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In Vitro Study of Passive Nitrate Transport by Native and Reconstituted Plasma Membrane Vesicles From Corn Root Cells

In Vitro Study of Passive Nitrate Transport by Native and Reconstituted Plasma Membrane Vesicles From Corn Root Cells

J P Grouzis, P Pouliquin, J Rigaud, C Grignon, R Gibrat

Biochim Biophys Acta. 1997 Apr 26;1325(2):329-42.

PMID: 9168158

Abstract:

Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H+)ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K+ diffusion gradient. Consequently, the H(+)-pumping activity of the (H+)ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO3- to electrically short-circuit the (H+)ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable (K(m) approximately 5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO3- > I- approximately ClO3- approximately Br- > Cl- approximately NO2- > Iminodiacetate approximately SO4(2-)). Passive NO3- transport was also determined, independently of the (H+)ATPase, from the NO3(-)-dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K(m) for NO3- and should be suitable to continue the functional and biochemical characterization of the NO3- transport system.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP1196157853	Nitrate Ionophore VI		1196157-85-3	Price

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