Solubilization and functional reconstitution of the human placental taurine transporter.

The taurine transporter from purified human placental brush-border membranes was solubilized and reconstituted into proteoliposomes in a functional form. Solubilization was done with 2.5% cholate in the presence of 4 M urea. The proteins in the solubilizate were precipitated with 6% poly(ethylene glycol) and the precipitated proteins were reconstituted into proteoliposomes with an asolectin/protein ratio of 10:1. Under these experimental conditions, the taurine transport activity in the proteoliposomes was maximal. SDS-PAGE analysis of proteins, however, revealed that the proteoliposomes still contained a majority of the proteins originally present in the brush-border membranes. Uptake of taurine in the reconstituted proteoliposomes was obligatorily dependent on the presence of Na+ as well as Cl-. Substitution of Na+ with other monovalent cations such as K+ and Li+ reduced the taurine transport activity drastically. Similarly, substitution of Cl- with other monovalent anions such as SCN-, F-, I- and NO3- could support the transport activity only to a maximum of 30% of the control activity. In the presence of Cl-, the uptake rate was sigmoidally related to Na+ concentration, resulting in a Na+/taurine coupling ratio of 2:1. The apparent dissociation constant for Na+ was about 195 mM. In the presence of Na+, the uptake rate was hyperbolically related to Cl- concentration, indicating a Cl-/taurine coupling ratio of 1:1. The apparent dissociation constant for Cl- was about 205 mM. The NaCl-dependent taurine uptake was stimulated by an inside-negative membrane potential, showing that the uptake process was electrogenic. The uptake system was specific for beta-amino acids. The affinity of the system for taurine was high with an apparent dissociation constant of 2.7 +/- 0.1 microM. It is concluded that the taurine transporter can be dislodged from the placental brush-border membranes and reconstituted in a catalytically active form in proteoliposomes with no significant change in its characteristics.