The ClpAP protease from E. coli is composed of a proteolytic component (ClpP) and a regulatory ATPase component (ClpA). Ultracentrifugation has been conducted at pH 7.5 (0.1-0.3 M KCl plus/minus 10% glycerol) and 4 or 20 degrees C to determine the macromolecular size and shape of the ClpAP components. Without nucleotide, ClpA (subunit MW of 84,150) exists in a monomer-dimer equilibrium with log K approximately 5 (apparent s/20,w = 8.7 S). With the non-hydrolyzable analogue adenosine-5'-O-(3- thiotriphosphate) (ATPtauS) present, ClpA associates to a hexamer with MW = 505,000 plus/minus 3000 and s/20,w = 17.2 S (frictional ratio = 1.4). The inactive pro-ClpP(SA) (13.2 S) and mature ClpP (12.2 S) have identical subunits of 23,164, and 21,558 MW, respectively, and appear as two rings of seven subunits in electron microscopic image reconstructions (M. Kessel et al., J. Mol. Biol. 250, 587-594, 1995). Sedimentation velocity studies confirm that both the pro-Clp(SA) mutant and ClpP are 14-mers with molecular weights of 324,000 and 302,000 plus/minus 5%, respectively. Pro-ClpP(SA) also behaves as an ideal tetradecameric species by sedimentation equilibrium analysis. Nucleotide-bound, hexameric ClpA is required for association with ClpP to form the active ClpAP protease; log K approximately 8.5 /(M ClpA hexamer or M ClpP tetradecamer) under assay conditions. Sedimentation velocity experiments in the presence of ATPtauS, Mg(II), and varying molar ratios of hexameric ClpA to tetradecamer ClpP show two complexes: s/20,w = 21.0 plus/minus 0.4 S and 27.3 plus/minus 0.7 S. These correspond to 1:1 and 2:1 complexes of hexameric ClpA and oligomeric ClpP, with MW about 807,000 (frictional ratio = 1.6) and MW about 1,310,000 (frictional ratio = 1.6), respectively. Both complexes are active in casein or propeptide degradation and both are seen in electron micrographs. (This project formerly part of Z01 HL 00204 LB.)
