Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions

Abstract

Glutamate decarboxylase (GAD) catalyzes the production of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter. The mammalian brain contains two forms of GAD, with Mrs of 67,000 and 65,000 (GAD67 and GAD65). Using a new antiserum specific for GAD67 and a monoclonal antibody specific for GAD65, we show that the two forms of GAD differ in their intraneuronal distributions: GAD67 is widely distributed throughout the neuron, whereas GAD65 lies primarily in axon terminals. In brain extracts, almost all GAD67 is in an active holoenzyme form, saturated with its cofactor, pyridoxal phosphate. In contrast, only about half of GAD65 (which is found in synaptic terminals) exists as active holoenzyme. We suggest that the relative levels of apo-GAD65 and holo-GAD65 in synaptic terminals may couple GABA production to neuronal activity.

FIG. 1.

Immunoblots of mouse brain extracts. Lane 1, preimmune serum from rabbit K-1; lane 2, preimmune serum from rabbit K-2; lane 3, immune serum from rabbit K-1; lane 4, immune serum from rabbit K-2; lane 5, sheep antiserum to GAD (Oertel et al., 1981a); lane 6, GAD-6 monoclonal antibody (Chang and Gottlieb, 1988). Similar results are obtained with extracts of rat and human brain.

FIG. 2.

Immunohistochemical localization of GAD₆₅ and GAD₆₇ in rat cerebellar cortex. A. With K-2 antiserum, immunoreactivity is present in the somata of Purkinje cells (P) and extends into the primary and secondary dendrites (arrows). Labeling is also present in the somata of stellate/basket cells (arrowheads) within the molecular layer (M). Labeled puncta (presumptive axon terminals) are evident in all layers. B. With the GAD-6 monoclonal antibody, immunoreactive puncta (arrows) are present around the cell bodies of Purkinje cells (P) and are highly concentrated near the axon initial segments at the base of these neurons. Presumptive axon terminals are also distributed throughout the molecular layer (M). Little immunoreactivity is evident in neuronal cell bodies of all layers. Scale lines = 25 μm.