Kinetics and Specificity of ATP-dependent Proton Translocation Measured with Acridine Orange in Microsomal Fractions from Green Suspension Cells of Chenopodium rubrum L.

From photoautotrophic cell suspension cultures of Chenopodium rubrum L. microsomal fractions were prepared by differential and isopycnic sucrose gradient centrifugation. ATP-generated proton-accumulation was studied on a microsomal fraction (MF) of proton-translocating vesicles with a density of 1.11 to 1.12 gem(-3). This activity did not overlap with mitochondrial or chloroplast markers. MF is presumably of tonoplast origin. The assay was based upon pH-dependent acridine orange (AO) absorption shifts at 490 nm, Δ(490), due to AO-accumulation and polymerization within the vesicles, and was calibrated using pK and K(D) for AO polymers given by Zanker (Z. physikal. Chemie 199, 225, 1952). Addition of 1 mM ATP to MF yields a transmembrane ΔpH of about 2 units within 10 min (kinetics with a single time constant, τ=400s) and collapses after addition of the protonophor CCCP (τ=22s). The proton pumping activity is insensitive to vanadate and highly specific for ATP compared with GTP, UTP, CTP, and ITP, and half-saturates at 0.38 to 0.46 mM ATP, indicated by both initial rate (1 min) and steady state value of Δ(490) (10 min). Proton pumping into and proton leakage from the vesicles was cast into a feedback-scheme showing that the pump is working at a constant rate, that is, independently of the size of the created ΔpH.