Immunological Localization and Tissue Distribution of Alkyldihydroxyacetonephosphate Synthase and Deficiency of the Enzyme in Peroxisomal Disorders

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Abstract
Alkyldihydroxyacetonephosphate synthase (alkylglycerone‐phosphate synthase) is a peroxisomal enzyme involved in ether phospholipid biosynthesis. The recent cloning of the cDNA encoding this enzyme from guinea pig liver enabled the raising of specific antisera against this enzyme. Both a synthetic peptide corresponding to a predicted epitope and a recombinant protein expressed in Escherichia coli were used for that purpose. Using western blot techniques, the solubilization of the enzyme from the peroxisomal membrane by Triton X‐100 in the presence of salt was confirmed. Neutral hydroxylamine treatment of peroxisomes resulted in almost no release of the protein from the membrane. The complete polypeptide chain of the enzyme was resistant to proteolysis by trypsin when intact peroxisomes were studied. Carbonate treatment released alkyldihydroxyacetonephosphate synthase from the membrane indicating that the enzyme is not an integral membrane protein. This idea is in accord with the absence of a clear hydrophobic transmembrane domain in the deduced amino acid sequence of the enzyme. Alkyldihydroxyacetonephosphate synthase, as well as its mRNA, could be detected in all five guinea pig tissues examined. When using the antiserum against guinea pig recombinant alkyldihydroxyacetonephosphate synthase, a cross‐reactive protein was detected in a human liver homogenate that runs at a slightly higher molecular mass. The absence of this band in liver of Zellweger syndrome and Rhizomelic chondrodysplasia punctata patients provides strong evidence that it represents the human homolog of this enzyme.