Binding of NAD+ by cholera toxin

Abstract

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.

Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

“…The 14 C label incorporation efficiency of cholera toxin, DRAT, dinitrogenase reductase, and altered dinitrogenase reductase were all in the same range, around 0.01 mol of NAD/mol of protein. The oxygen-denatured wild-type or R101F dinitrogenase reductase did not incorporate 14 C label (Fig. 4, lanes 8 and 9).…”

“…The ability of the DRAT-R101F dinitrogenase reductase complex to cleave NAD was tested by mixing the two proteins with [carbonyl- 14 C]NAD. The production of nicotinamide was tested by chromatography of the reaction mixture on thin-layer plates; various standards were cochromatographed to establish the chromatographic position of products.…”

“…4). The negative control in lane 1 shows that chicken egg albumin did not incorporate the 14 C label under the same conditions, while the positive control in lane 2 shows that cholera toxin A1 can incorporate the 14 C label. The 14 C label incorporation efficiency of cholera toxin, DRAT, dinitrogenase reductase, and altered dinitrogenase reductase were all in the same range, around 0.01 mol of NAD/mol of protein.…”

“…Then 32 g of dinitrogenase reductase was added to the anaerobic cuvette with a Hamilton syringe to bring the final volume to 100 l. The cuvette was placed on ice with the optical transmission side 4 cm away from a 25-W germicidal lamp (General Electric) for 1 h. Following UV irradiation, the proteins in the reaction mixture were precipitated by 5% TCA and were washed two times with 5% TCA to remove free [carbonyl- 14 C]NAD; the pellet was then resuspended in 80 l of SDS sample buffer. Then 20 l was loaded on a standard SDS-PAGE gel (24).…”

“…Most ADP-ribosyltransferases, such as diphtheria toxin, exotoxin A, cholera toxin, and pertussis toxin, can bind and glycohydrolyze NAD without the presence of target proteins (5,6,14,29). Unlike these other ADP-ribosyltransferases, however, the ability of DRAT alone to glycohydrolyze NAD is undetectable in vitro (31) and an affinity of DRAT for NAD has not so far been detected.…”