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Plant and Cell Physiology, 1985, Vol. 26, No. 3 397-405
© 1985

Article

Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

Masayuki Fujita1 and Tadashi Asahi2

Laboratory of Biochemistry, Faculty of Agriculture, Nagoya University Chikusa, Nagoya 464, Japan

2 To whom correspondence should be addressed

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450 reductase, was purified by chromatography through DEAE-cellulose, 2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilization from microsomes from Ceratocystis fimbriata-infected sweet potato root tissue with Emulgen 913. The enzyme existed in three forms after solubilization which migrated to positions corresponding to molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamide gel. Trypsin treatment of the enzyme species with the largest polypeptide yielded the species with the smallest one. After sucrose density gradient centrifugation of the pellet fraction obtained by centrifugation at 100,000×g of the crude extract, the enzyme species with the largest polypeptide was present in the particulate fractions, whereas that with the smallest one was only found at the top of the gradient. We conclude that the enzyme species with the largest polypeptide is in an intact, amphipathic form, whereas that with the smallest one, and probably also the other species, is its hydrophilic domain produced by an endogenous protease(s). The Km values of the enzyme in the intact form for NADPH and cytochrome c were 7.7 and 2.3 µM, respectively.

1 Present address: Laboratory of Food Hygienics, Faculty of Agriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07, Japan.

(Received September 6, 1984; Accepted December 27, 1984)
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