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Effects of Purine Nucleotides on the Stability of Glutamate Dehydrogenase Studied using a Fluorescence Based Thermal Shift Assay
Author(s) -
Smith Andrew,
Bell Jessica,
Bell Ellis
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.612.13
Subject(s) - nad+ kinase , chemistry , glutamate dehydrogenase , gtp' , nucleotide , biochemistry , cofactor , dehydrogenase , oxidative deamination , enzyme , stereochemistry , glutamate receptor , receptor , gene
Mammalian Glutamate Dehydrogenase is unique in that it utilizes both NAD+ and NADP+ in the oxidative deamination of L Glutamate, and like many higher eukaryote glutamate dehydrogenases is highly regulated with GTP being a potent inhibitor and ADP, depending upon pH and substrate concentrations being either an activator or an inhibitor. Previous studies using Thermal Melts followed by CD at 222nm have indicated that while GTP enhances global flexibility, ADP makes the enzyme more rigid. Due to absorbance in the 220–250nm region it is difficult to use these nucleotides at high concentrations or in combination with cofactors. To overcome this problem we have used a Fluorescence Based Thermal Shift assay to screen both the concentration dependence of the effects of ADP, GTP, NAD+, NADP+, NADH and NADPH individually and with various combinations of nucleotides, with and without glutamate or 2 oxoglutarate, on the stability of the enzyme to establish which binding events predominate effects on stability. More detailed information on the effects are being investigated using limited proteolysis with immobilized trypsin and chymotrypsin combined with tandem mass spectrometry to identify the time course of cleavage and the effects of purine ligands in the presence or absence of the substrate glutamate or 2 oxoglutarate.