l-leucine transport in human red blood cells: A detailed kinetic analysis

Abstract

The kinetic properties of L-leucine transport across the human red blood cell membrane was analyzed according to the simple pore and carrier theory of Lieb and Stein (Biochim. Biophys. Acta, 1974, 373: 165-177 and 178-196) at 25 degrees C, pH 7.4. Several methods were used in order to obtain a thorough kinetic description of L-leucine transport. A rejection of the simple pore model was suggested from the result of zero-trans influx and zero-trans and equilibrium-exchange efflux experiments. Several predictions from the simple carrier model, based on the requirement of consistency among different kinetic parameters, were tested in infinite experiments, i.e. experiments performed at a high concentration of substrate at one of the faces of the membrane. The simple pore model was rejected, but no crucial evidence against a simple carrier model, which displays symmetric properties at 25 degrees C, was found in the concentration range considered (0.002-68 mM). The relative magnitudes of the rate constants of the translocation process are discussed, and it is concluded (a) that both the dissociation and translocation of carrier-complex is faster than the translocation of the empty carrier, (b) that no translocation step is rate determining, and (c) that the carrier-complex is equally distributed across the membrane at equilibrium. The present work provides a unique example of a carrier-mediated transport mechanism which displays symmetric properties. L-leucine transport in red blood cells may be a convenient system for studying molecular mechanisms of facilitated transport.

Na‐independent and Na‐dependent transport of neutral amino acids in the human red blood cell

“…in the concentration range indicated. L-Leucine ,>arameters (-+SD) previously published (Rosenberg 1981 basic amino acids only had weak effect on the rate of L-leucine transport.…”

“…The transport experiments, the preparation of the cells, the calculation of the initial rate of the unidirectional flux and the statistical analysis of concentration dependence curves are described in detail elsewhere (Rosenberg 1981). In brief, heparinized blood from the same donor was washed 4 times in incubation medium (140 mM NaCI, 5 mM MgC12, 1 mM NalHP04, and 18 mM HEPES, pH 7.4, 25"C), discarding the buffy coat.…”

“…In zero-trans experiments only the initial part of the time course curve was applied for reasons discussed in detail by Rosenberg (1981).…”

“…All experiments were performed at 25°C a temperature previously applied in studies by Hoare (1972 a) and Rosenberg (1981) in order to measure initial rate of transport.…”

“…1, and results from non-linear regression analysis considering a 2and a 3-parameter model in Table 1. Kinetic parameters for L-leucine (Rosenberg 1981) have been included for comparison. The improvement of fit by increasing the number of kinetic parameters was evaluated by examining the reduction of the residual sum of squares.…”