Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Abstract

Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.

Identification of cysteine-319 as the target amino acid of 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate in bovine liver glutamate dehydrogenase

“…Two successive column centrifugations were carried out at 4 °C to remove the excess reagent, the first column being equilibrated with 0.1 M potassium phosphate buffer, pH 7.1, and the second column equilibrated with 0.1 M potassium phosphate buffer, pH 8.0. The "protected" enzyme was prepared under the same conditions except that 200 ^M GTP -I-5 mM NADH were included in the incubation mixture (Ozturk et al, 1990). A control enzyme was also prepared with neither ligands nor 8-BDB-TA-5'-TP present in the incubation mixture and was isolated in the same manner.…”

“…incorporation of 8-BDB-TA-5'-TP into Glutamate Dehydrogenase. The amount of 8-BDB-TA-5'-TP incorporated into the modified and the protected enzyme after 3-h incubation with 0.2 mM reagent was determined from the amount of organic phosphate incorporated into these enzyme samples, as described previously (Ozturk et al, 1990).…”

“…We have recently reported that bovine liver glutamate dehydrogenase reacts covalently with 8-[(4-bromo-2,3-dioxobutyl)thio] adenosine 5'-triphosphate (8-BDB-TA-5'-TP)' with incorporation of about 1 mol of reagent/mol of enzyme subunit (Ozturk et al, 1990). The modified enzyme is catalytically active, but exhibits decreased sensitivity to inhibition by GTP and by high concentrations of NADH.…”

Inhibition of ADP-induced Platelet Responses by Covalent Modification of Aggregin, a Putative ADP Receptor, by 8-(4-Bromo-2,3-dioxobutylthio)ADP

“…8-(4-Bromo-2,3-dioxobutylthio)ADP (8-BDB-TADP) (Fig. 1C) has been previously used as an ADP affinity label to probe the structure-function relationship of ADP-requiring enzymes such as pyruvate kinase (24) and glutamate dehydrogenase (25,26). 8-BDB-TADP has the characteristic diphosphate of the natural ligand ADP, and is both hydrophilic and negatively charged at neutral pH.…”

“…In order to further investigate and test our hypothesis that aggregin is a putative ADP receptor, we chose 8-BDB-TADP as a true ADP affinity ligand to label the ADP receptor and ascertain its effect on ADP-induced platelet responses. In this case the nature of the chemical reaction is well understood and chemically defined products have been isolated (26,27). This report describes in detail the effect of 8-BDB-TADP on ADP-induced platelet responses and the protein(s) modified on the surface of human platelets.…”