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Articles by K Ishii
Total Records ( 2 ) for K Ishii
  Y Iwase , S Honma , M Matsuzaki , Y Miyakawa , T Kanno , K Ishii , N Furuichi , K Furukawa and T. Horigome

For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including >90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.

  J Seino , K Ishii , T Nakano , N Ishida , M Tsujimoto , Y Hashimoto and S. Takashima

Using the basic local alignment search tool (BLAST) algorithm to search the Oryza sativa (Japanese rice) nucleotide sequence databases with the Arabidopsis thaliana UDP-galactose transporter sequences as queries, we found a number of sequences encoding putative O. sativa UDP-galactose transporters. From these, we cloned four putative UDP-galactose transporters, designated OsUGT1, 2, 3 and 4, which exhibited high sequence similarity with Arabidopsis thaliana UDP-galactose transporters. OsUGT1, 2, 3 and 4 consisted of 350, 337, 345 and 358 amino acids, respectively, and all of these proteins were predicted to have multiple transmembrane domains. To examine the UDP-galactose transporter activity of the OsUGTs, we introduced the OsUGTs’ expression vectors into UDP-galactose transporter activity-deficient Lec8 cells. Our results showed that transfection with OsUGT1, 2 and 3 resulted in recovery of the deficit phenotype of Lec8 cells, but transfection with OsUGT4 did not. The results of an in vitro nucleotide sugar transport assay of OsUGTs, carried out with a yeast expression system, suggested that OsUGT4 is a UDP-glucose transporter rather than a UDP-galactose transporter. Although plants have multiple UDP-galactose transporter genes, phylogenic analysis indicates that plant UDP-galactose transporter genes are not necessarily evolutionary related to each other.

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