Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis |
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| Authors: | Rea P A Britten C J Jennings I R Calvert C M Skiera L A Leigh R A Sanders D |
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| Affiliation: | Biochemistry and Physiology Department, Agricultural and Food Research Council Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, United Kingdom. |
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| Abstract: | The H+-translocating inorganic pyrophosphatase (H+-PPase) associated with vesicles of the vacuolar membrane (tonoplast) isolated from beet (Beta vulgaris L.) is subject to direct inhibition by Ca2+ and a number of other divalent cations (Co2+, Mn2+, Zn2+). By contrast, the H+-translocating ATPase (H+-ATPase) located on the same membrane is insensitive to Ca2+. Here we examine the mechanism and feasibility of regulation of the vacuolar H+-PPase by cytosolic free Ca2+ under the conditions thought to prevail in vivo with respect to Mg2+, inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca2+ or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg2PPi) at one site, inhibitory binding of Mg2PPi to a lower-affinity second site, binding of activator (Mg2+) at a third site, and direct binding of Ca2+ or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca2+ or CaPPi are explicable only if Ca2+, rather than CaPPi, is the inhibitory ligand. This conclusion is supported by the finding that CaPPi fails to mimic substrate in protection of the enzyme from inhibition by N-ethylmaleimide. Furthermore, the reaction scheme quantitatively and independently predicts the observed noncompetitive effects of free Ca2+ on the substrate concentration dependence of H+-PPase activity. The results are discussed in relation to the previous proposal that CaPPi is the principal inhibitory ligand of the vacuolar H+-PPase (M. Maeshima [1991] Eur J Biochem 196: 11-17) and the possibility that in vivo modulation of cytosolic free Ca2+ might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells. |
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