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PEPTIDE PLANT HORMONE, PHYTOSULFOKINE 71 of intact plants, indicating that PSK expression is not limited to regions in which cells actively divide and differentiate. (Yang H. et al. 2001) To study the function of PSK in plants, a large number of transformation experiments have been performed using Arabidopsis. However, loss-offunction techniques have not produced visible, directly informative phenotypes, suggesting functional redundancy between these 5 PSK genes in Arabidopsis. A definite picture will only emerge when combinations of all five knockouts are available. Overexpression of PSK slightly promotes callus formation in vitro in the presence of auxin/cytokinin, (Yang H. et al. 2001) but does not affect the growth of seedlings. Structure-activity relationships of PSK Derivatization of peptide hormones with biochemical tags such as photoactivatable groups has been used in characterization and purification of hormone receptors. (Hazum E. 1983) A key factor in the use of such functional groups is the ability to derivatize peptides without loss of binding activity or biological activity. To identify the active core of PSK, we synthesized several PSK analogs by solid phase peptide synthesis and direct sulfation of the peptide-resin using dimethylformamide-sulfurtrioxide complex. (Matsubayashi Y. et al. 1996) As shown in Fig. 5, N-terminal tetrapeptide and tripeptide of PSK retained 8% and 20% of the activity of the parent pentapeptide, respectively, but N- terminal dipeptide showed no activity. Deletion of the sulfate groups of Tyr 1 and Tyr 3 resulted in compounds with 0.6% and 4% of the activity of PSK, respectively, indicating that the sulfate group of Tyr 1 is more important than that of Tyr 3 for activity. In contrast, the N-terminal-truncated analog and an unsulfated analog exhibited no activity. Thus, the N-terminal tripeptide fragment Tyr(SO3H)- Ile-Tyr(SO3H) has been identified as the active core of PSK. A popular method for covalently linking functional groups to a peptide involves the use of activated esters of the functional groups, which react with primary amines to form amide bonds. However, modification of the N-terminal amino group of PSK by addition of Gly strongly decreases its biological activity. (Matsubayashi Y. et al. 1996) Thus, functional derivatization of PSK requires incorporation of an additional primary amino group at the C-terminal region, which is less involved in PSK activity than the N-terminal. To fulfill these requirements, several Alasubstituted PSK analogs were tested for activity, and the analog [Ala 5 ]PSK and [Lys 5 ]PSK was found to possess binding activity equal to that of PSK (Fig. 5). (Matsubayashi Y. et al. 1999) Interestingly, [Lys 5 ]PSK retained significant activity after derivatization of the side chain of Lys 5 by biotin, even when a very long spacer chain was inserted between the amino group of Lys 5 and the carboxyl group of biotin. This finding provided the breakthrough in a series of experiments conducted with the aim of visualization and purification of PSK receptors.
72 Yoshikatsu MATSUBAYASHI Fig. 5. Structure-activity relationships of PSK. PSK analogs were prepared by solid phase peptide synthesis and direct sulfation of the peptide-resin. Relative activity of each analog was determined by the bioassay using asparagus mesophyll cells or the competitive receptor binding assay (asterisk). ND=not determined. Among these, [ 125 I]-[N -(4-azidosalicyl)Lys 5 ]PSK was used for photoaffinity labeling experiments, and [Lys 5 ]PSK-Sepharose was used for purification of the PSK receptor. PSK receptor Because of the presence of highly hydrophilic sulfate groups in PSK molecules, it is unlikely that they pass through plasma membranes and directly interact with target molecules inside cells. To determine whether a cell surface receptor for PSK exists, radiolabeled PSK was synthesized by coupling [ 35 S]sulfuric acid with the phenolic groups of tyrosine. (Matsubayashi Y. et al. 1997) Binding of [ 35 S]PSK was detected on the surface of suspension cultured rice cells and in the plasma-membrane-enriched fractions. The binding is reversible and saturable, and only PSK analogs that possess biological activity can effectively displace the radioligand. To further characterize the PSK receptor, [ 3 H]PSK, which has higher specific radioactivity, was synthesized by catalytic reduction of a PSK analog containing tetradehydroisoleucine. (Matsubayashi Y & Sakagami Y. 1999) Ligand saturation analysis using [ 3 H]PSK revealed the existence of a high-affinity binding site in microsomal fractions derived from rice, maize,
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ISSN 0435-1096 Gamma Field Symposia
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The lecturers and the members of th
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KANEKO, T. KARITA, E. KASHIMA, M. K
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TANO, S. TSUCHIDA, Y. TSUTSUMI, N.
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The Symposium Committee Shigeki NAG
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CONTENTS Y. KAMIYA Biosyntheses and
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2 Yuji KAMIYA Fig. 1. Gibberellin b
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4 Yuji KAMIYA using the receptor pr
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6 Yuji KAMIYA Fig. 3. Origin of sid
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8 Yuji KAMIYA References AKIYOSHI,
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10 Yuji KAMIYA SHINOMURA, T. (1997)
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12 Yuji KAMIYA 8’ 8’ 273
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14 Motoyuki ASHIKARI, Hironori ITOH
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16 Motoyuki ASHIKARI, Hironori ITOH
Page 29 and 30: 18 Motoyuki ASHIKARI, Hironori ITOH
Page 37 and 38: 26 Atsuhiro OKA and how their signa
Page 39 and 40: 28 Atsuhiro OKA At almost the same
Page 41 and 42: 30 Atsuhiro OKA division for xylem
Page 43 and 44: 32 Atsuhiro OKA the RR domain in th
Page 45 and 46: 34 Atsuhiro OKA Fig. 4. Cross-talk
Page 47 and 48: 36 Atsuhiro OKA OKA, A., SAKAI, H.
Page 49 and 50: 38 Atsuhiro OKA ARR4 PHYB PHYBP
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Page 53 and 54: ETHYLENE-SIGNALING PATHWAY 43 signa
Page 55 and 56: ETHYLENE-SIGNALING PATHWAY 45 The f
Page 57 and 58: ETHYLENE-SIGNALING PATHWAY 47 Snf3p
Page 59 and 60: ETHYLENE-SIGNALING PATHWAY 49 In sp
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Page 65 and 66: MECHANISM OF BRASSINOSTEROID SIGNAL
Page 77 and 78: 68 Yoshikatsu MATSUBAYASHI are grow
Page 79: 70 Yoshikatsu MATSUBAYASHI Fig. 3.
Page 83 and 84: 74 Yoshikatsu MATSUBAYASHI Fig. 6.
Page 85 and 86: 76 Yoshikatsu MATSUBAYASHI 33. Yang
Page 87 and 88: 78 Yoshikatsu MATSUBAYASHI GUS
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Page 95 and 96: 86 PSK 23 BIL1/CFP
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