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KMID : 0614020020170010046
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Journal of Pharmaceutical Sciences (C.N.U.)
2002 Volume.17 No. 1 p.46 ~ p.54
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Purification and Characterization of 5’-Nucleotidase from Bovine Brain Membrane
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Liu-Xi-Wen
Sok Dai-Eun
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Abstract
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5’-Nucleotidase, bound to brain membranes as a glycosylphosphatidyl-inositol (GPI)-anchored protein, is responsible for the conversion of adenosine-5’-monophosphate into adenosine, which is an agonist in adenosine receptor signalling. Here, 5’-nucleotidase was isolated from bovine brain using PI-PLC treatment, and purified by concanavalin A sepharose chromatography, DEAE-sephacel chromatography, and finally AMP affinity chromatography. For higher yield of enzyme purification, Zn^2+ was Included in the elution buffer in DEAE-sephacel chromatography. Especially, NaCl was more favorable than MgCl_2 for the elution of 5’-nucleotidase, proper for inactivation study, from AMP affinity column. The purified 5’-nucleotidase was relatively pure on SDS-PAGE analysis, showing a specific activity of 30.27 μmole/min/㎎ (purification fold 19,000 fold). The purified enzyme, possessing a K_m value of 44μM and an optimum pH of 7.5, was inhibited competitively by ATP (K_i, 12 μM), and uncompetitively by cysteine (K_i, 0.32 mM). In addition, the enzyme was activated slighty (1.5-folds) by Mg^2+.
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KEYWORD
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5’-Nucleotidase, GPI-anchored protein, affinity chromatography, purification
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