatase [ATPase,** EC 3.6.1.3 [EC] ] of Escherichia coli was purified on a large scale. The purified ATPase contained 0.72 µg of total phosphorus per mg of protein. Less than 17% of the total phosphorus was extracted with chloroform-methanol and the purified enzyme contained an unknown phosphorus compound(s) which could not be extracted with chloroform-methanol and was not dialyzable.
We examined the subunits of ATPase. The pattern of the purified enzyme on SDS gel was very similar to that of beef heart mitochondrial ATPase. ATPase of Escherichia coli consisted of four subunits, 
, ß, 
and 
, with molecular weights of 54,000, 52,000, 33,000, and 11,000, respectively. ATPase purified by our method did not contain subunit 
reported by Futai et al.
An inactive fragment of ATPase was obtained, which did not contain subunit , suggesting requirement of this particular subunit for enzyme activity.
The isoelectric point of ATPase was estimated as pH 4.6 by electrophoresis on a column of Ampholine.
ATPase was cold-labile and inactivated by a high salt concentration. On inactivation it dissociated into small molecules.
*This work was supported in part by a grant from the Ministry of Education (C758034), Japan.