Shiro Nishikawa

Specific interaction of fused H protein of bacteriophage φX174 with receptor lipopolysaccharides

The DNA fragment encoding the spike H protein of bacteriophage phiX174 was amplified by polymerase chain reaction. The fragment was sub-cloned into pQE-30 to yield pQE-H. The histidine-tagged H protein (HisH) was obtained from the cell extract of Escherichia coli M15 (pREP4) harboring pQE-H and purified by nickel chelating and anion-exchange chromatographies. HisH was shown to bind dose-dependently to the lipopolysaccharides (LPSs) isolated from phiX174-sensitive strains, E. coli C or Salmonella typhimurium TV119 (Ra mutant). In sharp contrast, HisH did not bind to the LPSs from insensitive strains, E. coli F583 (Rd2 mutant) or E. coli O111:B4 (smooth strain). Since the same selectivity was observed in the plaque counting assay for in vitro inactivation of phiX174, the spike H protein was shown to recognize receptor LPS.

Different contributions of the outer and inner R-core residues of lipopolysaccharide to the recognition by spike H and G proteins of bacteriophage φX174

The binding of spike H and G proteins of bacteriophage phiX174 with lipopolysaccharides (LPSs) were evaluated by a competitive enzyme-linked plate assay using the biotin-labeled LPS of Escherichia coli C, one of a host strain, and the non-labeled LPSs having different R-core polysaccharide lengths. H protein promptly decreased its affinity when some saccharide residues were truncated from the outer R-core. However, G protein showed significant affinity to the LPSs lacking all the residues of the outer R-core and some of the inner R-core. Thus, G protein rather than H protein well recognized the residues of the inner R-core of LPS.

Contributions of polysaccharide and lipid regions of lipopolysaccharide to the recognition by spike G protein of bacteriophage Φx174

A histidine-tagged G protein of bacteriophage φX174 (HisG) bound strongly with lipopolysaccharide (LPS) of Escherichia coli C, one of a φX174-sensitive Ra strain. The dissociation constant, K d, was measured to be 0.16±0.04 μM by fluorometric titration. HisG showed slightly less affinity to LPSs of the insensitive Rc and Rd2 strains having shorter R-core polysaccharide sequences than that of the sensitive Ra strains. The difference between the two types of LPS was demonstrated by CD spectra; LPSs of the sensitive strains increased the signal intensity for β-sheet, while the insensitive strains decreased it. The chemically degraded LPS derivatives lacking a hydrophobic lipid region showed much less affinity to HisG, indicating the importance of the lipid region of LPS for strong binding with HisG. On the other hand, since the degraded derivatives increased the intensity of CD spectra, the polysaccharide region is thought to contribute to the conformation change of the protein.

Synthesis, crystal structure and cytokinin activities of β-substituted 6-styrylpurines

Abstract Conformationally restricted ( Z )- and ( E )-β-substituted 6-styrylpurines were synthesized by addition reaction to 6-phenylethynylpurine and photoisomerization. The crystalline ( Z )- and ( E )-β-chloro-substituted compounds exist in the anti - and syn -forms, respectively. While the ( Z )-β-chloro- and bromo-substituted compounds were almost as active as ( E )-6-styrylpurine and N 6 -benzyladenine (BA) in the Amaranthus betacyanin assay, they were more active than the latter in the tobacco callus assay. On the other hand, their ( E )-isomers showed activities almost comparable to that of kinetin in both assays. The crystal structure, as well as the high cytokinin activity of the conformationally restricted ( Z )-isomers strongly suggest that the anti-transoid form is most probably the active conformation of purine cytokinins.

1-O-nicotinoyl-β-d-glucopyranose in cultured tobacco cells

Abstract The structure of a new niacin metabolite in cultured tobacco cells grown in a high concentration of niacin (1 nM) was confirmed to be 1-O- nicotinoyl-β- d -glucopyranose . This glucoside is thought to be one of the detoxification forms of niacin in plants.