ATP-dependent proton transport into vesicles of microsomal membranes of Zea mays coleoptiles

Abstract

ATP-dependent proton pumps were found in the vesicles of microsomal membrane fractions of maize coleoptiles. Two membrane fractions isolated by density gradient centrifugation were identified by the aid of marker enzymes and electron microscopic analysis. Membrane fraction A largely consisted of vesicles of smooth ER and of the Golgi complex, fraction B predominantly of vesicles of plasmalemma and rough ER. The pH-indicator, neutral red, was used to measure changes in pH in the vesicles after ATP addition. Due to the binding of protonated neutral red molecules (NRH+) to negative charges of the energized membrane, a strong metachromasy of NRH+-absorption can be observed. Therefore, in order to accurately measure delta pH a pH-dependent change in absorption of neutral red covering the whole NR-spectrum was set up as difference spectra. The commonly employed method of measuring delta A of neutral red at just one wavelength (525 nm) leads to entirely incorrect results. It could be demonstrated that the ATP-dependent translocation of H+-ions into the interior of the vesicles was most efficient at pH 7. Acidification, which reaches its maximum 10--15 min after ATP addition, can be reverted by adding CCCP. An ATP-dependent proton-translocation into the vesicles of fraction B was also observed, however, the proton translocation is less than that found in fraction A in relation to the amount or protein found in each. The membrane fraction A displays a strong oxidation of NADH subsequently followed by an alkalization of the medium. This process cannot be reverted by adding CCCP. NADH oxidation at membranes of fraction A is consequently not an integral part of a redox-pump. A possible significance of the ATP-dependent proton pump in membranes of the ER and Golgi fraction of coleoptiles is discussed in connection with auxin induced elongation growth.