Effects of dicyclohexylcarbodiimide (DCCD) treatment on coupled activities of vanadate‐sensitive ATPase from plasma membrane of maize roots

Abstract

The presence of dicyclohexylcarbodiimide (DCCD) inhibited the activities of vanadate‐sensitive H+ ‐ATPase in both native and reconstituted plasma membrane of maize (Zea mays L. cv. WF9 × Mo 17) roots. Concentration dependence of DCCD inhibition on adenosine triphosphate (ATP) hydrolysis of native plasma membrane vesicles suggested that the molar ratio of effective DCCD binding to ATPase was close to 1. The DCCD inhibition of ATP hydrolysis could be slightly reduced by the addition of ATP, Mg:ATP, adenosine monophosphate (AMP), Mg:AMP and adenosine diphosphate (ADP). More hydrophilic derivatives of DCCD such as l‐ethyl‐N?‐3‐trimethyl ammonium carbodiimide (EDAC) or 1‐ethyl‐3‐3‐dimethyl‐aminopropyl carbodiimide (EDC) gave no inhibition, indicating that the effective DCCD binding site was located in a hydrophobic region of the protein. The proton transport activity of reconstituted plasma membrane at a temperature below 20°C or above 25°C was much sensitive to DCCD treatment. Build‐up of the proton gradient was analyzed according to a kinetic model, which showed that proton leakage across de‐energized reconstituted plasma membranes was not affected by DCCD, but was sensitive to the method employed to quench ATP hydrolysis. Reconstituted plasma membrane vesicles treated with DCCD exhibited a differential inhibition of the coupled H+‐transport and ATP hydrolysis. The presence of 50 μM DCCD nearly abolished transport but inhibited less than 50% of ATP hydrolysis. The above results suggest that the link between proton transport and vanadate‐sensitive ATP hydrolysis is indirect in nature.