Abstract
The ADP binding site within two types of bovine brain glutamate dehydrogenase isoproteins (GDH I and GDH II) was identified using photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-diphosphate (8N3ADP). 8N3ADP, without photolysis, mimicked the activatory properties of ADP on GDH I and GDH II activities, although maximal activity with 8N3ADP was about 75% of maximal ADP-stimulated activity. Saturation of photoinsertion with [alpha-32P]8N3ADP occurred at around 40 approximately 50 microM photoprobe with apparent Kd values near 25 and 40 microM for GDH I and GDH II, respectively. Photoinsertion of [alpha-32P]8N3ADP was decreased best by ADP in comparison with other nucleotides. With the combination of immobilized aluminum affinity chromatography and reversed-phase high performance liquid chromatography, photolabel-containing peptides generated by tryptic digestion were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as in the region containing the sequence, EMSWIADTYASTIGHYDIN. Photolabeling of the peptide was prevented over 90% by the presence of 1 mM ADP during photolysis, while other nucleotides could not reduce the amount of photoinsertion as effectively as ADP. These results demonstrate selectivity of the photoprobe for the ADP binding site and suggest that the photolabeled peptide with the residues Glu179-Asn197 is within the ADP binding domain of the brain GDH isoproteins.
Highlights
Glutamate dehydrogenase (GDH1; EC 1.4.1.3) is a family of enzymes that catalyze the reversible deamination of L-glutamate to 2-oxoglutarate using NADϩ, NADPϩ, or both as coenzyme [1]
The mutations identified in the patients with hyperinsulinism and hyperammonemia [23] exactly lie within a sequence of 15 amino acids that we previously suggested to contain GTP binding site of the brain GDH isoproteins [21]
These results were consistent with an earlier report showing interaction of 8-azido-ADP with bovine liver glutamate dehydrogenase [31]. These results show that the azidonucleotide, 8N3ADP, is able to elicit the similar biological effects on GDH isoproteins as the natural nucleotide, ADP
Summary
Materials—NADPϩ, NADϩ, NADH, 2-oxoglutarate, ADP, GTP, Lglutamate, aluminum chloride, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin were purchased from Sigma. 8N3ADP was purchased from RPI Corp. [␣-32P]8N3ADP was synthesized by the method as described previously [26, 27]. GDH I and GDH II (0.1 mg each) in 10 mM Tris acetate, pH 8.0, were separately incubated with various concentrations of [␣-32P]8N3ADP in Eppendorf tubes for 5 min with a hand-held UV lamp at a distance of 4 cm. 0.1 mg of GDH isoproteins were incubated with various concentrations of ADP for 10 min in the same buffer prior to the addition of 100 M [␣-32P]8N3ADP and allowed to incubate with the photoprobe for 5 min as described above. Tryptic Digestion of Photolabeled GDH Isoproteins—To determine the site modified by [␣-32P]8N3ADP, 2.0-mg samples of each GDH isoprotein in 10 mM Tris acetate, pH 8.0, were separately incubated with 100 M [␣-32P]8N3ADP for 5 min at 4 °C. HPLC fractions containing photolabeled peptides were pyridylethylated by the method described elsewhere [30] and sequenced by the Edman degradation method as described previously [17]
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