May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Fructose-3-phosphate (F3P) Inhibits the Phosphorylation of Protein-bound Fructoselysine by Fructosamine-3-kinase (FN3K)
Author Affiliations & Notes
  • B.S. Szwergold
    Dept of Medicine, Dartmouth Medical School, Hanover, NH, United States
  • Footnotes
    Commercial Relationships  B.S. Szwergold, None.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2345. doi:
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      B.S. Szwergold; Fructose-3-phosphate (F3P) Inhibits the Phosphorylation of Protein-bound Fructoselysine by Fructosamine-3-kinase (FN3K) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2345.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Fructosamine-3-kinase (FN3K), which was recently purified and cloned, is an enzyme that phosphorylates protein-bound fructoselysine (FL) to fructoselysine-3-phosphate (FL3P). This phosphorylation leads to the decomposition of FL and regeneration of unmodified lysine residues thereby reversing the initial steps of nonenzymatic glycation. FN3K is found in all mammalian tissues, including the rat lens where it was initially discovered. The widespread distribution of this kinase and its broad specificity towards glycated proteins suggest that it has an important cellular function, possibly as a defense mechanism against nonenzymatic glycation. The purpose of this investigation was to determine whether fructose and fructose-3-phosphate (F3P) which accumulate in the diabetic rat lens could inhibit the activity of FN3K vis-à-vis FL thereby allowing an enhanced glycation of lenticular proteins in diabetes. Methods: As an initial step in this study we investigated the effects of fructose and F3P on FN3K activity in lysates of rat lenses. Ten lenses from nondiabetic animals were homogenized in 2 ml of 100 mM TRIS, pH 7.5, containing 1-% protease inhibitor cocktail, 0.1 mM PMSF and 1 mM DTT. The homogenates were centrifuged and 200 microliter portions of the supernatant were incubated for 1 hr with glycated histones and 2 mM gamma-32P Mg-ATP in the presence or absence of fructose and F3P. Phosphorylation of the glycated histones was assessed by measuring the incorporation of 32P into histones bound to phosphocellulose paper. Results: At 5-mM concentration, F3P inhibited the phosphorylation of histone-bound FL by 50-60 %. By contrast, 20-mM fructose had little or no effect on the production of FL3P. The apparent Ki of F3P under these conditions was ~ 1mM which is close to the concentration of F3P attained in the diabetic rat lens Conclusions: Our results demonstrate that F3P inhibits the phosphorylation of fructoselysines on nonenzymatically glycated proteins. We believe that in diabetic lens this inhibition leads to diminished production of FL3P and contributes thereby to increased nonenzymatic glycation. Based on our studies we anticipate that ARI treatment of diabetic rats, which reduces the levels of lenticular F3P, should lead to an increase in FL3P production and consequent reduction in FL and AGE levels on crystallins.

Keywords: enzymes/enzyme inhibitors • diabetes • crystallins 

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