GIARDIA-INTESTINALIS - CHARACTERIZATION OF A NADP-DEPENDENT GLUTAMATE-DEHYDROGENASE

Glutamate dehydrogenase from Giardia intestinalis was purified 680-fol d to electrophoretic homogeneity with a 42% recovery through it two-st ep procedure. The most effective step in the purification was the use of CM-Trisacryl that eliminated nearly 99%, of the total proteins with 100% recovery. Matrix-assisted laser desorption ionization time-of-fl ight mass spectrometer was used to analyze the giardial glutamate dehy drogenase after deposition of the purified enzyme on a crystalline lay er of 3,5-dimethoxy-4-hydroxy-trans-cinnamic acid. Use of this sample preparation technique allowed the first successful determination of th e molecular mass of the enzyme (50, 120 +/- 75). Since the molecular w eight of the native enzyme was determined to be 270,000 by gel filtrat ion, the enzyme appears to be a hexamer. The enzyme was specific for N ADP(H) and functioned more favorably in the direction of glutamate for mation than catabolism. The pH optimum was 7.5 for reductive amination of 2-oxoglutarate and 9.3 for oxidative deamination of glutamate. The apparent K-m values were 0.28 mM for 2-oxoglutarate and 17 mu M for N ADPH. An unusual biphasic saturation curve characterized the effect of ammonium ion on the activity with a plateau between 40 and 55 mM. (C) 1998 Academic Press.