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 K_m of 0.24 mmole/liter and a V_max of 6.4 nmole/100 μg 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 K_m and V_max values, obtained by using a computer program, are a K_m of 15.0 mmole/liter and a V_max of 14.7 nmole/100 μg 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 K_m 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.