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. 1987 Jun;84(2):244–250. doi: 10.1104/pp.84.2.244

Formation of δ-Aminolevulinic Acid from Glutamic Acid in Algal Extracts 1

Separation into an RNA and Three Required Enzyme Components by Serial Affinity Chromatography

Jon D Weinstein 1,2, Sandra M Mayer 1, Samuel I Beale 1
PMCID: PMC1056564  PMID: 16665424

Abstract

Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to δ-aminolevulinic acid (ALA) in the first committed step of the tetrapyrrole biosynthetic pathway leading to chlorophylls, hemes, and bilins. The conversion requires ATP, Mg2+, and NADPH as cofactors. Soluble extracts from Chlorella vulgaris have now been resolved into four macromolecular fractions, all of which are required to reconstitute activity. One fraction contains a low molecular weight RNA which can be separated from the protein components in an active high-speed supernatant by treatment with 1 molar NaCl followed by precipitation of the proteins with (NH4)2SO4 at 70% saturation. The proteins recovered from the (NH4)2SO4 precipitate are reactivated by addition of a fraction containing tRNAs isolated from Chlorella by phenol-chloroform extraction and DEAE cellulose chromatography. Three required protein fractions were resolved from the RNA-depleted (NH4)2SO4 precipitate by serial affinity chromatography on Reactive Blue 2-Sepharose and 2′,5′-ADP-agarose. Glycerol was found to stabilize the enzyme activity during the separation process. The majority of the glutamate:tRNA ligase activity was associated with the fraction which was retained by Blue-Sepharose and not retained by ADP-agarose, in agreement with the reported properties of the affinity ligands. The active material in the fraction not retained by Blue-Sepharose eluted as a single component on gel filtration chromatography, with an apparent molecular weight of 67,000. The active component in the RNA fraction also eluted as a single component on gel filtration chromatography.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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