Studies of the M15 beta-galactosidase complementation process

M15 beta-Galactosidase was activated by heat-denatured wild-type beta-galactosidase, urea, and heat-denatured wild-type beta-galactosidase, a peptide made up of residues 6-44 of beta-galactosidase and CB2, the peptide that is normally used for complementation (residues 3-92 of beta-galactosidase). In each case roughly equal activation levels were attained. Heat-denatured wild-type beta-galactosidase was present as a finely divided visible white precipitate both before and after complementation. The heat-denatured protein by itself did not migrate on native PAGE and both the protein and the activity that occurred as a result of the complementation also remained at the point of application. The N-terminal ends of the heat-denatured wild-type beta-galactosidase must have been available for complementation and must have been mobile enough to allow tetramer to form despite being aggregated. Beta-galactosidase denatured by both urea and heat resulted in a streak of interacting protein on the native PAGE. Upon activation, a streak (indicating that interaction was still occurring) was still present, but it moves more slowly. Complementation using a peptide called XP (made up of residues 6-44 plus an additional nine C-terminal amino acids) resulted in three discrete forms of active enzyme at ratios of peptide to M15 beta-galactosidase monomer of less than 1:1. The fastest migrating of the three bands predominated at ratios near 1:1. A single active tetrameric form of M15 beta-galactosidase was formed with CB2. In both of these last two cases an active slow-moving diffuse band also formed (possibly a dimer of the tetramer). A quantitation of the amount of peptide bound to M15 beta-galactosidase by titration with XP and with CB2 and by using gel filtration after an excess of fluorescent-labeled XP was added showed that peptide bound in a 1:1 ratio (peptide/monomer) when full activity was achieved. These fluorescent studies also showed that peptide initially bound to dimer and that the tetramer was then formed.