Incorporation of labelled glycine into reduced glutathione of intact human erythrocytes by enzyme-catalysed exchange. A nuclear-magnetic-resonance study.
1H, 2H and 15N n.m.r. spectroscopy was used to monitor the incorporation of free glycine into the glycine residue of reduced glutathione (GSH) in suspensions of intact human erythrocytes. The following results were obtained. (i) By using 1H spin-echo n.m.r. the exchange reaction between [2H5]glycine and the protonated glycine residue of GSH was studied at various [2H5]glycine concentrations, thus enabling the calculation of an apparent Michaelis constant (Km) and maximal velocity (Vmax.) for the process. (ii) The reaction is catalysed by glutathione synthetase and proceeds most rapidly in the absence of glucose, which is the main physiological energy source of the erythrocyte. (iii) 15N n.m.r. spectroscopy, with a one-pulse sequence, and 2H n.m.r. spectroscopy, with an inversion recovery method, enabled demonstration of the incorporation of labelled glycine into an intra-erythrocyte peptide, consistent with incorporation into GSH. (iv) The exchange reaction, although inhibited by glucose, appeared not to be dependent on low ATP or 2,3-bisphosphoglycerate concentrations.
D003903:Deuterium; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D005978:Glutathione / Q000097:blood*; D005981:Glutathione Synthase / Q000097:blood; D005998:Glycine / Q000097:blood*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction
D009587:Nitrogen Isotopes; D003903:Deuterium; D005981:Glutathione Synthase; D005978:Glutathione; D005947:Glucose; D005998:Glycine