CHLOROPLAST BIOGENESIS. XXIII. THE CONVERSION OF EXOGENOUS PROTOCHLOROPHYLLIDE INTO PHOTOTRANSFORMABLE PROTOCHLOROPHYLLIDE <i>IN VITRO</i>*

The massive conversion of delta-aminolevulinic acid (ALA) to protochlorophyllide (Pchlide) and the massive conversion of chlorophyllide a (Chlide a) to chlorophyll a (Chl a) are two essential conditions for the ALA-dependent assembly of photosynthetic membranes in vitro. In this work, we describe the development of a cell-free system capable of the forementioned biosynthetic activities at rates higher than in vivo, for the first 2 h of dark-incubation. The cell-free system consisted of (1) etiochloroplasts prepared from kinetin and gibberellic-acid-pretreated cucumber cotyledons, and (2) cofactors and additives described elsewhere and which are needed for the massive conversion of ALA to Pchlide, (3) high concentrations of ATP, MgCl(2), and an isoprenol alcohol such as phytol, were required for the massive conversion of Chlide a to Chl a. An absolute and novel requirement of Mg(2+) for the conversion of Chlide a to Chl a was also demonstrated. In addition to the role of phytol as a substrate for the conversion of Chlide a to Chl a, the data suggested that this alcohol may also be involved in the regulation of the reactions between ALA and Pchlide. It is proposed that during greening, the conversion of Chlide a to Chl a may follow different biosynthetic rates, having different substrate and cofactor requirements, depending on the stage of plastid development.

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Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

Gómez-Silva

Timko

Schiff

1985

Planta

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Chloroplasts observed, by electron microscopy, to be intact and uncontaminated, with high rates of light-dependent protein synthesis and CO2 fixation were isolated from cells grown on low-vitamin-B12 medium in the light or from cells grown in the same medium in the dark and then exposed to light for 36 h. Both types of chloroplasts were active but less variability was encountered with developing chloroplasts from 36-h cells. The 36-h chloroplasts showed good light-dependent incorporation of 5-amino-levulinic acid (ALA) or L-glutamic acid into chlorophyll (Chl) a which was linear for approx. 1 h. The specific activity of the Chl a remained the same after conversion to pheophytin a, methylpheophorbide a or pyromethylpheophorbide a and rechromatography, indicating that the label was in the tetrapyrrole. Incorporation of ALA was inhibited by levulinic acid, and by chloramphenicol and other inhibitors of translation of 70S-type chloroplast ribosomes at concentrations which did not appreciably inhibit photosynthesis but which blocked plastid protein synthesis nearly completely. Cycloheximide, an inhibitor of translation on 87S cytoplasmic ribosomes of Euglena, was without effect. The 70S inhibitors did not block uptake of labeled ALA. Although labeled glycine was taken up by the plastids, no incorporation into Chl a was observed. Thus the developing chloroplasts appear to contain all of the enzymatic machinery necessary to convert glutamic acid to Chl via the C5 pathway of ALA formation but the Shemin pathway from succinyl coenzyme A and glycine to ALA appears to be absent. The requirement for plastid protein synthesis concomitant with Chl synthesis indicates a regulatory interaction and also indicates that at least one protein influencing Chl synthesis is synthesized on 70S-type plastid ribosomes and is subject to metabolic turnover.

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CHLOROPLAST BIOGENESIS. XXIII. THE CONVERSION OF EXOGENOUS PROTOCHLOROPHYLLIDE INTO PHOTOTRANSFORMABLE PROTOCHLOROPHYLLIDE *IN VITRO**

Cited by 11 publications

References 16 publications

Chlorophyll a Biosynthetic Heterogeneity

Chlorophyll a Biosynthetic Heterogeneity

Bioengineering of photosynthetic membranes. Requirement of magnesium for the conversion of chlorophyllide a to chlorophyll a during the greening of etiochloroplasts in vitro

Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

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CHLOROPLAST BIOGENESIS. XXIII. THE CONVERSION OF EXOGENOUS PROTOCHLOROPHYLLIDE INTO PHOTOTRANSFORMABLE PROTOCHLOROPHYLLIDE IN VITRO*

Cited by 11 publications

References 16 publications

Chlorophyll a Biosynthetic Heterogeneity

Chlorophyll a Biosynthetic Heterogeneity

Bioengineering of photosynthetic membranes. Requirement of magnesium for the conversion of chlorophyllide a to chlorophyll a during the greening of etiochloroplasts in vitro

Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

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Product

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CHLOROPLAST BIOGENESIS. XXIII. THE CONVERSION OF EXOGENOUS PROTOCHLOROPHYLLIDE INTO PHOTOTRANSFORMABLE PROTOCHLOROPHYLLIDE *IN VITRO**