| Autor: |
Lu, Hsieng S., Fausset, Patricia R., Narhi, Linda O., Horan, Thomas, Shinagawa, Kyoko, Shimamoto, Grant, Boone, Thomas C. |
| Zdroj: |
Archives of Biochemistry and Biophysics; February 1999, Vol. 362 Issue: 1 p1-11, 11p |
| Abstrakt: |
Chemical modification and mutagenesis of methionines in recombinant human granulocyte colony-stimulating factor (G-CSF) were investigated. Selective oxidation of G-CSF by H2O2andt-butyl hydroperoxide leads to generation of different oxidized forms. Four modified forms were isolated and shown to contain 1 to 4 oxidized methionyl residues. All methionines in G-CSF are reactive, with reaction kinetics following the order of Met1>Met138>Met127>>>Met122. H2O2oxidation of Met122is relatively slow and is biphasic with a faster second reaction phase being affected by the oxidation of Met127. All oxidized forms retain gross G-CSF conformation similar to that of the native molecule and are able to bind the soluble G-CSF receptor. However, G-CSF form oxidized at both Met127and Met122is unstable and exhibits decreased ability to dimerize the receptor after exposure to acid or elevated temperature. All modified forms, except Met1-oxidized G-CSF, also show significantly lower biological activity. Our data suggest that Met138is solvent accessible and its surrounding microenvironment may be critical for G-CSF function, whereas Met127is less accessible to solvent and Met122is near the hydrophobic core. Oxidation at both Met127and Met122results in alterations of G-CSF structure that affect the apparent molecular size, polarity, and stability and lead to the loss of G-CSF biological function. G-CSF variants with Leu replacement at Met127or at Met138are not completely resistant to oxidation-induced inactivation, while the variant with Leu replacement at both sites is more stable and can retainin vitrobiological activity following oxidation. |
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