Ether lipid synthesis: Purification and identification of alkyl dihydroxyacetone phosphate synthase from guinea-pig liver

Zomer, Anna W.M.; Weerd, Wim F.C. De; Langeveld, J.P.M.; Bosch, H. van den

doi:10.1016/0005-2760(93)90070-p

Cited by 44 publications

(29 citation statements)

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“…High enzymatic activities of 3.1 and 2.3 milliunits/mg were found in homogenates and supernatants, respectively, of IPTG-induced cells transformed with a vector carrying the synthase insert. For comparison, the specific activity of a guinea pig liver homogenate under these conditions amounts to 0.03 milliunits/mg protein (12). The specific synthase activity of 0.06 milliunits/mg in inclusion bodies suggests that the enzyme is not easily accessible for substrate in these precipitates or that it is present in a less active form.…”

Section: Resultsmentioning

confidence: 99%

“…The latter may hold for the expressed enzyme in general. When the amount of the 65-kDa protein in homogenate and supernatant was estimated by visual inspection of staining intensities against known amounts of bovine serum albumin, the soluble alkyl-DHAP synthase was estimated to have a specific activity amounting to about 15% of that previously determined for the enzyme purified from guinea pig liver (12). This lower value may be caused by the presence of the His-tag in the expressed enzyme or may result from less efficient folding in E. coli.…”

Section: Resultsmentioning

confidence: 99%

“…Enzyme Purification-Purification of alkyl-DHAP synthase from guinea pig liver was done as described before, with some changes (12). Rather than with a Triton X-100 gradient from 0.075 to 0.4% (w/v) in buffer C (50 mM Tris-HCl (pH 7.4), 20% (v/v) ethylene glycol, 0.5 M NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1 mM dithiothreitol), the protein was eluted batchwise with 0.4% (w/v) Triton X-100 in buffer C from the concanavalin A column.…”

Section: Methodsmentioning

confidence: 99%

“…The alkyl-DHAP synthase activity assay was performed as described previously (12). Protein was determined according to Bradford (18) with bovine serum albumin as the standard.…”

Section: ) (Cc(a/g)aa(t/c)tg(a/g)aa(t/c)tg(t/c)tg(a/g)-tt)mentioning

confidence: 99%

“…Partial purifications of alkyl-DHAP synthase from Ehrlich ascites cells (10) and from guinea pig liver (11) have been described, but no information on molecular weight was reported. Recently, we succeeded in the purification of alkyl-DHAP synthase from guinea pig liver to homogeneity and provided evidence that the enzyme consisted of a single polypeptide chain with a molecular mass of 65 kDa (12). Both N-terminal and internal amino acid sequences have now been obtained from this protein, and we here report on the cloning and expression of the cDNA …”

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confidence: 99%

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Polymerase Chain Reaction-based Cloning of Alkyl-dihydroxyacetonephosphate Synthase Complementary DNA from Guinea Pig Liver

Vet

Zomer

Lahaut

et al. 1997

Journal of Biological Chemistry

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Peroxisomes are indispensable organelles for ether lipid biosynthesis in mammalian tissues, and the deficiency of these organelles in a number of peroxisomal disorders leads to deficiencies in ether phospholipids. We have previously purified the committed enzyme for ether lipid biosynthesis, i.e. alkyl-dihydroxyacetonephosphate synthase, to homogeneity. We have now determined the N-terminal amino acid sequence, as well as additional internal sequences obtained after cyanogen bromide cleavage of the enzyme. With primers directed against the N-terminal sequence and against a cyanogen bromide fragment sequence, a 1100-bp cDNA fragment was obtained by conventional polymerase chain reaction using first-strand cDNA from guinea pig liver as a template. The 5 and 3 ends of the cDNA were obtained by rapid amplification of cDNA ends. The open reading frame encodes a protein of 658 amino acids, containing the N-terminal amino acid sequence as well as the cyanogen bromide cleavage fragment sequences. The derived amino acid sequence includes a mature protein 600 amino acids long and a presequence 58 amino acids long. The latter contains a stretch of amino acids known as peroxisomal targeting signal 2. The size of the mRNA was estimated to be around 4200 nucleotides. Recombinant His-tagged alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli was enzymatically active.Ether phospholipids constitute a special class of natural phospholipids. In mammals, these have either an alkyl or an alkenyl ether linkage at the sn-1 position and an acyl ester linkage at the sn-2 position of the glycerol backbone. Little is known about the specific functions of ether phospholipids. The only representative for which biological roles have clearly been established is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, better known by its trivial name, platelet activating factor (1). Other possible functions of ether phospholipids may include the protection of cells against certain types of oxidative stress by plasmalogens (2, 3).The biosynthesis of ether phospholipids requires the concerted action of two enzymes and starts with the acylation of dihydroxyacetonephosphate (DHAP) 1 by the enzyme DHAP acyltransferase (EC 2.3.1.42). The ether linkage is then introduced by a second enzyme, alkyl-DHAP synthase (EC 2.5.1.26), that catalyzes the exchange of the acyl chain in acyl-DHAP for a long chain fatty alcohol. Both enzymes are mainly, if not exclusively, located in peroxisomes (4).The importance of peroxisomes for human physiology was emphasized by the discovery of inherited diseases in humans caused by the loss of one or more peroxisomal functions (5). The prototypic Zellweger syndrome is a severe disorder characterized by a general loss of peroxisomal functioning, including impaired ether lipid synthesis, defective peroxisomal ␤-oxidation, and defective phytanic acid oxidation. A disorder with only a limited loss of peroxisomal functions is rhizomelic chondrodysplasia punctata, in which phytanic acid oxidase, DHAP acyltransferase, a...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The alkyl-DHAP synthase activity assay was performed as described previously (12). Protein was determined according to Bradford (18) with bovine serum albumin as the standard.…”

Section: ) (Cc(a/g)aa(t/c)tg(a/g)aa(t/c)tg(t/c)tg(a/g)-tt)mentioning

confidence: 99%

mentioning

confidence: 99%

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