Kinetic characteristics and regulation of hexose transport in a galactokinase-negative Chinese hamster fibroblast cell line: A good model for studies on sugar transport in cultured mammalian cells

We report the kinetic characteristics for D‐galactose, 2‐deoxy‐D‐glucose and 3‐0‐methyl‐D‐glucose transport in a galactokinase null‐allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D‐galactose, 2‐deoxy‐D‐glucose, and 3‐0‐methyl‐D‐glucose transport of 8.6 ± 2.6 mM and 26.1 ± 7.2nmol/mg p/min, 4.1 ± 1.2 mM and 40.3 ± 9.5 nmol/mg p/min, and 7.01 ±.85 mM and 11.6 ± 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 ± 3.7% of the galactose uptake was cytochalasin B inhibitable) and L‐glucose uptake (7.5% of the galactose uptake). D‐Galactose was not metabolized and effluxed rapidly from preloaded cells. The K1s for the inhibition of D‐galactose transport were 4.5 ± 2.5 mM for D‐glucose, 7.0 ± 2.0 mM for 2‐deoxy‐D‐glucose, 6 mM for 2‐deoxy‐D‐galactose and 6.0 ± 0.6 mM for 3‐0‐methyl‐D‐glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50‐60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum‐deprived cells. Hexose transport rates increased substantially in glucose‐deprived, D‐fructose‐ or D‐galactose‐fed cells as compared to glucose‐fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKI. The kinetic and regulatory characteristics of D‐galactose transport in the GalKI cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.