In this work [14 C]spermidine binding to total proteins solubilized from plasma membrane purified from zucchini (Cucurbita pepo L.) hypocotyls was investigated. Proteins were solubilized using octyl glucoside as a detergent. Specific polyamine binding was thermolabile, reversible, pH dependent with an optimum at pH 8.0, and had a K d value of 5 M, as determined by glass-fiber-filter assays. Sephadex G-25 M gel-filtration assays confirmed the presence of a spermidine-protein(s) complex with a specific binding activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis of collected fractions having the highest specific spermidine-binding activity, several protein bands (113, 75, 66, and 44 kD) were identified. The specificity of spermidine binding was examined by gel-filtration competition experiments performed using other polyamines and compounds structurally related to spermidine. Partial purification on Sephadex G-200 led to the identification of 66-and 44-kD protein bands, which may represent the putative spermidine-binding protein(s) on the plasmalemma.Polyamines are biologically ubiquitous compounds that are implicated in many aspects of growth and development in a wide range of organisms (microorganisms, animals, and plants) (Tabor and Tabor, 1985; Bagni, 1989;Persson et al., 1996), although their specific mechanism of action is not well understood. By virtue of their cationic nature at physiological pH, they can interact with several molecules and thereby affect their structure and function. Hydrogen binding, ionic and covalent linkages, and hydrophobic interactions may occur that involve the charged functional amino and imino groups and the tetra-and trimethylene chains (Feuerstein and Marton, 1989;Schuber, 1989; SerafiniFracassini et al., 1995).One possible mechanism by which polyamines might act as growth substances involves their binding to specific regulatory proteins, as has been observed in many organisms. In both animal and plant systems it has been postulated that these compounds may play a role in the posttranslational modifications of proteins (Ä schlimann and Paulsson, 1994;Serafini-Fracassini et al., 1995), as well as in the modulation of many enzyme activities such as protein kinases, phosphatases (Datta et al., 1986;Friedman, 1986), and 1,3-␤-glucan synthase (Kauss and Jeblick, 1985). Despite the large amount of information on the covalent interactions between polyamines and proteins, little is known about the noncovalent binding of polyamines to PM proteins, even though this may represent one of the first steps in their action at the cellular level.In Escherichia coli two periplasmic polyamine-binding proteins (PotD and PotF) have been isolated as part of two membrane transport systems, pPT104 and pPT79, respectively (Kashiwagi et al., 1993;Pistocchi et al., 1993b). In particular, the crystal structure of PotD, which strongly binds spermidine, was recently determined (Sugiyama et al., 1996). This crystallographic study led to the el...