Plant Physiology 100:1706-1715 (1992)
© 1992 American Society of Plant Biologists
Membranes and Bioenergetics
Regulation of Vacuolar H+-Pyrophosphatase by Free Calcium 1
A Reaction Kinetic Analysis
Philip A. Rea,
Christopher J. Britten,
Ian R. Jennings,
Caroline M. Calvert,
Lorna A. Skiera,
Roger A. Leigh and
Dale Sanders
Biochemistry and Physiology Department, Agricultural and Food Research Council Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, United Kingdom,
Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
Biology Department, University of York, York YO1 5DD, United Kingdom
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.
1 This work was supported by the Agricultural and Food Research Council (Grant PG87/501 to D.S.), the National Science Foundation (Grant DCB-9005330 to P.A.R.), and the Science and Engineering Research Council (Studentships to C.J.B. and C.M.C.)
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