AI Article Synopsis
- N-Glycoloylneuraminic acid (Neu5Gc) is produced by the enzyme CMP-N-acetylneuraminic acid hydroxylase, which has been highly purified from pig submandibular glands through a complex process, achieving significant purification with low yield.
- It is identified as a monomer with a molecular mass of approximately 65 kDa and is activated by FeSO4 while being inhibited by various iron-binding reagents.
- The enzyme's activity was studied with various substrates and the resulting antibodies from immunization of rabbits showed inhibition of its enzymatic function and a strong reaction with the hydroxylase in immunoblot assays.
Article Abstract
N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-glycoside by the action of CMP-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase. This enzyme is a soluble cytochrome b5-dependent monooxygenase and has been purified to apparent homogeneity from pig submandibular glands by precipitation with N-cetyl-N,N,N-trimethylammonium bromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose, Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12. This procedure resulted in an 8960-fold purification of the hydroxylase with a recovery of 0.8%. The molecular mass of this protein was shown to be 65 kDa on SDS-PAGE and approximately 60 kDa as determined by gel filtration on Superose S.12, which suggests that the enzyme is a monomer. The purified CMP-Neu5Ac hydroxylase is activated by FeSO4 and inhibited by iron-binding reagents such as o-phenanthroline, KCN, Tiron and ferrozine. An apparent Km of 11 microM was determined for the substrate CMP-Neu5Ac using purified hydroxylase in the presence of Triton X-100-solubilized microsomes. In a reconstituted system consisting of purified hydroxylase, cytochrome b5, cytochrome b5 reductase and catalase, an apparent Km of 3 microM was measured. The apparent Km for cytochrome b5 in this system was 0.24 microM. Immunization of a rabbit with enriched and purified hydroxylase led to an antiserum that inhibited CMP-Neu5Ac hydroxylase activity and reacted with the purified 65 kDa protein on a Western blot after SDS-PAGE. Antibodies specific for this 65 kDa protein were isolated and showed a strong reaction with the purified CMP-Neu5Ac hydroxylase from mouse liver after immunoblotting.(ABSTRACT TRUNCATED AT 250 WORDS)
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| http://dx.doi.org/10.1093/glycob/4.5.675 | DOI Listing |
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