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. 1985 Sep;79(1):159–164. doi: 10.1104/pp.79.1.159

Pyrophosphate-Driven Proton Transport by Microsomal Membranes of Corn Coleoptiles 1

Alain Chanson 1,2,2, Jenny Fichmann 1,2, David Spear 1,2, Lincoln Taiz 1,2
PMCID: PMC1074844  PMID: 16664362

Abstract

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATP-driven H+-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H+-transport. At saturating (3 millimolar) concentrations of Mg2+:ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H+-transport, but had no effect on ATP-driven transport. Moreover, PPi-dependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO3, unlike the ATP-dependent H+-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound H+-translocating pyrophosphatases. Both potassium and a permanent anion (NO3− > Cl−), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N′-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.

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Selected References

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