Sialyltransferases assay

NeuAc synthetase route [37]. The relative rates of these C9-modified CMP-NeuAc derivatives shown in Scheme 30 were compared against the natural donor CMP-NeuAc in sialyltransferase assays that utilized enzymes from different sources with their appropriate natural acceptors. The rat liver a-2,6-sialyltransferase tolerated a wide range of functional groups without signihcant decreases in the relative rates. Synthetically useful relative rates were observed for most of the CMP-NeuAc analogs with porcine sialyltransferase and rat liver a-2,3-sialyltransferase. The exception was the 9-amino analog, which was a poor substrate for both enzymes. Overall, these assays demonstrated that a wide variety of modihcations at the C9 position are tolerated by these sialyltransferases. 

The sialyltransferase assay is usually carried out with radioactive CMP-Neu5Ac and the sialic acid acceptor, followed by identification of the radioactive product. As this product is often membrane-bound and the radioactive cosubstrate is expensive, a non-radioactive assay was developed [625], in which CMP released from CMP-Neu5Ac by the transfer of Neu5Ac is determined with HPLC (see section 5.3.2). Since CMP-Neu5Ac can be analyzed in the same run, this test is also suited for the activity of the CMP-Neu5Ac synthase described above. 

A HeLa cells cultured in medium containing 5 mM sodium butyrate and 50 ixCi/mL N-[acetyl-sH]-D-mannosamine after 24 hr, one set of cells harvested and medium replaced with fresh medium with and without 0.5 pg/mL cycloheximide on the other two sets, which were harvested after another 19 hr cells then analyzed for radioactive GM3. B Essentially same as A except the cells were cultured in unlabeled medium and assayed for sialyltransferase activity. C same as B except the cells were assayed for GM3-... 

Kinetic Properties of Sialyltransferases. The sialyl-transferase activities with the endogenous glycoprotein and glycolipid acceptors in the standard assays (15) were linear with time for at least 60 min, while those with the exogenously added GMi and DS-fetuin were linear with time only for about 30 min (Figure 1). Activities were directly proportional to the amount of enzyme added up to 0.75 mg protein/assay (Figure 2). 

Sialyltransferases transfer sialic acid moieties as the terminal residues in oligosaccharide side chains of animal cell coat proteins and gangliosides. In this assay, CMP-7V-acetylneuraminic acid was reacted with asialoglycoprotein to form cytidfne 5 -monophosphate (CMP) and the O-acetylneuraminic acid derivative of the protein. 

The sialyltransferases investigated were found to accept many variations at C5 and C9 of the sialic acid moiety. Halcomb and Chappell confirmed this fact with studies of sialyltransferases from different sources [45]. The C9- and C5-modified substrates in Scheme 37 were good substrates. However no activity was detected when compounds 111 and 117 were assayed. In addition to these examples, other CMP-NeuAc analogs that have been prepared and successfully transferred include C9 fluorescent compounds, C9 thioacetyl, C5 thioacetyl, and C4 dexoy [37]. A particularly useful modification introduced by the Wong lab incorporates a mercury atom at C9 (Scheme 38) [48]. Transfer of this sialic acid to glycoproteins could gready aid in X-ray crystallographic analysis of these biomolecules. 

Rat liver was homogenized and fractionated by the method of Dallner (1963) and fractions were assayed for sialyltransferase, RNA, and protein. Results are expressed as a percentage of the homogenate assay. 

Lactosylceramide CMP-NANA sialyltransferase activity was assayed on whole ceU homogenates as described in Section II. 

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