Methylglyoxal can modify GAPDH activity and structure.
The activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can play an important role in regulating multiple upstream pathways relating to the development of diabetic complications. GAPDH can be modified by a number of metabolic factors, including oxidative and glycation products. To study the effect of glycation on GAPDH we have measured GAPDH structure and activity after exposure of the enzyme to the potent alpha dicarbonyl sugar methylglyoxal (MG). Rabbit GAPDH was incubated with 10-1000 microM MG for 96 hours, and enzyme activity was measured at intervals by a spectrophotometric assay. Isoelectric focusing of purified and cellular GAPDH was performed with a PROTEAN IEF system and the bands visualized by Western blotting. The mass of glycated and native GAPDH was determined by MALDI with a Applied Biosystems Voyager System 6235. GAPDH activity (at 96 h) was decreased by 20% with 1.0 micromolar MG and showed progressively greater suppression of activity with increasing concentrations up to 1 mM, where activity was decreased by 97%. Reduction in GAPDH activity was rapidly decreasing by 69.2% by two hours with 1 mM MG. IEF showed an isoelectric point (IEP) of 8.5 for native GAPDH, while measurable changes were seen with modification by MG levels of 1 mM (IEP 7.5) and 50 microM (IEP 8.0). With MALDI, GAPDH mass increased from 36.012 kDa to 37.071 after exposure to 50 microM MG and to 40.625 following 1 mM MG. This indicates addition of 12.75 and 55.6 MG residues, respectively, to GAPDH. GAPDH can be modified by methylglyoxal intracellular concentrations close to those previously observed in vivo, with measurable changes in isoelectric point and mass. These modifications can lead to decreased enzyme activity, suggesting that conditions associated with elevated intracellular MG could modify GAPDH activity in vivo.
Lee
Hyon Jae
HJ
Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, 1 Medical Center Drive, Lebanon, NH 03756, USA.
Howell
Scott K
SK
Sanford
Rebecca J
RJ
Beisswenger
Paul J
PJ
D003920:Diabetes Mellitus / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D005947:Glucose; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011765:Pyruvaldehyde / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011765:Pyruvaldehyde; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005947:Glucose