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Transport of tricarballylate by intestinal brush‐border membrane vesicles from steers
Author(s) -
Wolffram S,
Zimmermann W,
Scharrer E
Publication year - 1993
Publication title -
experimental physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.925
H-Index - 101
eISSN - 1469-445X
pISSN - 0958-0670
DOI - 10.1113/expphysiol.1993.sp003699
Subject(s) - brush border , chemistry , vesicle , differential centrifugation , biochemistry , citric acid , membrane transport , biophysics , membrane , chromatography , biology
Tricarballylic acid is a non‐metabolizable rumen bacterial fermentation product of the naturally occurring tricarboxylic acid trans‐aconitic acid. The aim of the present study was to investigate intestinal absorption of tricarballylate using brush‐border membrane vesicles (BBMVs) isolated from the proximal jejunum of steers by a Ca2+ precipitation method with subsequent differential centrifugation. Transport of tricarballylate was investigated indirectly (influence of tricarballylate on the uptake of 14C‐labelled citrate) as well as directly (uptake of 3H‐labelled tricarballylate). Citrate as well as tricarballylate uptake (at a concentration of 0.05 mmol l‐1) was strongly stimulated by an inwardly directed initial Na+ gradient. Furthermore, transport of both tricarboxylates under Na+ gradient conditions was clearly enhanced by lowering the extravesicular pH from 7.8 to 5.6. The imposition of an inwardly directed H+ gradient (pH(out)/pH(in) = 5.6/7.8) further enhanced the intravesicular accumulation of citrate as well as of tricarballylate compared with pH(out)/pH(in) = 5.6/5.6. Unequivocal evidence for a common transport site for tricarballylate and citrate was obtained from ‘cis‐inhibition’ and ‘trans‐stimulation’ of Na(+)‐dependent citrate uptake by tricarballylate. In further experiments the influence of different substances on the uptake of 3H‐labelled tricarballylate was evaluated. Unlabelled tricarballylate, citrate, succinate as well as trans‐ and cis‐aconitate significantly inhibited the accumulation of 3H‐labelled tricarballylate by BBMVs. Tricarballylate uptake as a function of the tricarballylate concentration revealed a Na(+)‐dependent saturable component (apparent kinetic parameters: maximal transport capacity (Vmax) = 119 pmol (mg protein)‐1 (3s)−1; affinity constant (Km) = 0.097 mmol l‐1) and a Na(+)‐independent diffusional component (diffusion constant: 169 nl (mg protein)‐1 (3s)−1). It is concluded that tricarballylate and citrate are transported across the intestinal brush‐border membrane by a common, Na(+)‐dependent transport mechanism. The stimulatory influence of a low extravesicular pH most probably indicates that the protonated forms of tricarboxylates are better transported than the trivalent species.