Measurement and Identification of NADPH : Protochlorophyllide Oxidoreductase Solubilized with Triton X-100 from Etioplast Membranes of Squash Cotyledons
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概要
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Etioplast membranes were solubilized with 1 mM Triton X-100 in the presence of excess NADPH and protochlorophyllide to isolate NADPH:protochlorophyllide oxidoreductase. The activity of this reductase was assayed as the formation of chlorophyllide by a single flash and was equivalent to the amount of photoactive protochlorophyllide-NADPH-enzyme complex present before illumination. The rate of regeneration of the phtoactive complex was estimated from the time course of chlorophyllide formation under a long flash. The highest rate was 651 nmol chlorophyllide formed min^<-1> mg^<-1> protein. Photoconversion of protochlorophyllide to chlorophyllide and regeneration of the photoactive protochlorophyllide-NADPH-enzyme complex were not much affected in a pH range from 6 to 8, at least for several minutes. The apparent dissociation constants of the photoactive complex were 0.039μM for protochlorophyllide and 0.44μM for NADPH. Triton-solubilized etioplast membranes were fractionated by glycerol density gradient centrifugation to isolate the NADPH:protochlorophyllide oxidoreductase. Most of the 36,000-dalton protein, the major protein of the prolamellar body was recovered in the fraction enriched by NADPH:protochlorophyllide oxidoreductase and protochlorophyllide. Protochlorophyll and carotenoids were present in different fractions. This is evidence that the 36.000-dalton protein has the activity of NADPH:protochloro-phyllide oxidoreductase and specifically binds protochlorophyllide. The most highly purified fraction of the enzyme showed an activity of 7.8 nmol chlorophyllide formed flash^<-1> mg^<-1> protein and bound 11.1 nmol protochlorophyllide mg^<-1> of protein.
- 日本植物生理学会の論文
著者
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MURAKAMI Satoru
Department of Applied Mathematics Okayama University of Science
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Ikeuchi Masahiko
Department Of Biology College Of General Education University Of Tokyo
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Murakami Satoru
Department Of Biology College Of General Education University Of Tokyo
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Ikeuchi Masahiko
Department Of Biological Science Graduate School Of Sciences The University Of Tokyo
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