Purification and functional characterisation of the pyruvate (monocarboxylate) carrier from baker's yeast mitochondria (Saccharomyces cerevisiae).
Isolated yeast mitochondria were subjected to solubilization by Triton X-114 and the detergent extract was subsequently chromatrographed on dry hydroxyapatite. Purification of the yeast monocarboxylate (pyruvate) carrier was achieved by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate, as described previously for bovine heart mitochondria (Bolli, R., Nałecz K.A. and Azzi, A. (1989) J. Biol. Chem. 264 18024-18030). The final preparation contained two polypeptides of apparent molecular mass 26 and 50 kDa. The yeast carrier appeared to be less abundant, but more active, than the analogous protein from higher eukaryotes. The carrier was able to catalyse the pyruvate / pyruvate and pyruvate / acetoacetate exchange reactions, both reactions being sensitive to cyanocinnamate and its derivatives, to phenylpyruvate and to mersalyl and p-chloromercuribenzoate. In the pyruvate / acetoacetate exchange reaction (200 mM internal acetoacetate, enzymatic assay), the Km value for external pyruvate was found to be 0.8 mM and the Vmax 135 mumol/min per mg protein. Among other substrates of the yeast carrier, all transported with similar affinity and identical maximal velocity against acetoacetate, we identified 2-oxoisocaproate, 2-oxoisovalerate and 2-oxo-3-methylvalerate. Lactate was not translocated by this carrier with a measurable rate, neither were di- or tricarboxylates.
Nałecz
M J
MJ
Institute of Biochemistry and Molecular Biology, University of Berne, Switzerland.
Nałecz
K A
KA
Azzi
A
A
D000818:Animals; D027321:Anion Transport Proteins; D001345:Autoradiography; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D027501:Monocarboxylic Acid Transporters; D009206:Myocardium / Q000737:chemistry; D017830:Octoxynol; D011092:Polyethylene Glycols; D011773:Pyruvates / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D003902:Detergents; C573715:FMP37 protein, S cerevisiae; D026901:Membrane Transport Proteins; D027501:Monocarboxylic Acid Transporters; D011773:Pyruvates; D029701:Saccharomyces cerevisiae Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40