Purification and Some Properties of Copper Reductase from Cell Surface of Debaryomyces hansenii

概要

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NAD(P)H-dependent copper reductase was solubilized with Zymolyase-100T in the presence of 5 mM phenylmethylsufonyl fluoride, 1 mM L-mannono-1,4-lactone, and 1 M sorbitol from yeast cell surface (cell wall and/or periplasmic space). The enzyme was unstable in all steps of purification. Addition of 0.1 M sorbitol and sucrose to the enzyme solution could prevent the loss of the activity caused by dialysis or ultrafiltration. When the enzyme was partially inactivated by changing to a buffer solution without these stabilizing agents, an addition of FMN restored the copper reducing activity. Copper reductase activities were found in three fractions (FI, FII, FIII) on DEAE-Sephacel column chromatography. Copper reductase FII was purified (about 8-fold) to give a single protein band on polyacrylamide gel electrophoresis. The molecular weight of this enzyme was about 100,000 by gel column chromatography. The values of K_m were 0.03 mM (Cu(II)), 0.98 mM (NADH), and 0.17 mM (NADPH), respectively. Fluorescence of purified enzyme FII was maximum at 340 nm (excitation at 285 nm) and quenched by Cu(II). Furthermore, FII had a FMN like fluorescence at 520 nm (excitation at 465 nm). From these results, it is strongly suggested that copper reductase is a glycoflavoprotein which requires FMN as a coenzyme and which catalyzes copper reduction from Cu(II) to Cu(I) with NADH or NADPH as an electron donor.
社団法人日本生物工学会の論文
1991-09-25