Abstract
The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNAGlu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a K m value of 2.3 μM ± 0.3 for glutamate.
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Abbreviations
- ALA:
-
5-aminolevulinic acid
- FPLC:
-
fast protein liquid chromatography
- Glu:
-
glutamate
- Hepes:
-
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- SDS:
-
sodium dodecylsulfate
- Tricine:
-
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine
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This work was supported by a grant of the Deutsche Forschungsgemeinschaft. U.C. Vothknecht is grateful for a Nachwuchs-förderungsstipendium des Landes Hessen. The authors want to thank Ms. B. Böhm, J. Gade and K. Eckhardt for skillful technical assistance. The authors also want to thank Dr. C.G. Kannangara (Carlsberg Institute, Kopenhagen, Denmark) for the donation of tRNA from barley and Dr. D. Jahn (FB Biology/Microbiology, Philipps-University, Marburg, FRG) for the tRNAGlufrom E. coli.
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Vothknecht, U.C., Senger, H. & Dörnemann, D. Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green alga Scenedesmus obliquus, mutant C-2A′. Planta 192, 256–260 (1994). https://doi.org/10.1007/BF00194460
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DOI: https://doi.org/10.1007/BF00194460