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 be ta-galactosidase). In each case roughly equal activation levels were a ttained. Heat-denatured wild-type beta-galactosidase was present as a finely divided visible white precipitate both before and after complem entation. The heat-denatured protein by itself did not migrate on nati ve PAGE and both the protein and the activity that occurred as a resul t of the complementation also remained at the point of application. Th e N-terminal ends of the heat-denatured wild-type beta-galactosidase m ust have been available for complementation and must have been mobile enough to allow tetramer to form despite being aggregated. beta-Galact osidase denatured by both urea and heat resulted in a streak of intera cting protein on the native PAGE. Upon activation, a streak (indicatin g that interaction was still occurring) was still present, but it move s more slowly. Complementation using a peptide called XP (made up of r esidues 6-44 plus an additional nine C-terminal amino acids) resulted in three discrete forms of active enzyme at ratios of peptide to M15 b eta-galactosidase monomer of less than 1:1. The fastest migrating of t he three bands predominated at ratios near 1:1. A single active tetram eric form of M15 beta-galactosidase was formed with CB2. In both of th ese last two cases an active slow-moving diffuse band also formed (pos sibly a dimer of the tetramer). A quantitation of the amount of peptid e bound to M15 beta-galactosidase by titration with XP and with CB2 an d by using gel filtration after an excess of fluorescent-labeled XP wa s added showed that peptide bound in a 1:1 ratio (peptide/monomer) whe n full activity was achieved. These fluorescent studies also showed th at peptide initially bound to dimer and that the tetramer was then for med.