THE CHARACTERIZATION OF BUTYRATE TRANSPORT ACROSS PIG AND HUMAN COLONIC LUMINAL MEMBRANE

1. Luminal membrane vesicles (LMV) were isolated from human and pig co lonic tissues. They were characterized in terms of purity and ability to transport [C-14]butyrate. 2. The activity of cysteine-sensitive alk aline phosphatase, and the abundance of villin, NHE2 and NHE3 proteins , markers of the colonic luminal membrane, were significantly enriched in the LMV compared with the original cellular homogenate. The LMV we re free from contamination by other cellular organelles and basolatera l membranes, as revealed by the negligible presence of either specific marker enzyme activity or characteristic immunogenic protein. 3. The transport of butyrate into the luminal membrane vesicles was enhanced 5-fold at pH 5.5 compared with pH 8.0. Butyrate transport was temperat ure dependent, and was stimulated in the presence of an outward-direct ed anion gradient in the order of butyrate > bicarbonate > propionate > chloride. Kinetic analysis of increasing substrate concentration sho wed saturation kinetics with an apparent K-m value of 14.8 +/- 3.6 mM and a V-max of 54 +/- 14 nmol min(-1) (mg protein)(-1). 4. Butyrate tr ansport was significantly reduced in the presence of short chain fatty acids (SCFA), acetate, propionate and other monocarboxylates (pyruvat e and L-lactate). Butyrate uptake was inhibited by several cysteine gr oup modifying reagents such as p-chloromercuribenzosulphonic acid (pCM BS), p-chloromercuribenzoate (pCMB), mersalyl acid and HgCl2, but not by the stilbene anion exchange inhibitors, 4,4'-diisothiocyanostilbene -2,2'-disulphonate (DIDS) and 4,4'-dinitrostilbene-2,2'-disulphonate ( SITS). 5. The described properties of butyrate transport across the lu minal pole of the colon suggest the involvement of a carrier protein, in the form of a pH-activated anion exchange process. The transporter is distinct from the erythrocyte band-3 type anion exchanger and may b elong to the monocarboxylate-type transport proteins (MCT1).