Journal Article

Purification and Characterization of Diacylglycerol Acyltransferase from the Lipid Body Fraction of an Oleaginous Fungus

Yasushi Kamisaka, Sanjay Mishra and Toro Nakahara

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 121, issue 6, pages 1107-1114
Published in print June 1997 | ISSN: 0021-924X
Published online June 1997 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/oxfordjournals.jbchem.a021702
Purification and Characterization of Diacylglycerol Acyltransferase from the Lipid Body Fraction of an Oleaginous Fungus

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Diacylglycerol acyltransferase (DGAT) [EC 2.3.1.20] was purified to apparent homogeneity from the lipid body fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the lipid body fraction with 0.1% Triton X-100, and purified by subsequent column chromatography on Yellow 86 agarose, Superdex-200, Heparin-Sepharose, second Superdex-200, and second Yellow 86 agarose. The enzyme activity was finally enriched 4,802-fold over that of the starting 1,500 × g supernatant. The apparent molecular mass of the enzyme was 53 kDa on SDS-polyacrylamide gel electrophoresis. The purified enzyme did not exhibit glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, lipase, transacylase, or acyl-CoA hydrolase activities, although 2-monoolein was acylated with about a half of the enzyme activity toward 1,2-diolein. The purified DGAT depended on exogenous sn-1,2-diolein and oleoyl-CoA, with the highest activity at about 200 and 20 μM, respectively. Purified DGAT utilized a broad range of molecular species of both diacylglycerol and acyl-CoA as substrates. The highest activity was observed with sn-1,2-diolein and lauroyl-CoA. Anionic phospholipids such as phosphatidic acid (PA) activated the purified enzyme, as found for the Triton X-100 extract. Sphingosine dose-dependently inhibited DGAT activity activated by PA and basal activity without PA. These results provide a basis for further studies on the molecular mechanism of triacylglycerol biosynthesis and lipid body formation, in which DGAT plays an important role.

Keywords: acyl-CoA; diacylglycerol; diacylglycerol acyltransferase; fungi; phosphatidic acid

Journal Article.  0 words. 

Subjects: Biochemistry

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