Sialyltransferases purification

In the earlier studies, complete purification of sialyltransferases had not been achieved, but this did not preclude the possibility of the presence of more than one sialyltransferase in an enzyme preparation, and it prevented detailed study of the molecular properties of these enzymes. This is why the results from the former studies on the specificity of substrates must be interpreted with care. Exact studies of si-... 

Our next objective was to solubilize and purify the sialyltransferases. We followed the procedure established (11) for the purification of fucosyltransferase. The details are outlined in Figure 5. 

Melkerson-Watson LJ, Sweeley CC. Purification to apparent homogeneity by immunoaffinity chromatography and partial characterization of the GM3 gangUoside-forming enzyme, CMP-sialic acid lactosylceramide alpha 2,3-sialyltransferase (SAT-1), from rat liver Golgi. J. Biol. Chem. 1991 266 4448-4457. 

Preuss U, Gu X, Gu T, Yu RK. Purification and characterization of CMP-N-acetylneuraminic acid lactosylceramide (alpha 2-3) sialyltransferase (GM3-synthase) from rat brain. J. Biol. Chem. 1993 268 26273-26278. 

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. 

Halcomb and Chappell developed a route to CMP-NeuAc 88 that promises to be general for the synthesis of virtually any derivative thereof [44,45]. The route (Scheme 36) utilizes a condensation of sialic acid derivative 99 with the phosphoramidite 112 to afford the phosphite 113 in 62% yield. Oxidation of the phosphite provided the phosphotriester 114 [46], which was taken directly to the next transformation without purification (owing to its instability to chromatography). Deal-lylation of the phosphate gave compound 115 (61% for two steps), which was stable to silica gel chromatography. Compound 115 was deacylated with methoxide, and its methyl ester was subsequently saponified with NaOH to provide CMP-NeuAc 88. The derivatives shown in Scheme 37 were synthesized according to this protocol and were investigated as substrates for sialyltransferases (see below). 

Liepkans, V., Alain, J., and Garan, L., 1988, Purification and characterization of a CMP-sialic LeOse4Cer sialyltransferase from human colorectal carcinoma cell membranes. Biochemistry 27 8683-8688. 

Paulson, J. C., Beranek, W. E., and Hill, R. L., 1977a, Purification of a sialyltransferase from bovine colostrum by affinity chromatography on CDP-agarose, J. Biol. Chem. 252 2356-2362. 

Sadler, J. E., Rearick, J. I., Paulson, J. C., and Hild, R. L., 1979, Purification and characterization of two sialyltransferase activities from porcine submamillary glands, in Glycoconjugate Research Proceedings of the Fourth International Symposium on Glycoconjugates, Vol. II (J. D. Gregory and R. W. Jeanloz, eds.), Academic Press, New York, pp. 763-766. 

Weinstein, J., de Souza-E-Silva, U., and Paulson, J. C., 1982, Purification of Galpl-4GlcNAca2-6 sialyltransferase and a Galpl-3(4)GlcNAca2-3 sialyltransferase to homogeneity from rat liver, J. Biol. Chem. 257 13835-13844. 

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