Extracellularly applied Br-TITU inhibits the Na+/K+ pump by interacting with Tryptophan/s at the entrance to the cation sites.

YUDOWSKI, GUILLERMO A.; BAR SHIMON, M.; RODOLFO MARTIN GONZALEZ LEBRERO; ROLANDO CARLOS, ROSSI; PATRICIO J, GARRAHAN; KARLISH, S.J.D.; BEAUGÉ, L

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES.

BLACKWELL PUBLISHING

Lugar: Oxford; Año: 2003 vol. 986 p. 287 - 289

0077-8923

P { margin-bottom: 0.21cm; direction: ltr; color: rgb(0, 0, 0); widows: 2; orphans: 2; }P.western { font-family: "Times New Roman",serif; font-size: 10pt; }P.cjk { font-family: "Times New Roman",serif; font-size: 10pt; }P.ctl { font-family: "Times New Roman",serif; font-size: 10pt; }A family of aryl isothiouronium derivatives was designed as molecular probes for the cation binding sites of Na,K‑pump. Previous work showed that Br‑TITU binds to renal Na,K‑ATPase with two distinct affinities: <1 µM on the inside and around 50 µM on the outside. Here we describe properties and kinetic characteristics of Br‑TITU as a cation antagonist at the extracellular surface. In partially purifies Na,K‑ATPase, Br‑TITU prevents K+‑stimulated dephosphorylation of the enzyme phosphorylated from ATP. In intact human and rat cells Br‑TITU applied externally inhibits the Na+ pump non‑competitively, K0.5  0.4 µM. An interesting property of Br‑TITU is that it reacts irreversibly with Trp residues of renal Na,K‑ATPase at alkaline pH and inactivates Rb occlusion. After preincubation with red cells at pH 9 Br‑TITU, also irreversibly inhibits a large fraction of the ouabain sensitive [22Na]Na+ measured at pH 7.4 in 140 mM Na+, K+‑free medium. When presnt with Br‑TITU, K+ antaggonizes this effect, as do Cs+ and Li+, and with the same apparent affinity despite their different affinities for the Na+ pump. Furthermore, Br‑TITU quenches Trp fluorescence at pH 9 and this quenching is prevented by K+ regardless of the conformational state of the pump. Our data suggest that Br‑TITU may bind a Trp residue at the extracellular entrance port of the Na,K‑ATPase which acts as a cation concentrating region. P { margin-bottom: 0cm; direction: ltr; color: rgb(0, 0, 0); text-align: center; widows: 2; orphans: 2; }P.western { font-family: "Times New Roman",serif; font-size: 12pt; }P.cjk { font-family: "Times New Roman",serif; font-size: 12pt; }P.ctl { font-family: "Times New Roman",serif; font-size: 10pt; }A:link { color: rgb(0, 51, 204); }