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Purification of the active mitochondrial phosphate carrier by affinity chromatography with an organomercurial agarose column
Author(s) -
de Pinto V.,
Tommasino M.,
Palmieri F.,
Kadenbach B.
Publication year - 1982
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(82)81252-0
Subject(s) - chemistry , agarose , chromatography
The general procedure for the isolation of the phosphate carrier from mitochondria involves solubilization with non-ionic detergents and chromatography on hydroxylapatite [l-3]. With high resolution SDS-gel electrophoresis this preparation can be separated into 4-5 protein bands [2]. Further purification, in particular removal of the ADP/ATP-carrier, was obtained by chromatography on Celite [2], on Mersalyl-Ultrogel [3,4] and by using Triton X-l 14 instead of Triton X-100 [5]. However, after all these procedures the purified phosphate carrier fraction still contained 4-5 protein bands in the &.-region of 30 000-35 000 [5]. Affi-Gel 501 (an organomercurial agarose gel), Dowex AG l-X8 and hydroxylapatite (Bio-Gel HTP) were obtained from Bio-Rad; [32P]phosphate (carrier free) from Amersham; [3H]NEM from New England Nuclear; acrylamide, N,N’methylenebisacrylamide, SDS, Triton X-100 and NEM from Serva; scintillation liquid (Maxifluor) from J. Baker; L-cY-phosphatidylcholine (from egg yolk, Type X-E) from Sigma and cardiolipin from Serdary.