Colorimetric assay for aminoacyl-tRNA synthetases

Chang, Gu‐Gang; Pan, Foo; Yeh, Chun Chang; Huang, Tsai-Wang

doi:10.1016/0003-2697(83)90665-6

Cited by 13 publications

(4 citation statements)

References 18 publications

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“…The activity of mt-ArgRS (EC 6.1.1.19) was assayed in mitochondria purified from cultured skin fibroblasts. The mt-ArgRS enzyme activity was measured by a modification of the method previously described (Chang et al 1983(Chang et al , 1984. Briefly, 100 μg of mitochondrial proteins were incubated in 50 mM Tris-HCl buffer (pH 7.5), 1.25 mM MgAc 2 , 1.2 mM ATP, and 6 mM L-arginine.…”

Section: Arginyl-trna Synthetase Enzyme Activitymentioning

confidence: 99%

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients

Cassandrini

Cilio

Bianchi

et al. 2012

J of Inher Metab Disea

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Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.

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Section: Arginyl-trna Synthetase Enzyme Activitymentioning

confidence: 99%

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients

Cassandrini

Cilio

Bianchi

et al. 2012

J of Inher Metab Disea

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“…As examples, mutations L200V and V368L (HARS2) and I329T (FARS2) were shown to affect ATP-PPi exchange ability of mt-HisRS [90] and mt-PheRS [104], respectively. As an alternative procedure of the ATP-PPi exchange experiment, Cassandrini and co-workers applied a colorimetricbased measurement of Pi production [105]. The authors demonstrated that crude mitochondrial extracts, extracted from cultured skin fibroblasts of patients harboring either R245Q/R469H or W241R/Q12R mutations on mt-ArgRS, have residual activities in Pi formation of only 33% and 19%, respectively.…”

Section: Impact On Mt-aarss Enzyme Activitiesmentioning

confidence: 99%

Pathogenic Implications of Human Mitochondrial Aminoacyl-tRNA Synthetases

Schwenzer

Zoll

Florentz

et al. 2013

Topics in Current Chemistry

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Mitochondria are considered as the powerhouse of eukaryotic cells. They host several central metabolic processes fueling the oxidative phosphorylation pathway (OXPHOS) that produces ATP from its precursors ADP and inorganic phosphate Pi (PPi). The respiratory chain complexes responsible for the OXPHOS pathway are formed from complementary sets of protein subunits encoded by the nuclear genome and the mitochondrial genome, respectively. The expression of the mitochondrial genome requires a specific and fully active translation machinery from which aminoacyl-tRNA synthetases (aaRSs) are key actors. Whilst the macromolecules involved in mammalian mitochondrial translation have been under investigation for many years, there has been an explosion of interest in human mitochondrial aaRSs (mt-aaRSs) since the discovery of a large (and growing) number of mutations in these genes that are linked to a variety of neurodegenerative disorders. Herein we will review the present knowledge on mt-aaRSs in terms of their biogenesis, their connection to mitochondrial respiration, i.e., the respiratory chain (RC) complexes, and to the mitochondrial translation machinery. The pathology-related mutations detected so far are described, with special attention given to their impact on mt-aaRSs biogenesis, functioning, and/or subsequent activities. The collected data to date shed light on the diverse routes that are linking primary molecular possible impact of a mutation to its phenotypic expression. It is envisioned that a variety of mechanisms, inside and outside the translation machinery, would play a role on the heterogeneous manifestations of mitochondrial disorders.

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“…The resulting pyrophosphate reacts with ammonium molybdate and 2-mercaptoethanol, which are usually used for the detection and measurement of phosphate (Eq. 2 ) 23 . Finally, the resulting blue shade is quantified, and the amino acid concentrations are calculated 19 .…”

Section: Methodsmentioning

confidence: 99%

“…aaRSs exist for each of the 20 corresponding amino acids and are involved in the biosynthesis of proteins and peptides in the body. Thus, aaRS could serve as a recognition material for amino acid analysis 20 – 23 .…”

Section: Introductionmentioning

confidence: 99%

Development of a one-step analysis method for several amino acids using a microfluidic paper-based analytical device

Kugimiya

Wakimoto

Kohda

et al. 2022

Sci Rep

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A one-step analysis method was developed for four types of amino acids using a microfluidic paper-based analytical device fabricated from chromatography filtration paper and laminate films. Aminoacyl-tRNA synthetase was used to detect each amino acid. The obtained laminated paper-based analytical device (LPAD) contained four enzymatic reaction areas. Colorimetric detection was performed based on the molybdenum blue reaction. A model method for the simple, easy, and simultaneous detection of several amino acid concentrations was suggested, in contrast to the conventional methods such as HPLC or LC–MS. The method provided a selective quantification at the ranges of 3.6–100 μM for tryptophan, 10.1–100 μM for glycine, 5.9–100 μM for histidine and 5.6–100 μM for lysine with a detection limit of 1.1 μM, 3.3 μM, 1.9 μM and 1.8 μM, respectively. LPAD fabrication was considerably simple, and the subsequent detection process was easy and required a short period of time (within 15 min).

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Colorimetric assay for aminoacyl-tRNA synthetases

Cited by 13 publications

References 18 publications

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients

Pontocerebellar hypoplasia type 6 caused by mutations in RARS2: definition of the clinical spectrum and molecular findings in five patients

Pathogenic Implications of Human Mitochondrial Aminoacyl-tRNA Synthetases

Development of a one-step analysis method for several amino acids using a microfluidic paper-based analytical device

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