Biosynthesis of Δ-aminolevulinate in greening barley leaves IV. Isolation of three soluble enzymes required for the conversion of glutamate to Δ-aminolevulinate

Abstract

The soluble enzymes converting glutamate into b-aminolevulinate and subsequently into uroporphyrinogen were partially purified from the stroma of greening barley plastids using Sephacryl S-300 gel filtration. By affinity chromatography employing sequentially Blue Sepharose, Matrex Gel Red A and heme-Sepharose the partially purified enzymes were separated into three fractions which together are required to catalyze the synthesis of b-aminolevulinate from glutamate: proteins binding to Blue Sepharose, proteins binding to hemeSepharose and run-off proteins not retained by the three columns. By analysing the characteristics of these fractions the following conclusions are reached:I. Conversion of glutamate into glutamate-l-semialdehyde in the presence of ATP, Mg2 § and NADPH requires at least two proteins, one binding to heme-Sepharose and one binding to Blue Sepharose.2. Glutamate-l-semialdehyde is converted into b-aminolevulinate by glutamate-l-semialdehyde aminotransferase, which is not retained on the affinity columns.3. The run-off protein fraction also contains 5-aminolevulinate dehydratase and porphobilinogen deaminase. 4. The heme-Sepharose bound protein(s) probably converts glutamate to glutamate-l-phosphate in the presence of ATP and Mg2+ and the Blue-Sepharose bound protein(s), glutamate-l-phosphate to glutamate-l-semialdehyde, in the presence of NADPH.

Purification and partial amino acid sequence of the glutamate 1-semialdehyde aminotransferase of barley and Synechococcus

“…Svalrfs Bonus), which were illuminated for 12 h. Stroma proteins were prepared and subjected to serial affinity chromatography (Fig. l) as described in (6,15). Fifteen g wet weight synechococcal cells were suspended in 50 ml 0.7 M sodium phosphate buffer, pH 7.5 and disrupted by passing them twice through a French pressure cell.…”

“…Finally, the precipitated peptides were dissolved in sample buffer for electrophoresis. For the antibody preparation aminotransferase was purified as previously described (6,15). An active protein from a non-denaturing gel was injected into rabbits.…”

“…The separation of the glutamate 1-semialdehyde aminotransferase from the other enzymes and the tRNA ~ of the pathway was performed by passing the proteins obtained by solubilization with the French pressure cell through the Sephacryl S-300 column and the different affinity columns as described previously (6,15).…”

Biosynthesis of Δ-aminolevulinate in greening barley leaves. IX. Structure of the substrate, mode of gabaculine inhibition, and the catalytic mechanism of glutamate 1-semialdehyde aminotransferase

“…Most experiments were performed with enzyme partially purified by passing chloroplast stroma through columns of Sephacryl S-300, Cibacron blue-Sepharose, Procion red-agarose and chlorophyllin-Sepharose as described previously (14,30). Because this preparation contained porphobilinogen synthase and porphobilinogen deaminase, assays for the formation of 5-aminolevulinate included l0 mM-levulinate to block utilization of the product.…”

“…The product of this second reaction is glutamate 1-semialdehyde (8,15,19). Finally, 5-aminolevulinate is formed from glutamate 1-semialdehyde by an isomerization that is catalyzed by an aminotransferase (12,13,30).…”