A proton n.m.r. study of iminodipeptide transport and hydrolysis in the human erythrocyte. Possible physiological roles for the coupled system.
The first description of a saturable iminodipeptide transport system present in human erythrocytes is given. The 1H-n.m.r. spectra of glycyl-L-proline and those of free glycine and L-proline are significantly different. This enabled the non-invasive monitoring by 1H-n.m.r. spectroscopy of the hydrolysis of the dipeptide in human erythrocytes and their lysates. The concentration-dependence of the rate of glycyl-L-proline hydrolysis by haemolysates was described by the Michaelis-Menten expression with Km = 14.1 +/- 2.4 mmol/litre and Vmax. = 130 +/- 10 mmol/h per litre of cell water. At concentrations of the dipeptide that saturated prolidase, hydrolysis of glycyl-L-proline by whole cells was approximately 130 times slower than by lysates. This rate difference indicated that transport is the rate-determining step in peptide hydrolysis by whole cells, and thus the concentration-dependence of the transport rate was determined. The membrane transport system was found to be saturable and could be described by the Michaelis-Menten expression with Kt = 4.7 +/- 0.4 mmol/litre and Vmax. = 0.997 +/- 0.026 mmol/h per litre of cell water. Numerical integration of a consistent set of differential rate equations that described a minimal model of the coupled transport-hydrolysis system successfully described prolonged time courses of peptide hydrolysis by whole cells. The simulations showed very low steady-state levels of dipeptide in the erythrocyte and very small lag periods (less than 5 min) in the progress curve describing the appearance of free amino acid inside the cells. The rates of transport of glycyl-L-proline into erythrocytes and kidney proximal-tubular epithelium were compared and the possible importance of erythrocyte prolidase in whole-body prolyl-peptide turnover is discussed.
D001692:Biological Transport; D004150:Dipeptidases / Q000097:blood; D004151:Dipeptides / Q000097:blood*; D004912:Erythrocytes / Q000378:metabolism*; D005998:Glycine / Q000097:blood; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D011392:Proline / Q000097:blood
D004151:Dipeptides; C015248:glycylproline; D011392:Proline; D004150:Dipeptidases; C022449:proline dipeptidase; D005998:Glycine