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Plant Physiol, February 2002, Vol. 128, pp. 714-725
Purification and Identification of a 42-Kilodalton Abscisic
Acid-Specific-Binding Protein from Epidermis of Broad Bean
Leaves1
Da-Peng
Zhang,*
Zhong-Yi
Wu,
Xi-Yan
Li, and
Zhi-Xin
Zhao
Laboratory of Molecular Developmental Biology of Fruit Trees, China
National Key Laboratory of Plant Physiology and Biochemistry, China
Agricultural University, 100094 Beijing, Peoples Republic of
China
Purification of abscisic acid (ABA)-binding proteins is considered
to constitute a major step toward isolating ABA receptors. We report
here that an ABA-binding protein was for the first time, to our
knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, isoelectric focusing
electrophoresis, and isoelectric focusing/sodium dodecyl
sulfate-polyacrylamide gel electrophoresis two-dimensional
electrophoresis of the purified ABA-binding protein all identified a
single protein band with a molecular mass of 42 kD and an isoelectric
point 4.86. The Scatchard plot for the purified protein showed a linear
function with a maximum binding activity of 0.87 mol mol 1
protein and an equilibrium dissociation constant of 21 nM,
indicating that the purified protein may be a monomeric one, possessing
one binding site. The ABA-binding protein was enriched more than
300-fold with a yield of 14%. ( )ABA and trans-ABA were substantially
incapable of displacing 3H-(±)ABA bound to the ABA-binding
protein, and (±)ABA was less effective than (+)ABA in the competition.
These findings allow establishment of the stereospecificity of the
42-kD protein and suggest its ABA receptor nature. Pretreatment of the
guard cell protoplasts of broad bean leaves with the monoclonal
antibody raised against the 42-kD protein significantly decreased the
ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.
1
This work was supported by the National Natural
Science Foundation of China (grant nos. 39730340, 39870487, and
30070532) and a grant from the China National Key Basic Research
Program (grant no. G1999011700).
*
Corresponding author; e-mail zhangdp{at}95777.com; fax
86-10-62891899.
© 2002 American Society of Plant Physiologists
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