CHYMOTRYPTIC CONVERSION OF BACTERIAL-MEMBRANE ATPASE TO AN ACTIVE FORM WITH MODIFIED ALPHA-CHAINS AND DEFECTIVE MEMBRANE BINDING PROPERTIES

The effect of chymotrypsin on the structure, catalytic activity and membrane binding properties of the energy transducing ATPase in Streptococcus faecalis was examined. Chymotrypsin caused a limited cleavage of the solubilized ATPase producing a fully active protein which migrated as a single faster moving band in gel electrophoresis. The modified ATPase, designated CHY-ATPase, was relatively resistant to further chymotryptic alteration. It retained the full complement of tightly bound nonexchangeable nucleotide present in the native ATPase. The CHY-ATPase contained modified .alpha. chains, designated .alpha.'', which were about 2000 daltons smaller than the native 55,000-dalton .alpha. chains. However, no alterations in the other subunits, .beta., .gamma., .delta. or .epsilon., were detected. The subunit composition of the native ATPase, .alpha.3.beta.3 .gamma. .delta. .epsilon., was apparently changed to .alpha.''3.beta.3 .gamma. .delta. .epsilon.. Unlike the native ATPase, the CHY-ATPase failed completely to reattach to depleted membranes. Short chymotrypsin-sensitive .alpha. chain tails protrude from the ATPase surface, and these peptide segments are needed for membrane attachment. The .delta. chain was also shown to be required for attachment, confirming previous work. The 3 .alpha. chains and the .delta. chain act together as a device to ensure firm association of the ATPase with the membrane.