Measurement and Identification of NADPH:Protochlorophyllide Oxidoreductase Solubilized with Triton X-100 from Etioplast Membranes of Squash Cotyledons

Etioplast membranes were solubilized with 1 mM Triton X-100in the presence of excess NADPH and protochlorophyllide to isolateNADPH:protochlorophyllide oxidoreductase. The activity of thisreductase was assayed as the formation of chlorophyllide bya single flash and was equivalent to the amount of photoactiveprotochlorophyllide-NADPH-enzyme complex present before illumination.The rate of regeneration of the phtoactive complex was estimatedfrom the time course of chlorophyllide formation under a longflash. The highest rate was 651 nmol chlorophyllide formed min^–1mg^–1 protein. Photoconversion of protochlorophyllide to chlorophyllide andregeneration of the photoactive protochlorophyllide-NADPH-enzymecomplex were not much affected in a pH range from 6 to 8, atleast for several minutes. The apparent dissociation constantsof the photoactive complex were 0.039 µM for protochlorophyllideand 0.44 µM for NADPH. Triton-solubilized etioplast membraneswere fractionated by glycerol density gradient centrifugationto isolate the NADPH:protochlorophyllide oxidoreductase. Mostof the 36,000-dalton protein, the major protein of the prolamellarbody was recovered in the fraction enriched by NADPH:protochlorophyllideoxidoreductase and protochlorophyllide. Protochlorophyll andcarotenoids were present in different fractions. This is evidencethat the 36,000-dalton protein has the activity of NADPH:protochlorophyllideoxidoreductase and specifically binds protochlorophyllide. Themost highly purified fraction of the enzyme showed an activityof 7.8 nmol chiorophyllide formed flash^–1 mg^–1 proteinand bound 11.1 nmol protochlorophyllide mg^–1 of protein. (Received April 28, 1982; Accepted June 29, 1982)