Copurification of dihydroxyacetone-phosphate acyl-transferase and other peroxisomal proteins from liver of fenofibrate-treated rats.
Dihydroxyacetone-phosphate acyl-transferase (DHAP-AT), a peroxisomal membrane-bound enzyme that catalyzes the first step of ether-glycerolipid synthesis, was purified from liver of rats treated with fenofibrate, a peroxisome proliferator. The protocol first included isolation of peroxisomes, their purification through a discontinuous gradient and solubilization of membranes in CHAPS. DHAP-AT was further purified by four chromatographic steps, namely low-pressure size-exclusion, cation-exchange, hydroxylapatite and chromatofocusing. The chromatofocusing step led to a 4000-fold increase in the specific activity of DHAP-AT with respect to the liver homogenate with a yield of about 0.2%. Trypsin digestion of a 64-kDa protein band upon SDS-PAGE resulted in a peptide sequence unknown in databases. A corresponding degenerated oligonucleotide was used as a probe in Northern blotting, and a transcript of 3.3 kb was detected in some rat tissues. Moreover, the overall procedure allowed co-purification of four major peroxisomal enzymes: urate-oxidase, catalase, multifunctional enzyme and palmitoyl-CoA oxidase, respectively.
Causeret
C
C
Laboratoire de Biologie Moléculaire et Cellulaire, Faculté des Sciences Mirande, Université de Bourgogne, Dijon, France.
Bentejac
M
M
Albet
S
S
Teubner
B
B
Bugaut
M
M
D000217:Acyltransferases / Q000187:drug effects / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D011345:Fenofibrate / Q000494:pharmacology*; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008565:Membrane Proteins / Q000187:drug effects* / Q000302:isolation & purification*; D008830:Microbodies / Q000737:chemistry / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D017421:Sequence Analysis; D012995:Solubility
D008565:Membrane Proteins; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase; D011345:Fenofibrate