Phylogenesis of Brain Glutamic Acid Decarboxylase from Vertebrates: Immunochemical Studies

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Abstract
Brain high-speed supernatants from various lower and higher vertebrates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblot on nitrocellulose membranes, and immunolabelling using an anti-glutamic acid decarboxylase (anti-GAD) antiserum prepared from rat antigen. Rat brain extracts showed two distinct immunolabelled bands (MW 59,000 and 62,000 daltons). The molecular weight of the native enzyme was 120,000 daltons. The immunoblot pattern was not affected by a 3-h incubation of the homogenate. In the substantia nigra, the decrease in the immunolabelling of both bands corresponded very closely to the decrease of GAD activity following lesioning of the striato-nigral pathway. Moreover, experiments with preadsorbed antiserum showed that both subunits have common antigenic determinants. The immunolabelling was consistently more intense over the lightest band. The autoradiography of immunoprecipitated rat brain GAD, iodinated prior to electrophoresis, revealed two radiolabelled bands corresponding to the two immunolabelled ones. Their radioactivity was found in a one-to-five ratio which closely paralleled their respective immunolabelling intensity. Thus, the two subunits recognized by the antiserum are not present in stoichiometric proportions in the rat brain high-speed supernatant. These findings suggest the existence of two homodimeric GAD with common antigenic determinants which are present in different amounts. Immunoprecipitation curves of brain GAD from rat, mouse, rabbit, monkey, human, quail frog, and trout were similar, with a < 10-fold maximum shift in affinity for GAD. GAD immunoblots from the various higher vertebrates showed a pattern similar to that obtained in rat. In contrast, in lower vertebrates (trout, frog) only one band was detected, the molecular weight of which was consistently found to be between the molecular weight values of the two bands immunodetected in the brains of higher vertebrates. The SDS-electrophoresis of iodinated immunoprecipitated GAD from trout brain showed only one radiolabelled band which corresponded to the immunolabelled one. Native GAD from rat and trout brain exhibited an identical molecular weight (MW 120,000). These findings suggest that GAD exists in a unique homodimeric form in the brain of lower vertebrates. With antiserum preadsorbed on trout GAD, the immunolabelling of the two bands disappeared on rat brain immunoblots. All the antigenic determinants present on either subunit of the rat brain GAD are also present on the unique GAD subunit of the trout brain. Our results show that, although all immunogenic determinants have been highly conserved, a limited phylogenetic evolution on brain GAD has occurred among the vertebrates. This might have taken place at the posttranslational processing level, or alternatively at the genomic level, through, for example, gene duplication and subsequent mutations.