Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies.

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.

Application of the magnetization-inversion-transfer technique to the transport of 7Li+, 23Na+, and 39K+ ions through the gramicidin channel and the M2 delta transmembrane domain of the nicotinic acetylcholine receptor.

The magnetization-inversion-transfer NMR technique has been used to determine the activation enthalpy (delta H not equal to) for the transport of the 7Li+, 23Na+, and 39K+ ions through the gramicidin channel incorporated into vesicle membranes. The activation enthalpy obtained for the overall transport of the monovalent cations was found to be in agreement with the selectivity order and single-channel ion conductance values. Furthermore, the magnetization-inversion-transfer technique was also found to be applicable to the channel formed from a synthetic peptide having the same amino acid sequence as the M2 delta transmembrane segment of the nicotinic acetylcholine receptor.

23Na-NMR study of ion transport across vesicle membranes facilitated by phenylalanine analogs of gramicidin.

The transport of Na+ ions across phosphatidylcholine/phosphatidylglycerol large unilamellar vesicle membranes facilitated by phenylalanine analogs of gramicidin A has been studied using 23Na-NMR spectroscopy. The four analogs studied were Phe9-, Phe11-, Phe13-and Phe15-gramicidin A. These analogs were found to transport Na+ ions in the following order Phe15 > Phe13 > Phe11 > Phe9. The entropy and enthalpy of activation for the transport of Na+ ions were determined for each analog. A correlation is made between the activation enthalpies and the single channel conductance values of the analogs.

Kinetics of channel formation of gramicidins A and B in phospholipid vesicle membranes.

The thermal incorporation and channel formation of gramicidins A and B into phosphatidylcholine/phosphatidylglycerol large unilamellar vesicle membranes was studied using 23Na NMR. Delta H and delta S of activation for channel formation for gramicidin A are 11.8 kcal/mol and -11 e.u., respectively. For gramicidin B, delta H and delta S of activation are 14.6 kcal/mol and -4 e.u., respectively. Possible reasons for the differences in delta H and delta S of activation between the two analogues are discussed.