Publications

• Purification of peroxisomal acyl-CoA: dihydroxyacetonephosphate acyltransferase from human placenta.

  Ofman R., Wanders R.J.

  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 t ... >> More

  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. << Less

  Biochim Biophys Acta 1206:27-34(1994) [PubMed] [EuropePMC]

• Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes.

  Biermann J., Just W.W., Wanders R.J., Van Den Bosch H.

  Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by ... >> More

  Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-acyltransferase in a heterotrimeric complex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex. << Less

  Eur J Biochem 261:492-499(1999) [PubMed] [EuropePMC]

• The native molecular size of alkyl-dihydroxyacetonephosphate synthase and dihydroxyacetonephosphate acyltransferase.

  Biermann J., Schoonderwoerd K., Hom M.L., Luthjens L.H., Van den Bosch H.

  Dihydroxyacetonephosphate acyltransferase (DHAP-acyltransferase) and alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) are the first two enzymes involved in the biosynthesis of ether phospholipids. Both peroxisomal enzymes have recently been purified to homogeneity and their molecular ... >> More

  Dihydroxyacetonephosphate acyltransferase (DHAP-acyltransferase) and alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) are the first two enzymes involved in the biosynthesis of ether phospholipids. Both peroxisomal enzymes have recently been purified to homogeneity and their molecular weights under denaturing conditions were reported. To determine the in situ functional size of both enzymes, radiation inactivation experiments were performed. Alkyl-DHAP synthase showed single exponential decays, both when enzymatic activity and when immunoreactive protein levels were measured, from which target sizes of 79+/-2 kDa and 78+/-4 kDa, respectively, were calculated. DHAP-acyltransferase activity increased at lower doses and decayed upon further irradiation with an apparent target size of 62+/-7 kDa. We conclude from these data that the functional unit sizes for both enzymes in situ are represented by their single polypeptide chains. << Less

  Biochim Biophys Acta 1393:137-142(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Role of dihydroxyacetonephosphate acyltransferase in the biosynthesis of plasmalogens and nonether glycerolipids.

  Liu D., Nagan N., Just W.W., Rodemer C., Thai T.P., Zoeller R.A.

  The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese ham ... >> More

  The variant CHO-K1 cell line, NRel-4, is unable to synthesize plasmalogens because of a severe reduction in dihydroxyacetonephosphate acyltransferase (DHAPAT) activity (Nagan, N., A. K. Hajra, L. K. Larkins, P. Lazarow, P. E. Purdue, W. B. Rizzo, and R. A. Zoeller. 1998. Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol. Biochem. J. 332: 273-279). Northern analysis demonstrated that the loss of this activity was attributable to a severe reduction in mRNA levels for DHAPAT. Transfection of NRel-4 cells with a plasmid bearing the human DHAPAT cDNA recovered DHAPAT activity and plasmalogen biosynthesis. Examination of clonal isolates from the transfected population showed that recovery of as little as 10% of wild-type DHAPAT activity restored plasmalogen levels to 55% of normal, whereas in one isolate, NRel-4.15, which overexpressed DHAPAT activity by 6-fold over wild-type cells, plasmalogen levels were returned only to wild-type values. Although the rate of plasmenylethanolamine biosynthesis was restored in NRel-4.15, the biosynthesis of nonether glycerolipids was either decreased or unaffected, suggesting that peroxisomal DHAPAT does not normally contribute to nonether glycerolipid biosynthesis. These data demonstrate that a defect in the gene that codes for peroxisomal DHAPAT is the primary lesion in the NRel-4 cell line and that the peroxisomal DHAPAT is essential for the biosynthesis of plasmalogens in animal cells. << Less

  J. Lipid Res. 46:727-735(2005) [PubMed] [EuropePMC]