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. 1980 Aug;66(2):308–312. doi: 10.1104/pp.66.2.308

Isolation and Characterization of Thiamin Pyrophosphotransferase from Glycine max Seedlings 1

William T Molin 1,2, Roger C Fites 1,3
PMCID: PMC440588  PMID: 16661427

Abstract

Thiamin:ATP pyrophosphotransferase (EC2.7.6.2) activity from soybean (Merr.) seedlings grown for 48 hours was determined by measuring the rate of [2-14C]thiamin incorporation into thiamin pyrophosphate. With partially purified (11-fold) enzyme, optimal activity occurred between pH 7.1 and 7.3, depending on the buffer system that was used. Assays were routinely conducted at a final pH of 8.1 in order to minimize interference from competing reactions. Enzyme activity required the presence of a divalent cation, and a number of nucleoside triphosphates proved to be active as pyrophosphate donors. Apparent Km values of 18.3 millimolar and 4.64 micromolar were obtained for Mg·ATP and thiamin, respectively. Among the compounds tested, pyrithiamin and thiamin pyrophosphate were most effective in inhibiting thiamin pyrophosphotransferase activity. Based on Sephadex G-100 gel filtration, soybean thiamin pyrophosphotransferase has a molecular weight of 49,000.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Itokawa Y., Cooper J. R. The enzymatic synthesis of triphosphothiamin. Biochim Biophys Acta. 1968 Apr 16;158(1):180–182. doi: 10.1016/0304-4165(68)90093-7. [DOI] [PubMed] [Google Scholar]
  3. Johnson L. R., Gubler C. J. Studies on the physiological functions of thiamine. 3. The phosphorylation of thiamine in brain. Biochim Biophys Acta. 1968 Feb 1;156(1):85–96. doi: 10.1016/0304-4165(68)90107-4. [DOI] [PubMed] [Google Scholar]
  4. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  5. Mitsuda H., Takii Y., Iwami K., Yasumoto K. Enzymic formation of thiamine pyrophosphate in plants. J Nutr Sci Vitaminol (Tokyo) 1975;21(1):19–26. doi: 10.3177/jnsv.21.19. [DOI] [PubMed] [Google Scholar]
  6. Mitsuda H., Takii Y., Iwami K., Yasumoto K. Mechanism and regulation of thiamine pyrophosphokinase from parsely leaf. J Nutr Sci Vitaminol (Tokyo) 1975;21(3):189–198. doi: 10.3177/jnsv.21.189. [DOI] [PubMed] [Google Scholar]
  7. Mitsuda H., Takii Y., Iwami K., Yasumoto K. Purification and properties of thiamine pyrophosphokinase from parsely leaf. J Nutr Sci Vitaminol (Tokyo) 1975;21(2):103–115. doi: 10.3177/jnsv.21.103. [DOI] [PubMed] [Google Scholar]
  8. Molin W. T., Wilkerson C. G., Fites R. C. Thiamin Phosphorylation by Thiamin Pyrophosphotransferase during Seed Germination. Plant Physiol. 1980 Aug;66(2):313–315. doi: 10.1104/pp.66.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. SILIPRANDI D., SILIPRANDI N. Separation and quantitative determination of thiamine and thiamine phosphoric esters and their preparation in pure form. Biochim Biophys Acta. 1954 May;14(1):52–61. doi: 10.1016/0006-3002(54)90129-8. [DOI] [PubMed] [Google Scholar]
  10. Sanemori H., Egi Y., Kawasaki T. Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans. J Bacteriol. 1976 Jun;126(3):1030–1036. doi: 10.1128/jb.126.3.1030-1036.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Voskoboev A. I., Chernikevich I. P., Ostrovskii Iu M. Substratnaia spetsifichnost' tiaminpirofosfokinazy iz pivnykh drozhzhei. Dokl Akad Nauk SSSR. 1975 May 11;222(2):486–488. [PubMed] [Google Scholar]

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