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.

The site of synthesis of the plastid membrane-located enzyme, protochlorophyllide reductase, has been determined. Plastid ribosome-deficient and normal rye (Secale cereake L., cv Rheidol) plants were grown in darkness at 33°C and 22°C, respectively. Extracts from these plants were analyzed for the levels of different ribosomal RNAs and cytochrome f and the activity of a number of enzymes with well-established sites of synthesis. The results confirmed that the higher temperature had induced a specific inhibition of protein synthesis in the plastids. The activity and level of protochlorophyllide reductase was unaffected by growth at the higher temperature, suggesting it to be a cytoplasmically synthesized enzyme.NADPH-Pchlide oxidoreductase is an enzyme of the Chl biosynthetic pathway and is responsible for the light-dependent reduction of Pchilde in higher plants. The enzyme is present at high activity in etiolated tissue (13) and has recently been purified and shown to be one of the major proteins of the etioplast internal membrane system (2, 21). Light exerts an interesting regulatory effect on the enzyme in that illumination ofpreviously dark-grown plants results in a decrease in both the activity (19) and amount (2) of the enzyme. To characterize this light effect, it is obviously essential to gain information about the synthesis of the enzyme, and in this paper the site of synthesis of the enzyme is reported.In an attempt to determine whether the enzyme is synthesized in the cytoplasm or within the plastid, it was decided to make use of the observation that a deficiency of plastid ribosomes can be induced in certain higher plants by growth at high temperatures, a phenomenon first described and extensively investigated by Feierabend (11,12). Rye was chosen for study because in this species an elevated growth temperature sufficient to bring about an almost total lack of plastid ribosomes has little effect on cytoplasmic or mitochondrial protein synthesis (12). An alternative approach to answer the question might have been the analysis of plastid ribosome-deficient mutants (5); however, in view of the findings of Bradbeer et at. (7) that certain cytoplasmically synthesized plastid enzymes can still occur, but in greatly reduced amounts, in plastid ribosome-free mutants, it was felt that measurement of Pchilde reductase activity in plants with a heatinduced plastid ribosome deficiency would yield more reliable data regarding the site of synthesis of the enzyme. A previous study of the enzymes of Chl synthesis failed to detect a significant level of Pchilde reductase, measured as photoconvertible Pchilde, in such high-temperature-grown plants (10), but in the present work use was made of a much more sensitive assay of enzymic activity (13). A growth temperature of 33°C was adopted here to 'Supported by the Science and Engineering Research Council.

The membranes from oat etioplasts have been separated by sucrose density gradient centrifugation into a heavy fraction of density 1.20 mg/ml (prolamellar body fraction) and a light fraction of density 1.12 mg/ml (prothylakoid fraction). The light fraction was shown to be enriched in protein, protochlorophyllide and the chloroplast enzyme CF1‐ATPase, but to be deficient in saponins. In the electron microscope this fraction appeared as swollen vesicles. In contrast the heavy fraction appeared considerably enriched in crystalline tubular material and was on analysis found to contain most of the etioplasts' saponin but with reduced protein and pigment. In the same experiments the enzyme NADPH:protochlorephyllide oxidoreductase showed the same distribution pattern as the CF1‐ATPase suggesting its location on the prothylakoids.