Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches

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Abstract
An energy-transducing ATPase (EC 3.6.1.3) that contains an extra polypeptide (.delta.) and 3 intrinsic subunits (.alpha., .beta., .gamma.) was purified from M. lysodeikticus luteus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is .alpha.3.beta.3.gamma..delta.. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its .alpha., .beta. and .gamma. subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(.alpha.):3.00(.beta.):0.17(.gamma.) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of .alpha. subunits and absence of .gamma. subunit are not active. Of the antisera to subunits, only anti-(.beta.-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(.alpha.-subunit) or anti-(.gamma.-subunit) serum increases the effect. Apparently .beta. subunit is required for full ATPase activity, although a minor proportion of .alpha. and perhaps .gamma. subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in M. lysodeikticus ATPase had a MW of 20,000 and was involved in ATPase binding to membranes. This 20,000-dalton component can be equated with the .delta. subunit of other energy-transducing ATPases; its association with the (.alpha., .beta., .gamma.) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The .gamma. subunit may also play a role in ATPase binding, in which the major subunits do not seem to play a role.

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