Abstract
Acetyl-coenzyme A: 13-sophorosyloxydocosanoic acid (Glc2HDA) acetyltransferase was purified 14-fold in low yield from Candida bogoriensis cells. The enzyme catalyzes acetylation of the 6' and 6" positions of the sophorosyl group, producing the 13-[2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyloxy]-docosanoic acid 6',6"-diacetate (Ac2Glc2HDA) and monoacetate (AcGlc2HDA) in a product ratio of 5:1. Neither the purification steps nor heat denaturation studies indicated separation of the first and second acetylation steps. The acetyltransferase has a molecular weight of about 500,000 as determined by gel filtration on a Sepharose 4-B column. It shows a pH optimum range from 7 to 9, is strongly inhibited by 1 mM concentrations of the sulfhydryl reagents N-ethylmaleimide, p-hydroxymercuribenzoate, and 5,5'-dithiobis(2-nitrobenzoic acid), but only partly inhibited by 10 mM iodoacetamide. It has an apparent Km of 30 muM for acetyl-CoA, utilizes propionyl-CoA at 45% the rate of acetyl-CoA, and utilizes longer chain acyl-CoA derivatives much less efficiently. The critical micelle concentrations of the C. bogoriensis glycolipids in pH 7.7 phosphate buffer were estimated by pinacyanol chloride binding as follows: Glc2HDA, 50 mum; AcGlc2HDA, 30 muM; Ac2Glc2HDA, 12 muM. The Stokes radius of Ac2Glc2HDA micelles was 22 A as estimated by gel filtration on Bio-Gel P-150. Glc2HDA was a much better acceptor than its methyl ester in the acetyltransferase assay. A plateau in the Glc2HDA saturation curve at 50 muM and a corresponding break in the reciprocal plot at this concentration indicate the enzyme utilizes the monomeric form of this lipid as substrate.
Highlights
In the paper (12) we report the isolation, purification, and partial characterization of an acetylesterase from C. bogoriensis which could be involved in this later metabolism of Ac,Glc,HDA
Activity is expressed as acetate incorporated into ether-soluble form, isolated, and assayed as described under “Experimental
Summary
Radioactive samples were anlayzed in 10 ml of a dioxane scintillator solution as previously described (4). Radioactive areas on thin-layer plates were determined by scanning with a Varian. In some cases radioactive samples were scraped from thin layer plates, suspended in scintillator solution with Cab-0-Sil, and assayed in the scintillation spectrometer. Lipids on thin layer plates were visualized either by exposure to iodine vapor or by spraying with a 3% phosphomolybdic acid solution in ethanol, followed by heating at 100” for 10 min. Protein was determined by the method of Lowry et al (5) using bovine serum albumin as a standard. Glucose was determined by the phenolsulfuric method (6)
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