Functional properties of purified and reconstituted mitochondrial metabolite carriers
- 1 October 1993
- journal article
- review article
- Published by Springer Nature in Journal of Bioenergetics and Biomembranes
- Vol. 25 (5) , 525-535
- https://doi.org/10.1007/bf01108409
Abstract
Eight mitochondrial carrier proteins were solubilized and purified in the authors' laboratories using variations of a general procedure based on hydroxyapatite and Celite chromatography. The molecular mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatography on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties. A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asymmetric orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution. All carriers which have been investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport; i.e., a ternary complex, made up of two substrates and the carrier protein, is involved in the catalytic cycle. The only exception was the carnitine carrier, where a ping-pong mechanism of transport was found. By reaction of particular cysteine residues with mercurial reagents, several carriers could be reversibly converted to a functional state different from the various physiological transport modes. This “unphysiological” transport mode is characterized by a combination of channel-type and carrier-type properties.Keywords
This publication has 67 references indexed in Scilit:
- Transmembrane topology, genes, and biogenesis of the mitochondrial phosphate and oxoglutarate carriersJournal of Bioenergetics and Biomembranes, 1993
- The mitochondrial transport protein superfamilyJournal of Bioenergetics and Biomembranes, 1993
- Solute carriers involved in energy transfer of mitochondria form a homologous protein familyFEBS Letters, 1987
- Purification of the active mitochondrial phosphate carrier by affinity chromatography with an organomercurial agarose columnFEBS Letters, 1982
- The mitochondrial phosphate carrier has an essential requirement for cardiolipinFEBS Letters, 1982
- Isolation and reconstruction of the phosphate‐transport system from pig heart mitochondriaFEBS Letters, 1981
- Enhancement of reconstituted ADP,ATP exchange activity by phosphatidylethanolamine and by anionic phospholipidsFEBS Letters, 1980
- Isolation of the uncoupling protein from brown adipose tissue mitochondriaFEBS Letters, 1980
- Reconstitution of adenine nucleotide transport with purified ADP, ATP‐carrier proteinFEBS Letters, 1977
- Purification of the carboxy‐atractylate binding protein from mitochondriaFEBS Letters, 1975