Abstract
Glutamate was converted to the chlorophyll and heme precursor delta-aminolevulinic acid in soluble extracts of Euglena gracilis. delta-Aminolevulinic acid-forming activity depended on the presence of native enzyme, glutamate, ATP, Mg2+, NADPH or NADH, and RNA. The requirement for reduced pyridine nucleotide was observed only if, prior to incubation, the enzyme extract was filtered through activated carbon to remove firmly bound reductant. Dithiothreitol was also required for activity after carbon treatment. delta-Aminolevulinic acid formation was stimulated by RNA from various plant tissues and algal cells, including greening barley leaves and members of the algal groups Chlorophyta (Chlorella vulgaris, Chlamydomonas reinhardtii), Rhodophyta (Cyanidium caldarium), Cyanophyta (Anacystis nidulans, Synechocystis sp. PCC 6803), and Prochlorophyta (Prochlorothrix hollandica), but not by RNA derived from Escherichia coli, yeast, wheat germ, bovine liver, and Methanobacterium thermoautotrophicum. E. coli glutamate-specific tRNA was inhibitory. Several of the RNAs that did not stimulate delta-aminolevulinic acid formation nevertheless became acylated when incubated with glutamate in the presence of Euglena enzyme extract. RNA extracted from nongreen dark-grown wild-type Euglena cells was about half as stimulatory as that from chlorophyllous light-grown cells, and RNA from aplastidic mutant cells stimulated only slightly. delta-Aminolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidic mutant and dark-grown wild-type cells. Gabaculine inhibited delta-aminolevulinic acid formation at submicromolar concentration. Heme inhibited 50% at 25 microM, but protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide inhibited only slightly at this concentration.
Highlights
Euglena is unusual among eukaryotic photosynthetic organisms in that viable aplastidic strains exist which appear to lack all chloroplast components, and are thusunable to carry out photosynthesis or chloroplast biosynthetic reactions, but which are capable of vigorous heterotrophic growth [13, 14]
Several of the RNAs that aminolevulinic acid that serves as precursor to chloroplast did not stimulate 6-aminolevulinic acid formation tetrapyrroles is derived from glutamate, whereas that which became acylated when incubated with forms mitochondrial hemes arises by the 6-aminolevulinic glutamate in the presence of Euglena enzyme extract. acid synthase reaction [15]
Euglena cells was about half as stimulatory as that to study the function, organization, and regulation of both from chlorophyllous light-grown cells, and RNA from routes of 6-aminolevulinic acid biosynthesis and thepossible aplastidic mutantcells stimulated only slightly. &Ami- interaction of the two separately compartmented tetrapyrrole nolevulinic acid-forming enzyme activity was present in extracts of light-grown wild-type cells, but undetectable in extracts of aplastidicmutantand darkgrown wild-type cells
Summary
Incubation Conditions-Soluble extracts of light-grown wild-type Euglena cells formed 6-aminolevulinic acid when prepared and incubated as described under “Experimental Procedures.’’ The amount of 6-aminolevulinic acid that accumulated in the incubation mixture increased with increasing incubation time for at least 120 min, whether the incubation temperature was 25, 30, or 35 “C (Fig. 2). Similar resultswere obtained inall cases whether the source of the RNA component in the incubation mixture was lightgrown wild-type Euglena or Chlorella (see below and Ref. 11). The amount of 6-aminolevulinic acid accumulated increased with increasing content of RNA up to at least 50 AZWunits (Fig. 5). Substrateand Cofactor Requirements-6-Aminolevulinic acid formation in cell extract that had been depleted of low molecular weight materials by passage through Sephadex G25 was absolutely dependent on added ATP and Mg2+ (Table I).
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