Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast.
OBJECTIVE
Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65.


METHODS
Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera.


RESULTS
The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C.


CONCLUSIONS
Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.
Tong
Jonathan C
JC
Department of Biochemistry and Molecular Biology, Monash University, Clayton Vic. 3800, Australia.
Mackay
Ian R
IR
Chin
Judy
J
Law
Ruby H P
RH
Fayad
Karine
K
Rowley
Merrill J
MJ
D002384:Catalysis; D004795:Enzyme Stability; D015870:Gene Expression; D005968:Glutamate Decarboxylase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D005680:gamma-Aminobutyric Acid / Q000737:chemistry
D007527:Isoenzymes; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D011732:Pyridoxal Phosphate; D005968:Glutamate Decarboxylase