A membrane form of L-glutamate decarboxylase (GAD) was identified and purified to apparent homogeneity from hog brain. The purified GAD was established as an integral membrane protein by phase-partitioning assay, charge-shift electrophoresis, and chromatography on a hydrophobic interaction column. This membrane GAD has a native molecular mass of 96 ± 5 kDa and is a homodimer of 48 ± 3-kDa subunits. Immunoprecipitation and immunoblotting tests revealed the presence of antibodies against this membrane GAD in sera from patients with insulin-dependent diabetes meilitus. Since this form of GAD appears to be an integral membrane protein and is presumed to have extracellular domains exposed, it seems reasonable to suggest that membrane GAD is more likely than soluble GAD to be involved in the pathogenesis of insulin-dependent diabetes and related autoimmune disorders such as stiff-man syndrome.y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (1) and also serves signaling (2) and trophic (3) roles in several neuronal and non-neuronal tissues. The rate-limiting step in GABA biosynthesis is the decarboxylation of L-glutamate by L-glutamate decarboxylase (GAD; EC 4.1.1.15). GAD has been implicated in several neuronal disorders, such as epilepsy (4), schizophrenia (5), and stiff-man syndrome (6), and has been identified as the autoantigen in insulin-dependent diabetes mellitus (IDDM) (7) and stiff-man syndrome (8). Several forms of soluble GAD (sGAD), notably (65 and 67 kDa, respectively), have been extensively studied, ranging from kinetic studies, to studies of regional and cellular/subcellular distribution, to structural analysis (for review, see ref. 9). From sequence information, it is clear that none of the sGADs, including GAD-65 and GAD-67, contains a stretch of hydrophobic amino acids long enough to span the membrane (about 20 residues), a typical feature for integral membrane proteins, or contains the appropriate consensus sequences for the attachment of GAD to membranes through fatty acylation via esterification, N-myristoylation, or glypiation (9-11). Unlike sGAD, little is known with certainty about the structure and function ofmembrane GAD (mGAD) despite the fact that about 50%6 of the total GAD activity in the brain is attributable to mGAD (12,13). GAD can interact with membranes by ionic or hydrophobic mechanisms. It was reported that GAD could become associated with membranes in the presence of Ca2+ (14). Covarrubias and Tapia (15,16) showed that this Ca2+-induced binding of GAD to the membranes occurred predominantly with pyridoxal 5'-phosphate (PLP)-dependent GAD, suggesting that mGAD might be dif-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.ferent from sGAD in terms ofaffinity toward its cofactor, PLP. Martin and Martin (17) have shown that apoGAD has a strong affinity for pol...
