PLANT PHYSIOLOGY , Vol 114, Issue 2 605-613, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
ATP Hydrolysis Activity and Polymerization State of Ribulose-1,5-Bisphosphate Carboxylase Oxygenase Activase (Do the Effects of Mg2+, K+, and Activase Concentrations Indicate a Functional Similarity to Actin?)
R. M. Lilley and A. R. Portis Jr
Department of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong 2522, Australia (R.M.L.)
The ATPase activity and fluoresence of ribulose-1,5-bisphosphate
carboxylase oxygenase (Rubisco) activase were determined over a range of
MgCl2, KCl, and activase concentrations. Both salts promoted ADP release
from ATP and intrinsic fluorescence enhancement by adenosine
5[prime]-[[gamma]-thio] triphosphate, but Mg2+ was about 10 times more
effective than K+. ATPase and fluorescence enhancement both increased from
zero to saturation within the same Mg2+ and K+ concentration ranges. At
saturating concentrations (5 mM Mg2+ and 22 mM K+), the specific activity
of ATPase (turnover time, about 1 s) and specific intrinsic fluorescence
enhancement were maximal and unaffected by activase concentration above 1
[mu]M activase; below 1 [mu]M activase, both decreased sharply. These
responses are remarkably similar to the behavior of actin. Intrinsic
fluorescence enhancement of Rubisco activase reflects the extent of
polymerization, showing that the smaller oligomer or monomer present in
low-salt and activase concentrations is inactive in ATP hydrolysis.
However, quenching of 1-anilinonapthaline-8-sulfonate fluorescence revealed
that ADP and adenosine 5[prime]-[[gamma]-thio] triphosphate bind equally
well to activase at low- and high-salt concentrations. This is consistent
with an actin-like mechanism requiring a dynamic equilibrium between
monomer and oligomers for ATP hydrolysis. The specific activation rate of
substrate-bound decarbamylated Rubisco decreased at activase concentrations
below 1 [mu]M. This suggests that a large oligomeric form of activase,
rather than a monomer, interacts with Rubisco when performing the release
of bound ribulose-1,5-bisphosphate from the inactive enzyme.
