Regulation of vacuolar h-pyrophosphatase by free calcium : a reaction kinetic analysis.
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 Ca(2+) and a number of other divalent cations (Co(2+), Mn(2+), Zn(2+)). By contrast, the H(+)-translocating ATPase (H(+)-ATPase) located on the same membrane is insensitive to Ca(2+). Here we examine the mechanism and feasibility of regulation of the vacuolar H(+)-PPase by cytosolic free Ca(2+) under the conditions thought to prevail in vivo with respect to Mg(2+), inorganic pyrophosphate (PPi), and pH. The minimal reaction scheme that satisfactorily describes the effects of elevated Ca(2+) or CaPPi on the enzyme is one that invokes equilibrium binding of substrate (Mg(2)PPi) at one site, inhibitory binding of Mg(2)PPi to a lower-affinity second site, binding of activator (Mg(2+)) at a third site, and direct binding of Ca(2+) or CaPPi to a fourth site. Changes in enzyme activity in response to selective manipulation of either Ca(2+) or CaPPi are explicable only if Ca(2+), 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 Ca(2+) 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 Ca(2+) might constitute a specific mechanism for selective regulation of this enzyme, and consequently for stabilization of PPi levels in the cytoplasm of plant cells.
Rea
P A
PA
Biochemistry and Physiology Department, Agricultural and Food Research Council Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, United Kingdom.
Britten
C J
CJ
Jennings
I R
IR
Calvert
C M
CM
Skiera
L A
LA
Leigh
R A
RA
Sanders
D
D