Abstract
Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at −20° C decreased both the number of the low-affinity sites and their dissociation constant (KD); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [3H]dihydroFC and the competition between [3H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H.
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Abbreviations
- Bmax :
-
maximal number of binding sites
- FC:
-
fusicoccin
- KD :
-
dissociation constant
- PIM:
-
protein-ase-inhibitor mixture
- PMSF:
-
phenylmethylsulfonyl fluoride
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The authors are indebted to Dr. L.M. Krasnopolskaya (Institute of Agricultural Biotechnology, Moscow, Russia) for fusicoccins A, C, J, and H, and to Dr. A.V. Galkin (Institute of Agricultural Biotechnology, Moscow, Russia) for valuable comments and ren dering the paper into English.
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Abramycheva, N.Y., Nesterenko, M.V. & Babakov, A.V. Factors affecting the binding between fusicoccin and plasma membranes from maize roots. Planta 189, 301–305 (1993). https://doi.org/10.1007/BF00194425
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DOI: https://doi.org/10.1007/BF00194425