Characterization of three kinetically distinct forms of glutamate decarboxylase from pig brain

466

317

Section: Discussionmentioning

confidence: 92%

Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene.

Erlander

Hitz

et al. 1992

466

317

“…This raises the important question "What is the relationship of these bands to each other?" GAD may exist as a number of structural and kinetic variants (2)(3)(4). One interesting possibility is that these bands represent different variants.…”

Section: Discussionmentioning

confidence: 99%

“…1). Subsequent work has raised the possibility that important kinetic variant forms of GAD exist (2)(3)(4). Purification of GAD by conventional biochemical methods is beset with a number ofproblems, including a relatively low starting concentration in the brain, a tendency of the enzyme to aggregate, and the necessity of multiple column separations to resolve GAD from other proteins.…”

mentioning

confidence: 99%

Monoclonal antibodies to glutamic acid decarboxylase.

Gottlieb¹,

Chang²,

Schwob³

1986

111

Five monoclonal antibodies that recognize chicken brain glutamic acid decarboxylase (GAD) have been selected and designated GAD-1 to -5. GAD-1 to -5 were selected on the basis of their ability to immunoprecipitate active GAD from crude brain extracts. GAD-1 recognizes an epitope that is conserved in many vertebrates; the epitope recognized by GAD-5 is restricted to the chicken. Radioimmunoassays with GAD-1 indicate that GAD is highly enriched in brain relative to other tissues. GAD was localized immunocytochemically with GAD-1 and GAD-2 in rat cerebellum, spinal cord, and retina. The staining pattern is in agreement with that obtained previously with polyclonal antisera to GAD. GAD from the chicken brain was purified by chromatography on an immunoaffinity column made of GAD-1. NaDodSO4/PAGE analysis of the immunoafflity-purified GAD fractions shows a major band of 59 kDa and minor bands at 63 and 54 kDa.The enzyme glutamic acid decarboxylase (GAD) catalyzes the conversion of glutamic acid to y-aminobutyric acid (GABA) and CO2. The tissue and cellular distribution of GAD is highly restricted. GAD is localized largely to neurons and in particular to those that appear to use GABA as a neurotransmitter. Thus, the expression of GAD plays a key role in determining a neuron's transmitter phenotype.Many of the important kinetic and chemical properties of mammalian GAD were elucidated in the fundamental and pioneering studies of Roberts and his colleagues (reviewed in ref. 1). Subsequent work has raised the possibility that important kinetic variant forms of GAD exist (2-4). Purification of GAD by conventional biochemical methods is beset with a number ofproblems, including a relatively low starting concentration in the brain, a tendency of the enzyme to aggregate, and the necessity of multiple column separations to resolve GAD from other proteins. As a consequence, certain basic questions remain unanswered. Wu et al. (5) have purified mouse brain GAD to apparent homogeneity. However, NaDodSO4/PAGE analysis of the pure preparation reveals multiple bands ranging from 15 to 118 kDa (6). The relationship of these multiple bands to the active form(s) of the enzyme has not been elucidated. Antisera to this preparation have been extensively used to map the distribution of GAD in the brain (1). The exact range of polypeptides recognized by such antisera remains to be defined. No data on the amino acid sequence of GAD have been published.Monoclonal antibodies (mAbs) to GAD could greatly facilitate research on many aspects of the enzyme and the neurons that contain it. In this report, we describe five mAbs (GAD-1 to -5) that recognize GAD. GAD-1 and -2 are useful for immunohistochemical localization of the enzyme. Immunoaffinity purification with GAD-1 has given new data on the molecular nature of GAD. MATERIALS AND METHODSPartial Purification of GAD from Chicken Brains. GAD was partially purified from chicken brains by methods adapted from those of Wu et al. (5) and Oertel et al. (7). Frozen adult chicken brains were obt...

“…However, there is also a lack of concordance between the biochemical properties of GAD (19)(20)(21)(22) and those reported for the 64-kDa antigen (23,24). The deduced molecular masses ofthe recombinant GAD isoforms range from 64 to 67 kDa (25)(26)(27)(28) and native GAD is not a single species that migrates uniquely at 64 kDa (19,20). Furthermore, analysis of tryptic fragments of the 64-kDa antigen (29) reveals a GAD-associated 50-kDa product that is immunologically distinct from a 37-or 40-kDa fragment.…”

mentioning

confidence: 99%

Glutamic acid decarboxylase autoantibodies in preclinical insulin-dependent diabetes.

Aizpurua

Wilson

Harrison

1992

Insulin-dependent diabetes mellitus (IDDM) is associated with serum antibodies that precipitate a 64-kDa pancreatic islet cell protein reported to be glutamic acid decarboxylase (GAD; glutamate decarboxylase, EC 4.1.1.15). Previously, antibodies to GAD were found in the rare neurological disorder stiff man syndrome. To demonstrate directly antibodies to GAD, enzymatically active GAD was first purified from fresh human cerebellum. Brain GAD activity was precipitated by noninhibitory antibodies in the sera of 16/26 (62%) subjects defined as having preclinical IDDM (islet cell antibody-positive first-degree relatives of a person with IDDM), 3/13 (23%) with recent-onset IDDM, and 3/3 with the stiff man syndrome. In addition, sera of 5/26 (19%) preclinical and 2/13 (15%) recent-onset IDDM subjects contained antibodies that precipitated GAD but inhibited its activity. Thus, overall, 21/26 (81%) preclinical and 5/13 (38%) recent-onset IDDM subjects had antibodies that precipitated GAD activity.Antibodies to GAD were not detected in sera from subjects with other autoimmune diseases (n = 29) or healthy controls (n = 14). GAD affinity-purified to homogeneity (specific activity, 58 units/mg) was specifically immunoprecipitated as a single 60-kDa species by the IDDM sera. In an ELISA incorporating whole mouse brain GAD captured by the GAD-6 monoclonal antibody the frequencies of GAD antibodies for all subject groups were indistinguishable from those found by precipitation of human brain enzymatic activity. We conclude that (i) GAD is an (auto)antigen in a majority of subjects operationally dermed as having preclinical IDDM, (u) pancreatic islet and brain GAD are likely to be cross-reactive, and (Ui) the majority of GAD antibodies are directed away from the catalytic site of the brain enzyme. The lower frequency of GAD antibodies in recent-onset IDDM subjects indicates either that immunoreactivity is lost with near-total (3-cell destruction or that GAD antibodies denote a low risk of progression to clinical disease.