In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor.
Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme involved in the biosynthesis of ether phospholipids, is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2. The human alkyl-dihydroxyacetonephosphate synthase precursor produced in vitro or expressed in Escherichia coli could be processed to a lower molecular weight protein by incubation at 37 degrees C with a guinea pig liver fraction, enriched in mitochondria, lysosomes, and peroxisomes. This lower molecular weight protein was identified as the mature human alkyl-dihydroxyacetonephosphate synthase by radiosequencing, indicating that the processing protease is present in this organellar fraction. Characterization of the processing protease indicated that it is a cysteine protease with a pH optimum of 6.5. Furthermore, it was demonstrated that exogenously added pre-alkyl-dihydroxyacetonephosphate synthase was imported and processed in purified peroxisomes in vitro. Processing of alkyl-dihydroxyacetonephosphate synthase did not increase the activity of the enzyme. This indicates that the presence of the presequence does not affect the activity of the enzyme.
Institute for Biomembranes, Utrecht University, Utrecht, The Netherlands.
J
J
Biermann
H
H
van den Bosch
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase; D003546:Cysteine Endopeptidases