Assimilation of alpha-glutamyl-peptides by human erythrocytes. A possible means of glutamate supply for glutathione synthesis.
Human erythrocytes are essentially impermeable to glutamate and yet there is a continual requirement for the amino acid for glutathione synthesis. In addition, the intracellular glutamate concentration is approximately five times that of plasma. We present evidence that glutamate enters the red cell as small peptides which are rapidly hydrolysed by cytoplasmic peptidase(s) and that with the estimated physiological levels of plasma glutamyl-peptides the rate of inward flux would be adequate to maintain the glutamate pool at its observed level. Experimentally, we used 1H spin-echo n.m.r. spectroscopy to follow peptide hydrolysis, since peptide spectra are different from those of the free amino acids and the spin-echo sequence enables the monitoring of reactions in concentrated lysates and whole cell suspensions. Thus, the system was studied under near-physiological conditions. Weighted non-linear regression analysis of progress curves using the integrated Michaelis-Menten equation was used to obtain estimates of Km and Vmax. for the hydrolysis of alpha-L-glutamyl-L-alanine and L-alanyl-alpha-L-glutamate in lysates and whole cell suspensions; the values for lysates were Km = 3.60 +/- 0.29 and 5.4 +/- 0.4 mmol/l and Vmax. = 120 +/- 4 and 46.7 +/- 1.7 mmol/h per 1 of packed cells respectively. In whole cell suspensions the rate of peptide hydrolysis was much slower and dominated by the transmembrane flux-rate. The estimates of the steady-state kinetic parameters for the transport were Kt = 2.35 +/- 0.41 and 11.2 +/- 1.0 mmol/l and Vmax. = 3.26 +/- 0.13 and 19.7 +/- 0.7 mmol/h per 1 of packed cells respectively for the previously mentioned peptides. Using the n.m.r. procedure we failed to detect any glutaminase activity in whole cells or lysates; thus, we exclude the possibility that glutamate gains entry to the cell as glutamine which is subsequently hydrolysed by glutaminase.
King
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Kuchel
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D000409:Alanine / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D004151:Dipeptides / Q000097:blood*; D004912:Erythrocytes / Q000378:metabolism*; D005971:Glutamates / Q000097:blood*; D018698:Glutamic Acid; D005972:Glutaminase / Q000097:blood; D005978:Glutathione / Q000096:biosynthesis* / Q000097:blood; D006461:Hemolysis; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D011817:Rabbits
D004151:Dipeptides; D005971:Glutamates; C004276:glutamylalanine; D018698:Glutamic Acid; C045560:alanylglutamic acid; D005972:Glutaminase; D005978:Glutathione; D000409:Alanine