PLANT PHYSIOLOGY , Vol 112, Issue 1 343-351, Copyright © 1996 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Purification and Characterization of Pyrophosphate-Dependent Phosphofructokinase from Phosphate-Starved Brassica nigra Suspension Cells
M. E. Theodorou and W. C. Plaxton
Departments of Biology and Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6
Previously, we reported that inorganic phosphate (Pi) deprivation of
Brassica nigra suspension cells or seedlings leads to a progressive
increase in the [alpha]:[beta]-subunit ratio of the inorganic pyrophosphate
(PPi)-dependent phosphofructokinase (PFP) and that this coincides with a
marked enhancement in the enzyme's activity and sensitivity to its
allosteric activator, fructose-2,6-bisphosphate (Fru-2,6-P2). To further
investigate the effect of Pi nutrition on B. nigra PFP, the enzyme was
purified and characterized from Pi-starved B. nigra suspension cell
cultures. Polyacrylamide gel electrophoresis, immunoblot, and
gel-filtration analyses of the final preparation indicated that this enzyme
exists as a heterooctamer of approximately 500 kD and is composed of a 1:1
ratio of immunologically distinct [alpha] (66 kD) and [beta] (60 kD)
subunits. The enzyme's [alpha] subunit was susceptible to partial
proteolysis during purification, but this was prevented by the presence of
chymostatin and leupeptin. In the presence and absence of 5 [mu]M
Fru-2,6-P2, the forward activity of PFP displayed pH optima of pH 6.8 and
7.6, respectively. Maximal activation of the forward and reverse reactions
by Fru-2,6-P2 occurred at pH 6.8. The potent inhibition of the forward
activity by Pi (concentration of inhibitor producing 50% inhibition of
enzyme activity [I50] = 1.3 mM) was attributed to a marked Pi-dependent
reduction in Fru-2,6-P2 binding. The reverse reaction was
substrate-inhibited by Pi (I50 = 13 mM) and product-inhibited by PPi (I50 =
0.9 mM). The kinetic data are consistent with the hypothesis that PFP may
function to bypass the ATP-dependent PFP in Pi-starved B. nigra. The
importance of the Pi nutritional status to the regulation and predicted
physiological function of PFP is emphasized.
