PLANT PHYSIOLOGY , Vol 110, Issue 3 867-874, Copyright © 1996 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
Identification and Preliminary Characterization of a Ca2+- Dependent High-Affinity Binding Site for Inositol-1,4,5-Trisphosphate from Chenopodium rubrum
C. H. Scanlon, J. Martinec, I. Machackova, C. E. Rolph and P. J. Lumsden
Department of Applied Biology, University of Central Lancashire, Preston, PR1 2HE, United Kingdom (C.H.S., C.E.R., P.J.L)
Using a radioligand-binding assay we have identified a Ca2+- dependent
high-affinity D-myo-inositol-1,4,5-trisphosphate (InsP3) binding site in a
membrane vesicle preparation from Chenopodium rubrum. Millimolar
concentrations of Ca2+ were required to observe specific binding of
[3H]InsP3. A stable equilibrium between bound and free ligand was
established within 5 min and bound [3H]InsP3 could be completely displaced
by InsP3 in a time- and concentration-dependent manner. Displacement assays
indicated a single class of binding sites with an estimated dissociation
constant of 142 [plus or minus] 17 nM. Other inositol phosphates bound to
the receptor with much lower affinity. The glycosaminoglycan heparin was an
effective competitor for the binding site (inhibitor concentration for 50%
displacement = 534 nM). ATP at higher, although physiologically relevant,
concentrations (inhibitor concentration for 50% displacement = 241 [mu]M)
also displaced [3H]InsP3 from the receptor. Recent studies in animals have
highlighted the importance of Ca2+ regulation of InsP3-induced Ca2+
release. The potential for the operation of similar regulatory mechanisms
in plants is discussed.
