| Abstract: | Antibodies raised against wild type β-galactosidase of Escherichiu coli activate certain inactive mutant β-galactosidases to enzymic activity. We have developed a technique to detect 7 S activating antibody after isoelectric focusing in thin layers of polyacrylamide gels. Enzymic activity bound to antibodies is detected using the histochemical substrate 5-bromo-4-chloro-3-indoxyl-β-D-galacto-pyranoside. When split, this substrate produces insoluble, blue-green indigo stain at the sites of enzymic reaction. With this technique antisera raised in five rabbits against the wild type enzyme have been analyzed for their capacity to activate inactive mutant enzyme. Each rabbit produces a characteristic spectrum of activating antibodies when tested with a given mutant enzyme, which is different from that produced by another rabbit. A restricted population of antibodies, e.g. the product of one to five clones of antibody-forming cells activate a mutant enzyme. The spectrum of activating antibodies obtained with a mutant enzyme can be correlated to the position of the mutation in the gene for β-galactosidase which gives rise to this inactive enzyme. Three genetically different groups of mutant enzymes are activated by three distinct populations of antibodies within the serum of one rabbit. Mutation at a fourth site within the gene yields a mutant enzyme which is activated by one of the three populations of specific antibodies. This indicates that one antibody, in binding to one antigenic site, can restore the activity of two mutant enzymes altered at two different sites within the polypeptide chain of the enzyme. |
