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0344193512
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34
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85030817244
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note
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4, 300 mM NaCl, 10 mM imidazole, pH 8.0) per gram of cells containing 200 μg/mL lysozyme. The suspension was incubated on ice for 30 min and treated with a sonicator (six times for 10 s with 10 s cooling time in between). After centrifugation at 10,000g for 30 min at 4°C the cleared lysate was incubated with Ni-NTA suspension (Qiagen). The expressed protein was purified according to the manufacturer's protocol. The isolated hCA IV was further purified by Sephadex G25 chromatography (Amersham). The purified enzyme had a catalytic activity very similar to that described in Ref. 8
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35
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0015239422
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2 solutions in water at 20°C were used as substrate. Stock solutions of inhibitors were prepared at a concentration of 10-50 mM (in the assay buffer) and dilutions up to 0.01 nM done with the assay buffer mentioned above. Enzyme concentrations were 0.09 μM for hCA I, 0.06 μM for hCA II, 1 μM for hCA IV and bCA IV. Inhibition constants were calculated as described by R.G. Khalifah J. Biol. Chem. 246 1971 2561 2573
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Khalifah, R.G.1
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39
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0142095047
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B. Alvarez, F.B. Loiselle, C.T. Supuran, G.J. Schwartz, and J.R. Casey Biochemistry 42 2003 12321 12329
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Alvarez, B.1
Loiselle, F.B.2
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Schwartz, G.J.4
Casey, J.R.5
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