Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves.
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 (3)H-(+/-)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.
Zhang
Da-Peng
DP
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. zhangdp@95777.com
Wu
Zhong-Yi
ZY
Li
Xi-Yan
XY
Zhao
Zhi-Xin
ZX
D000040:Abscisic Acid / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D007887:Fabaceae / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010739:Phospholipase D / Q000187:drug effects / Q000378:metabolism; D019441:Plant Epidermis / Q000737:chemistry; D010936:Plant Extracts / Q000302:isolation & purification; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D010936:Plant Extracts; D010940:Plant Proteins; D000040:Abscisic Acid; D010739:Phospholipase D