カツオ筋肉のNAD pyrophosphorylaseの精製およびその性質について

概要

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Attempts were made to isolate NAD pyrophosphorylase from the nuclear fraction of skipjack muscle. After extraction with 0.1 M phosphate buffer (pH 7.0) containing 0.15 M NaCl, the enzyme was purified by ammonium sulfate fractionation and several types of column chromatography on DEAE- and CM-cellulose, Sepharose-4B, and hydroxylapatite. The enzyme thus obtained showed a specific activity about 600 times as high as that of the homogenate. The molecular weight was estimated to be about 200, 000 by Sepharose-4B gel filtration. The isoelectric point was found to be 4.2 by isoelectric focusing. The optimum pH of this enzyme was very wide, ranging from 7 to 10. The activity was strongly inhibited by NBS and NQS and also by several metal ions, such as Zn2+, Cd2+, and Cu2+. The presence of bivalent ions was quite essential for the enzyme to exhibit any activity; Mg2+, Co2+, and Ni2+ were particularly effective. The apparent Km values for NMN, ATP, NAD, and pyrophosphate were 0.08, 0.09, 0.25, and 0.22x 10-3 M, respectively.
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