Biosynthesis of Δ-aminolevulinate in greening barley leaves. VIII: Purification and characterization of the glutamate-tRNA ligase

Bruyant, P.; Kannangara, C. Gamini

doi:10.1007/bf02910432

Cited by 48 publications

(18 citation statements)

References 17 publications

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“…A partially purified pea chloroplast enzyme preparation was obtained by sequential affinity chromatography on Blue SepharoseR and Salicylate SepharoseR [18]. The fraction bound to Salicylate SepharoseR (Sal fraction) was used for aminoacylation experiments.…”

Section: Preparation Of a Chloroplast Fraction Containing Glutamyl-trmentioning

confidence: 99%

Discrimination against misacylated tRNA by chloroplast elongation factor Tu

Stanzel

Schön

Sprinzl

1994

European Journal of Biochemistry

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Chloroplast elongation factor Tu was purified from Pisum sativum and the binding properties of glutamylated chloroplast tRNAs were studied by gel‐permeation chromatography. Whereas chloroplast Glu‐tRNAGlu is efficiently bound by this factor, the misacylated Glu‐tRNAGin does not interact with chloroplast elongation factor Tu · GTP and is thus efficiently excluded from protein synthesis. Comparison with the behaviour of Escherichia coli elongation factor Tu · GTP shows that this factor, which is not confronted with the in vivo misacylation phenomenon of organelles, binds both Glu‐tRNAGlu and Glu‐tRNAGln from chloroplasts with approximately equal efficiency.

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Section: Preparation Of a Chloroplast Fraction Containing Glutamyl-trmentioning

confidence: 99%

Discrimination against misacylated tRNA by chloroplast elongation factor Tu

Stanzel

Schön

Sprinzl

1994

European Journal of Biochemistry

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“…In the first step of this pathway, glutamate is esterified to tRNA Cju by glutamic acid-tRNA ligase, which also provides glutamyl-tRNA for protein synthesis (4,14,26). Glutamyl-tRNA then is reduced in an NADPH-requiring reaction.…”

Section: Introductionmentioning

confidence: 99%

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

Hoober

Kahn

Ash

et al. 1988

Carlsberg Res. Commun.

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101

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Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by ~H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 rain. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate l-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the abs~ence of glutamate I-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.

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“…The 100 ,uL reaction mixture contained 1 mm ATP, 1 mM MgCl2, 1 mm NADPH, 2 mM 4,6-dioxoheptanoic acid, 5 MCi of [3H]glutamic acid, and enzyme preparation.…”

Section: Methodsmentioning

confidence: 99%

“…Glu RS activity involved in ALA synthesis has been reported in barley (13), Chlamydomonas (24), and Chlorella (25). The enzyme has been partially purified from chloroplasts of wheat (18) and barley (5 The Glu RS from Chiamydomonas has a native mol wt of about 60,000. This value was obtain by analyzing the purified enzyme with native PAGE followed by Ferguson plots and with a HPLC gel filtration column.…”

Section: Antibody Preparationmentioning

confidence: 99%