Measurement of Mitochondrial ATPase Activity in Maize Root Tips by Saturation Transfer 31P Nuclear Magnetic Resonance

Abstract

We show that saturation transfer ³¹P nuclear magnetic resonance can be used to measure the activity of the mitochondrial ATPase of maize (Zea mays L. hybrid WW × Br38) root tips in vivo. Unidirectional rates of ATP synthesis were determined in the steady state (i.e. ATP and cytoplasmic orthophosphate constant) under various conditions. These measurements, and determinations of oxygen consumption, give a P/O ratio (measured in the living tissue) close to 3. In succinate-fed root tips the P/O ratio is approximately 2. Cyanide inhibits the rate of ATP synthesis by two-thirds (P/O ratio ∼1), with an effective inhibitor constant of ∼35 micromolars. We show that the alternative electron pathway cannot make ATP, and does not normally operate in this tissue. This method of studying plant mitochondrial metabolism avoids potential artifacts encountered in studies using isolated and purified mitochondria. The method also allows, for the first time, direct and simultaneous examination of the relationship between the rate of ATP synthesis and levels of metabolites such as ATP, and derived parameters such as phosphorylation potential.