Autor: |
Allen, Rosamund E., Lo, Theodore W. C., Thornalley, Paul J. |
Zdroj: |
Journal of Protein Chemistry; April 1993, Vol. 12 Issue: 2 p111-119, 9p |
Abstrakt: |
Glyoxalase I (EC 4.4.1.5) was purified from human red blood cells by a simplified method using S-hexylglutathione affinity chromatography with a modified concentration gradient of S-hexylglutathione for elution. The pure protein had a specific activity of 1830 U/mg of protein, where the overall yield was 9%. The pure protein had a molecular mass of 46,000 D, comprised of two subunits of 23,000 D each, and an isoelectric point value of 5.1. TheKMvalue for methylglyoxal-glutathione hemithioacetal was 192±8 µM and thekcatvalue was 10.9±0.2 × 104min−1(N= 15). The glyoxalase I inhibitor S-p-bromobenzylglutathione had aKivalue of 0.16±0.04 µM and S-p-nitrobenzoxycarbonylglutathione, previously thought to inhibit only glyoxalase II, also inhibited glyoxalase I with aKivalue of 3.12±0.88 µM. Reduced glutathione was a weak competitive inhibitor of glyoxalase I with aKivalue of 18±8 mM. The polyclonal antibodies were raised to the purified enzyme and were found to react specifically with glyoxalase I antigen by immunoblotting. This procedure gave a protein of high purity with simple low pressure chromatographic techniques with a moderate but adequate yield for small-scale preparations. |
Databáze: |
Supplemental Index |
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