Sialyltransferase membranes

Sialyltransferase activity has also been described in plasma membranes from different cells, including blood platelets.285 The function of the enzyme on cell surfaces is unknown, as resialylation of membrane components directly on the cell surface is improbable, owing to the presence of CMP-Neu5Ac hydrolase in the plasma membrane, and the lack of CMP-sialic acids in the extracellular space. Furthermore, evidence has never been obtained for a role of cell-surface gly-cosyltransferases in cellular interaction according to a hypothesis of Roseman s.281... 

A unique, membrane-associated, sialyltransferase system in E. coli has been described that catalyzes the synthesis of sialic acid polymers with the aid of sialyl-monophosphonoundecaprenol.293 In mammalian systems, no evidence was obtained lor the involvement of lipid intermediates in sialic acid transfer, or in the fonnation of sialyl-(2— -8) link-... 

Keenan, T. W. 1974B. Membranes of mammary gland. IX. Concentration of glyco-sphingolipid galactosyl and sialyltransferases in Golgi apparatus from bovine mammary gland. J. Dairy Sci. 57, 187-192. 

Figure 6. Effect of CMP-NeuAc concentration (V/S), of pH (V/pH), of enzymatic protein concentration (V/protein), and of incubation time (V/t) on the activity of synaptosomal membrane-bound sialyltransferase. Calf brain cortex. Acceptor substrates for sialyltransf erase (if) lactosylceramide ( ) desialylated fetuin (%) endogenous glycoprotein (endogenous glycolipids.

Figure 8. Location of gangliosides, sialidase, and sialyltransferase at the nerveending membrane...

Specificity and Membrane Properties of Young Rat Brain Sialyltransferases... 

It was apparent, however, that the rat brain sialyltransferases have not been sufficiently characterized for the postulation of a biological role for the sialylation-desialylation cycle. Consequently, we concentrated our efforts to characterizing the general behaviors of the sialyltransferases in their membrane environments in the rat brain. What follows is a summary of our... 

The basis for the multiplicity of the sialyltransferase activities remains to be elucidated. We plan to purify these enzyme species to homogeneity, using isoelectric focusing columns of smaller pH ranges in conjunction with affinity chromatography which has been successfully used to purify the soluble sialyl-transferases from bovine colostrum (57). Possibility exists that the heterogeneity of sialyltransferase activities as observed is due to differences in polypeptide sequences, carbohydrate content, or non-covalent interactions with other membrane components, and these possibilities can be clarified only with highly purified enzyme preparations. 

A continuous sucrose gradient of the total homogenate from young rat brain and electron microscopic examination of these fractions found most of the sialyltransferase activities to be localized in smooth microsomal membrane and Golgi complex derivatives and not associated with synaptosomes. 

A simplified one-pot two-step enzymatic approach was used by James Paulson s group to produce sialoside derivatives (23). Instead of recycling the CMP-sialic acid, the sialyltransferase donors with modifications at C-5 or C-9 position of NeuSAc were enzymatically synthesized from ManNAc or its C-2 or C-6 modified derivatives, pyruvate, and CTP using a sialic acid aldolase and a CMP-Neu5Ac synthetase/sialyltransferase fusion protein. After removing the protein by membrane filtration, the filtrate containing produced CMP-sialic acid... 

The sialyltransferases are membrane-bound proteins located in the endoplasmic reticulum (ER) and in the Golgi apparatus. Information about their sequence homology is limited, but they do appear to share a common topography [35]. A catalytic domain resides at the C-terminus followed by an N-terminal segment that anchors the enzyme into the ER or Golgi membrane. Soluble, catalytically active sialyltransferases that lack the anchor segment have been isolated from milk, serum, and other body fluids, suggesting that this N-terminal anchor is not necessary for the enzyme to retain catalytic activity. However, the ability to obtain from natural sources quantities of most sialyltransferases that would be needed for synthesis applications is hampered by low tissue concentrations and difficult purifications. 

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. 

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