Automated pre-column amino acid analyses by reversed-phase high-performance liquid chromatography

Winspear, Michael J.; Oaks, Ann

doi:10.1016/s0021-9673(01)96389-7

Cited by 33 publications

(5 citation statements)

References 4 publications

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“…The initial fluorescence readings were taken within 1 min after mixing to initiate the reaction. The fluorescence intensities are not strictly comparable since the data were collected using slightly different wavelength settings depending upon the thiol employed in Roth's reagent (emission and excitation wavelengths are given in the Experimental and Table 1 primary amino compounds (11,22,33,34,37,41,67,70,71,101,108,115,137,145). The relatively stable isoindole formed from OPA/2-ME and leucyltryptophan observed in this study is also in accord with a previous report (76).…”

Section: Effect Of Thiol In Roth's Reagent In Aqueous Buffered Mediasupporting

confidence: 84%

Update and Evaluation of the Effectiveness of Different Thiols and Micellar Media in Roth's Fluorimetric Method for the Determination of Primary Amino Compounds

Dai

Burkert

Singh

et al. 1997

Microchemical Journal

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Section: Effect Of Thiol In Roth's Reagent In Aqueous Buffered Mediasupporting

confidence: 84%

Update and Evaluation of the Effectiveness of Different Thiols and Micellar Media in Roth's Fluorimetric Method for the Determination of Primary Amino Compounds

Dai

Burkert

Singh

et al. 1997

Microchemical Journal

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“…Levels of amino acids in the mitochondria were determined by reversed phase chromatography on an Altex Ultrasphere-ODS (5 AM) column as described by Winspear and Oaks (31 To estimate the equilibrium of the GDH reaction in corn mitochondria, purified GDH was incubated in the 0.2 M TrisMes buffer at a pH range from 6.0 to 9.0 with the estimated in vivo levels of substrate (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

Characteristics of Glutamate Dehydrogenase in Mitochondria Prepared from Corn Shoots

Yamaya¹,

Oaks²,

Matsumoto³

1984

Plant Physiol.

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The amination of a-ketoglutarate (a-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca2+. The NADH-GDH purified 161-fold with ammonium sulfate, was also activated by Ca2" in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca2 had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca2".About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca2" had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca2". In a simulated in vitro system using concentrations of 6A millimolar NAD, 0.21 millimolar NADH, 5 millimolar a-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca2", 10 and 50 millimolar NH4', and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.NADH-GDH2 is easily solubilized from mitochondria from roots (30), shoots (4, 25), and seeds (28) of various plants. Although it is the only mitochondrial enzyme with the potential for the assimilation of NH44, it has been suggested by Miflin and Lea (21) that the NH44 released by the conversion of glycine to serine in the mitochondria (3) is reassimilated via GS in the cytosol. They have also suggested that the principal function of mitochondrial GDH is the oxidation ofglutamate (21). However, a part of intramitochondrially generated NH44 from glycine is incorporated into glutamate by mitochondria isolated from pea shoots (I 1). Chou and Splittstoesser (7), Joy (14), and recently Furuhashi and Takahashi (10) Isolation of Mitochondria by Density Gradient Centrifugation. All reagents and buffer solutions were prepared with double glass-distilled H20 to minimize Ca24 contamination, and all operations were carried out at 4°C. Whole shoots of 5-d seedlings were harvested, washed in water, blotted dry with filter paper, and weighed. The shoots (40 to 60 g in fresh weight) were chopped with razor blades in extraction buffer consisting of 0.4 M mannitol, 0.1 M Hepes-KOH buffer (pH 7.5), 1 mM EDTA, 0.1% (w/v) BSA, and 0.6% (w/v) insoluble PVP, at a ratio of 1 g fresh weight per 3 ml of buffer. The chopped materials were ground with motar and pestle.The mitochondrial fraction was isolated by modification of the method described by Nishimura et al. (26) as described previously by us (32). The purified mitochondria after Percoll discontinuous density gradient were able to oxidize both malate and succinate with a high P/O and RCR ratios and appear to...

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“…An alternative to the above approach is to use an automated precolumn derivatization and injection method. This method has been successfully applied to the chromatographic determination of amino acids with OPA (17,18) and therefore should be easily adapted to the present thiol method.…”

Section: Methodsmentioning

confidence: 99%

Trace determination of biological thiols by liquid chromatography and precolumn fluorometric labeling with o-phthalaldehyde

Mopper¹,

Delmas²

1984

Anal. Chem.

119

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Automated pre-column amino acid analyses by reversed-phase high-performance liquid chromatography

Cited by 33 publications

References 4 publications

Update and Evaluation of the Effectiveness of Different Thiols and Micellar Media in Roth's Fluorimetric Method for the Determination of Primary Amino Compounds

Update and Evaluation of the Effectiveness of Different Thiols and Micellar Media in Roth's Fluorimetric Method for the Determination of Primary Amino Compounds

Characteristics of Glutamate Dehydrogenase in Mitochondria Prepared from Corn Shoots

Trace determination of biological thiols by liquid chromatography and precolumn fluorometric labeling with o-phthalaldehyde

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