Purification and partial characterization of CMP-Neu5Ac synthetase from rat brain |
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Authors: | J.C. Feo-Manga,L.B. Rodrí guez-Aparicio,M.A. Ferrero,A. Reglero |
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Affiliation: | Department of Biochemistry, Molecular Biology and Analytical Chemistry, University of León, 24071 León, Spain |
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Abstract: | N-Acetyl-neuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase), which catalyzes the formation of cytidine-5′-monophospho-N-acetyl-neuraminic acid (CMP-Neu5Ac) from cytidine-5′-triphosphate (CTP) and N-acetyl-neuraminic acid (Neu5Ac), was purified from rat brains aged 8-9 days, which presented the highest specific activity, and partially characterized. Partial protein fractionation in the crude extract was achieved by using 40-60% ammonium sulphate. Subsequently, CMP-Neu5Ac synthetase was purified by column chromatography on Sephacryl S-200 (gel filtration), Yellow-86-Agarose (affinity) and Phenyl-Sepharose (hydrophobic affinity). The pure enzyme had a specific activity of 3.6555 U/mg of protein and was purified 1662-fold, with an 18% yield. The purified CMP-Neu5Ac synthetase had a molecular weight of about 46 ± 1 kDa. Its purity was confirmed by sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC). The active enzyme chromatographed on a gel filtration column at 190 kDa, suggesting it exists in its native form as a tetramer. The greatest activity of enzyme was observed a temperature of 40 °C for a period of 45 min of incubation, revealing a certain thermal stability. The enzyme was found to remain stable in the pH range 8.5-9.5 at 40 °C, specifically at pH 9.0 for a 45 min incubation period. The enzyme was blocked by thiol-modifying reagents and such heavy metal cations as Mn2+, Cu2+, Sn2+, Co2+, Zn2+ and Hg2+, but was not inhibited by thiol-containing reagents like reduced glutathione (GSH), mercaptoethanol and cysteine. Finally, in the presence of 0.01 M of dithiothreitol (DTT) or 0.06 M of NaF, the enzyme showed activity losses of approximately 20 and 17%, respectively. |
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Keywords: | CMP-sialic acid synthetase Rat brain Enzyme purification pH stability Thermal stability |
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