Reconstitution of chlorophyllide formation by isolated etioplast membranes

Abstract

1. The reconstitution of chlorophyllide biosynthesis by barley etioplast membranes is described. 2. The process is dependent on the addition of NADPH and protochlorophyllide and on illumination, which can be either continuous or intermittent. 3. The reconstituted process involves spectroscopically similar intermediates to the native reaction in whole leaves. 4. Steps in the process are an initial enzymic formation in the dark of a photoactive complex, P638/652 (probably a ternary protochlorophyllide-NADPH-enzyme complex), followed by a very rapid light-dependent hydrogen transfer from the NADPH to the protochlorophyllide giving chlorophyllide, finally releasing the enzyme for repeating the process. 5. A continuous assay for the system regenerating complex P638/652 was devised on the basis of monitoring chlorophyllide formation.6. The pH optimum of the reaction is at 6.9 and Km values for protochlorophyllide and NADPH are 0.46 and 35,uM respectively. 7. The reaction is associated specifically with the etioplast membrane fraction. 8. Activities of the system assayed in vitro are more than adequate to account for rates of chlorophyll formation in vivo.

Regulation of photosynthesis by reversible phosphorylation of the light-harvesting chlorophyll a/b protein

“…This NADPH-linked enzyme requires an excited protochlorophyllide substrate as part of its reaction mechanism (Griffiths, 1978). Thus, continuous illumination is required for continuous chlorophyll(ide) synthesis.…”

The effects of low pH on the properties of protochlorophyllide oxidoreductase and the organization of prolamellar bodies of maize (Zea mays)

“…Griffiths and his coworkers [4,5] successfully used this sensitivity to establish the basic framework of relationships existing between the different ternary complexes formed between POR, PChlide/Chlide and NADP(H). The proposed relationship between the different POR complexes referred to in this paper, based on the generally accepted scheme of Oliver & Griffiths [5], is summarized in Fig.…”

“…The NADPH binding properties of the photoconverted enzyme at pH 7.5 were investigated using the red-shift in the Chlide absorption maximum accompanying the displacement of bound NADP + by NADPH [4,5]. The samples were first photoconverted in the absence of excess NADPH to form the NADP + -bound enzyme.…”

“…Isolated PLB show a similar pattern of spectroscopic changes immediately following illumination. The presence of excess NADPH, however, is required to ensure the replacement of the NADP + by NADPH and drive the absorption peak shift from 678 to 684 nm [4,5]. Under these conditions, no Shibata shift occurs and the PLB lack the ability to undergo the compositional and morphological changes seen in vivo.…”

The dark side of green fluorescent protein

“…To determine the effect of chlorophyll on GFP fluorescence, 1 mg of high-performance liquid chromatography (HPLC)-purified chlorophyll a or b (Sigma, St Louis, MO, USA) was dissolved in 2 ml methanol containing 20 mg ml − 1 sodium (Na) cholate (Griffiths, 1978), dried in a Speed Vac Concentrator (Savant, Milford, MA, USA), and resolublized in 2 ml TE buffer. Various amounts of chlorophyll solution were then mixed with 1 µg of standard enhanced green fluorescent protein (EGFP) (Clontech) in a total volume of 1 ml TE buffer containing 20 mg ml − 1 Na cholate.…”