Identification and purification of a functional amine transporter from bovine chromaffin granules

Yael Stern-Bach*, Noa Greenberg-Ofrath, Iancu Flechner, Shimon Schuldiner

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

Abstract

The amine transporter from bovine chromaffin granules has been purified in a functional state. Two isofor ms with different pI values have been separated and shown to be active. One with an unusually acidic pi (∼3.5) has been shown to be a glycoprotein with an apparent Mr of 80,000. The purified polypeptide catalyzes transport of serotonin upon reconstitution with an apparent Km of 2 μM and a Vmax of 140 nmol/mg/ min, 150-200-fold higher than the one determined in the native system. Transport is inhibited by reserpine and tetrabenazine, ligands which bind to two distinct sites on the transporter. These findings suggest that the binding sites for both drugs reside on a single polypeptide. The reconstituted purified transporter binds [3H]reserpine with a biphasic kinetic behavior, KD values of 0.3 and 30 nM and Bmax of 310 and 4200 pmol/mg protein, respectively. In addition, binding of [3H]reserpine is accelerated upon imposition of a pH gradient across the proteoliposome. From these findings it is evident that a single polypeptide catalyzes the various functions of the transporter.

Original languageAmerican English
Pages (from-to)3961-3966
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number7
StatePublished - 5 Mar 1990

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