Characterization of the D-glucose/Na+ cotransport system in the intestinal brush-border membrane by using the specific substrate, methyl alpha-D-glucopyranoside.
By using isolated membrane vesicles, we have investigated the tenet that D-glucose transport across the intestinal brush-border membrane involves at least two distinct, Na+-activated agencies (D-glucose transport systems S-1 and S-2), only one of which (S-1) can use methyl alpha-D-glucopyranoside (methyl alpha-glucoside) as a substrate. Our results with this glucose analogue show that: (a) As a function of time, methyl alpha-glucoside uptake exhibits a typical overshoot, similar to but smaller than that given by D-glucose with the same vesicle batch. (b) Nonlinear regression analysis of substrate-saturation curves reveals that, contrary to D-glucose, methyl alpha-glucoside transport involves a single transport system which we have identified as S-1. (c) Methyl alpha-glucoside exhibits an apparent affinity (defined as the reciprocal of Km) 4-times smaller than that of D-glucose for S-1 (Km(Dglucose) = 0.5 mM; Km(methyl alpha-glucoside) = 2 mM). However, methyl alpha-glucoside has a Vmax (230 pmol/mg protein per s) identical to that characterizing D-glucose transport by this system. (d) In the absence of Na+, methyl alpha-glucoside uptake is indistinguishable from simple diffusion, confirming that Na+ is an obligatory activator of S-1. (e) Phlorizin behaves as a fully competitive inhibitor of methyl alpha-glucoside transport (Ki = 18 microM), again indicating that S-1 is involved. (f) Neither phloretin nor cytochalasin B affects methyl alpha-glucoside uptake. We conclude that methyl alpha-glucoside is a substrate specific for S-1, which permits study of the properties of this system without interference by substrate fluxes taking place through any other channel.
Brot-Laroche
E
E
Centre National de la Recherche Scientifique, Meudon, France.
Supplisson
S
S
Delhomme
B
B
Alcalde
A I
AI
Alvarado
F
F
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003571:Cytochalasin B / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000378:metabolism*; D006168:Guinea Pigs; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials; D008757:Methylglucosides / Q000378:metabolism*; D008759:Methylglycosides / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D010695:Phlorhizin / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology
D008757:Methylglucosides; D008759:Methylglycosides; D003571:Cytochalasin B; C027020:methylglucoside; D012964:Sodium; D010695:Phlorhizin; D005947:Glucose