Abstract
We have developed a sensitive chromatographic method using fluorescence detection for the rapid analysis of monobromobimane-derivatized cysteine and cysteine-containing peptides. The method allows determination of these substances and assay of the enzymes involved in their formation and breakdown: γ-glutamylcysteine synthetase (γ-Glu-Cys synthetase), GSH synthetase, γ-glutamyl transpeptidase (γ-Glu transpeptidase) and dipeptidase. In the assay of GSH synthetase, 90% of the endogenous interfering GSH can be removed by preincubation with thiopropyl Sepharose resin. The distribution of the four enzymes listed above has been measured in liver, heart, lung and kidney of the rat. Some kinetic constants are: hepatic γ-Gly-Cys synthetase: Km (glutamate) 3.36 ± 0.33 mM, Vmax 85.0 ± 2.3 nmol/min/mg protein (Ki for buthionine sulfoximine 1.84 ± 0.11 μM); hepatic GSH synthetase: high affinity Km (glycine) 0.67 ± 0.30 mM, Vmax 7.86 ± 1.17 nmol/min/mg protein; renal γ-Glu transpeptidase: Km (glycylglycine) 8.63 ± 0.07 mM, Vmax 23.6 ± 0.7 μmol/min/mg protein (Ki for AT-125 2.64 ± 0.17 mM); renal dipeptidase: Km (cysteinylglycine) 0.55 ± 0.06 mM, Vmax 100.6 ± 4.5 nmol/min/mg protein.
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Yan, C.C., Huxtable, R.J. (1998). Determination of Cysteinyl-Containing Peptides and Associated Enzyme Activities in Rat Tissues by Reverse Phase HPLC. In: Schaffer, S., Lombardini, J.B., Huxtable, R.J. (eds) Taurine 3. Advances in Experimental Medicine and Biology, vol 442. Springer, Boston, MA. https://doi.org/10.1007/978-1-4899-0117-0_6
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DOI: https://doi.org/10.1007/978-1-4899-0117-0_6
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