Purification of peroxisomal acyl-CoA: dihydroxyacetonephosphate acyltransferase from human placenta.
The peroxisomal enzyme acyl-CoA:dihydroxyacetonephosphate acyltransferase (DHAPAT) was extracted from human placental membranes using CHAPS as a detergent in the presence of 1 M KCl. Prior to assay dipalmitoylphosphatidylcholine was added to the sample as eluted from the various columns in order to stabilize the protein for subsequent enzyme activity measurements at 37 degrees C. The enzyme was purified from the placental membrane using ocytl-Sepharose CL-4B chromatography, Hydroxyapatite HTP chromatography, CM-Sepharose CL-6B, PBE 94 chromatofocusing and TSK G3000 SW size exclusion chromatography. A final purification of more than 8000-fold with respect to the placental membranes was achieved with a final yield of about 5%. Upon chromatofocusing the peak of activity eluted at a pH of 5.1-5.3 indicating a low isoelectric point. A native M(r) of 60-80 kDa was calculated from HPLC size exclusion chromatography. SDS-PAGE of the final purified fraction showed one major band with a M(r) of 65 kDa. These results suggest that DHAPAT is a monomeric protein. A polyclonal antiserum raised against the purified fraction was prepared in rabbits. Immunoprecipitation experiments showed complete precipitation of DHAPAT activity in fractions prepared from human placenta, liver and skin fibroblasts. Immunoprecipitation was also used to determine the residual amount of DHAPAT protein in liver from a patient with the Zellweger syndrome. A value of about 10% was found, which closely corresponds to the residual amount of enzyme activity.
Ofman
R
R
Department of Clinical Biochemistry, University Hospital Amsterdam, AMC, The Netherlands.
Wanders
R J
RJ
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000494:pharmacology; D000217:Acyltransferases / Q000172:deficiency / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis; D008830:Microbodies / Q000201:enzymology*; D010920:Placenta / Q000201:enzymology*; D011817:Rabbits; D013379:Substrate Specificity
D007106:Immune Sera; D015060:1,2-Dipalmitoylphosphatidylcholine; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase