Separation and Characterization of Prolamellar Bodies and Prothylakoids from Squash Etioplasts

Intact etioplasts of squash cotyledons, which had been preparedby Percoll density gradient centrifugation, were ruptured hypotonicallyin the presence of deoxyribonuclease I then fractionated intoprolamellar bodies and prothylakoids by differential and Percolldensity gradient centrifugations. This procedure provided ahighly purified prolamellar body fraction that was composedmainly of a 36,000-dalton protein. This protein was identifiedas NADPH:protochIorophyllideoxidoreductase [Ikeuchi and Murakami(1982) Plant & Cell Physiol. 23: 1089]. The fraction alsohad a high content of protochlorophyllide that absorbed at 648nm and its NADPH:protochlorophyllide oxidoreductase had highactivity. When the fraction was illuminated, a chlorophyllidethat absorbed at 684–685 nm formed. In contrast, the prothylakoid fraction, which showed high activityfor the Ca²⁺-dependent ATPase of coupling factor 1, containedonly a small amount of the 36,000-dalton protein and showedvery low NADPH:protochlorophyllide oxidoreductase activity.The protochlorophyllide content of this fraction also was low,and the ratio of protochlorophyll to protochlorophyll(ide) high.The absorption peak in the prothylakoids was at 633–635nm, and after a brief illumination a chlorophyllide that absorbedat 672–673 nm formed. These results indicate that thephotoactive protochlorophyllide-NADPHreductase complex in etioplastsis concentrated in the prolamellar body and that the physicalstate of protochlorophyll(ide) in the prolamellar body differsfrom that of the prothylakoid. (Received April 28, 1982; Accepted November 15, 1982)