Abstract
Creatine kinase (EC 2.7.3.2) of rabbit brain (BB) was purified to hemogeneity as judged by sedimentation velocity analysis in the ultracentrifuge. Nevertheless, the purified enzyme contained as many as seven extraneous protein species when examined by disc electrophoresis in polyacrylamide gel. The purified preparation also showed antigenic heterogeneity when it reacted in agar gel double immunodiffusion analysis with antibodies produced against it in the rooster. At least some of the extraneous proteins appeared to arise from BB during storage. Subsequently BB was purified to hemogeneity by disc electrophoresis in polyacrylamide gel. It was excised from the gel and injected repeatedly with Freund's complete adjuvant into roosters. A single immunoprecipitin line was noted in agar gel double immunodiffusion analysis when partially pure enzyme reacted with the antibodies raised to the electrophoretically pure protein. These antibodies inhibited the enzymatic activity of BB in both soluble and insoluble immune complexes. They did not, however, form immunoprecipitates with or inhibit rabbit muscle (MM) or rabbit hybrid (MB) creatine kinases. The antibodies were therefore specific for BB and did not react with MM or MB.
Highlights
From the Neurosciences Division, Research Institute, The Hospital for Sick Children, Montreal Neurological Institute, Montreal, Canada
The purified enzyme contained as many as seven extraneous protein species when examined by disc electrophoresis in polyacrylamide gel
The purified preparation showed antigenic heterogeneity when it reacted in agar gel double immunodiffusion analysis with antibodies produced against it in the rooster
Summary
From the Neurosciences Division, Research Institute, The Hospital for Sick Children, Montreal Neurological Institute, Montreal, Canada. The purified enzyme contained as many as seven extraneous protein species when examined by disc electrophoresis in polyacrylamide gel. The purified preparation showed antigenic heterogeneity when it reacted in agar gel double immunodiffusion analysis with antibodies produced against it in the rooster. BB was purified to homogeneity by dis’c electrophoresis in polyacrylamide gel. A single immunoprecipitin line was noted in agar gel double immunodiffusion analysis when partially pure enzyme reacted with the antibodies raised to the electrophoretically pure protein. These antibodies inhibited the enzymatic activity of BB in both soluble and insoluble immune complexes. Several methods for purification of creatine kinase’ (EC 2.7.3.2) from mammalian brain have been reported (l-5). $ During the course of this research, Fellow of the Medical Research Council of Canada, and subsequently, of the Muscular Dystrophy Association of Canada
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