Transport of L‐alanine in cultured human fibroblasts: Evidence for two kinetically distinguishable systems

Abstract

The transport of L-alanine in human diploid fibroblasts was investigated. Transport measurements were performed on subcultures between the third and eighth passages with subconfluent cells growing on glass coverslips. Kinetic analysis of approximate initial rates of transport at substrate concentrations from 0.05 to 10 mmole/liter indicate the presence of two distinguishable systems. The high affinity system has a Km of 0.24 mmole/liter and a Vmax of 6.4 nmole/100 micrograms protein/2 min. For the low affinity system, the contribution of the high affinity system to the uptake must absolutely be taken into account. The Km and Vmax values, obtained by using a computer program, are a Km of 15.0 mmole/liter and a Vmax of 14.7 nmole/100 micrograms protein/2 min. For alanine concentrations below 1 mmole/liter, the contribution of the Na+-independent uptake is less than 10%, and the kinetic constants of the high affinity system are in the same range if this contribution is taken into account. On the contrary the influence of a diffusion-like process is more significant on the low affinity system whose Km is about 49 mmole/liter after subtraction of the Na+-independent uptake from the experimental velocities. Inhibition studies were performed with NCH3-alanine. They permitted us first to confirm the existence of system A in cultured human fibroblasts in agreement with two recent works and second to show how this system contributes to L-alanine uptake. This contribution seems very small in low concentrations but it rises as the concentrations increase.

The regulation of neutral amino acid transport in mammalian cells

“…Cysteine appears to serve as a specific substrate in the rat hepatocyte [28], and threonine may serve as an appropriate substrate in HTC cells [29], but the lack of an acceptable model substrate for System ASC in other cell types complicates the characterization of this system in the presence of other neutral amino acid transport systems. In some cases, System ASC activity has been operationally defined as the sodium-dependent uptake of an amino acid that is not inhibited by excess MeAIB [14,15] or N-methylalanine [33], although kinetic criteria of identification have also 269 been applied [13]. More extensive discussions of the difficulty in distinguishing System ASC from System A have recently been reported [ 13,30].…”

Evidence that cycloleucine affects the high-affinity systems of amino acid uptake in cultured human fibroblasts

“…For the last passage ofthe experiment, cells were allowed to grow as monolayers on glass coverslips by the method of Foster & Pardee (1969). As we previously described (Feneant et al, 1981), after 1 h of incubation in phosphate-buffered saline solution, pH7.45, containing 0.1% glucose, each coverslip was transferred into an incubation vial containing 1.Oml of buffer and the appropriate concentration of 14C-labelled amino acid (0.5yCi/ml incubation mixture) in the absen9e or presence of 5 mM-cycloleucine. The concentrations of the amino acid tested were 2mM for time-course determination, and 0.05 or 0.066 to 10mM for kinetic studies.…”

“…In the first mathematical procedure, previously described in detail (Feneant et al, 1981), lines were plotted by the double-reciprocal method (Lineweaver & Burk, 1934), by weighted linear regression, with v2 as the weighting factor of reciprocal velocity 1/v. When the presence of two systems with different affinities was shown by a break in the plot, the contribution of the highaffinity system to the uptake of high substrate concentrations was necessarily taken into account when calculating the kinetic constants of the lowaffinity system.…”

“…In our preliminary report (Moatti et al, 1978), the kinetic parameters of uptake were not defined. To gain new insight into the effects of cycloleucine, a study on the mechanisms of amino acid transport in this cell type was first carried out (Feneant et al, 1981;Corriat et al, 1983). In the last few years, considerable progress has been made in the characterization of the amino acidtransport systems in human fibroblasts.…”