Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool

Journal Article · Tue Dec 05 00:00:00 EST 1989 · Journal of Biological Chemistry; (USA)

OSTI ID:5031698

Muchmore, E A; Milewski, M; Varki, A; Diaz, S ^[1]

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1. San Diego Veterans Administration Medical Center, CA (USA)

N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor (6-3H)N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc.

• OSTI ID: 5031698
• Journal Information: Journal of Biological Chemistry; (USA), Vol. 264:34; ISSN 0021-9258
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
ANTIGENS
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HYDROXYLASES
ENZYME ACTIVITY
AMINES
BIOLOGICAL PATHWAYS
CARBON ISOTOPES
ISOTOPE DILUTION
MICE
SIALIC ACID
TRITIUM COMPOUNDS
TUMOR CELLS
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ISOTOPE APPLICATIONS
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MAMMALS
MONOSACCHARIDES
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OXIDOREDUCTASES
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VERTEBRATES
550201* - Biochemistry- Tracer Techniques