Kinetic characterization of Na+/D-mannose cotransport in dog kidney: comparison with Na+/D-glucose cotransport.
Brush-border membrane vesicles (BBMV) prepared from whole dog kidney cortex, or separately from outer cortex (OC) and outer medulla (OM), were used to study the kinetics and inhibition specificity of Na(+)-dependent D-mannose cotransport. In BBMV from whole cortex the measured parameters for Na+/D-mannose uptake were Km = 0.07 +/- 0.01 mM and Vmax = 4.19 +/- 0.24 nmol/mg protein per min (n = 36). In OC BBMV the Km for Na+/D-mannose was 0.04 mM, Vmax = 3.41 nmol/mg per min. In OM the Km was 0.06 +/- 0.02 mM Vmax = 0.18 nmol/mg per min. Thus only about 5% of Na+/D-mannose activity occurs in OM. Both mannoheptulose (Ki = 5.6 mM) and methyl alpha-D-mannoside (Ki = 0.05 mM) are competitive inhibitors of Na+/D-mannose uptake, but at comparable concentrations have little effect on Na+/D-glucose uptake. Phlorizin is a noncompetitive inhibitor of Na+/D-mannose uptake (Ki = 4.45 microM) but a more potent and competitive inhibitor (Ki = 0.58 microM) of Na+/D-glucose uptake. Phloretin (Ki = 104 microM) is a noncompetitive inhibitor of Na+/D-mannose uptake in BBMV. We conclude that Na+/D-mannose uptake is mediated by a unique high-affinity carrier located in the OC presumably at the luminal surface of the proximal convoluted tubule, with strong specificity requirements for sugars with mannose-like structures (i.e., axial C-2 hydroxyl group). Phlorizin is an inhibitor of both Na+/D-mannose and Na+/D-glucose cotransporters but is approx. 10 times less potent for the Na+/D-mannose system and also has a different mode of inhibition (i.e., noncompetitive vs. competitive). The different phlorizin inhibitory mechanisms on the Na+/D-glucose and Na+/D-mannose cotransporters may be mediated by distinct hydrophobic and sugar binding sites that characterize phlorizin-carrier interaction.
Silverman
M
M
Department of Medicine, University of Toronto, Ontario, Canada.
Ho
L
L
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D004285:Dogs; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D008356:Mannoheptulose / Q000737:chemistry / Q000378:metabolism; D008358:Mannose / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000187:drug effects / Q000378:metabolism; D010693:Phloretin / Q000494:pharmacology; D010695:Phlorhizin / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*
D009004:Monosaccharide Transport Proteins; D008356:Mannoheptulose; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose; D008358:Mannose; D010693:Phloretin