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Metabolism and Enzymology

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

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

Division of Biology and Medicine, Brown University, Providence Rhode Island 02912

Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to -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.

¹ Supported by National Science Foundation Grants DMB-8213948 and DMB-8518580.