Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.
A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.
D002499:Centrifugation, Density Gradient; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D003624:Darkness; D002523:Edible Grain / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010446:Peptide Fragments / Q000032:analysis
D010446:Peptide Fragments; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase