Sialyltransferases soluble

Sialyltransferases can be solubilized from their subcellular site by using detergents, and be purified by affinity chromatography on, for example, CDP-6-aminohexanol-agarose,267 as already described. Solubilization of frog- and rat-liver sialyltransferases by means of Triton X-100 has been described.27 Soluble sialyltransferase occurs in colostrum, and is also present in small quantities in normal blood-serum. From the latter source, the enzyme was purified 300-fold by poly (acrylamide) gel-electrophoresis.2 ... 

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. 

Our group has reported a novel multifunctional bacterial enzyme, Pasteurella multocida sialyltransferase (PmSTl). PmSTl is an easy to express extremely soluble, and highly active sialyltransferase. Routinely, about 100 mg of PmSTl can be purified from one liter E. coli cell culture. PmSTl has four different activities including a2,3-sialyltransferase (optimal activity at pH 7.5-9.0), a2,6-sialyltransferase (optimal activity at pH 4.5-7.0), a2,3-sialidase (optimal activity at pH 5.0-5.5), and trans-sialidase (optimal activity at pH 5.5-6.5) activities. Among them, a2,3-sialyltransferase activity is the major function of the PmSTl with a specific activity of 60 U/mg protein (24),... 

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. 

At present little detailed evidence is available for sialyltransferases specifically involved in membrane glycoprotein biosynthesis. It is probable that the membrane-bound sialyltransferase specificities described for glycoproteins (sections III. 3 and III.4) and glycolipids (section III. 5) will be similar, as the structures of membrane complex carbohydrates show the same features found in soluble molecules (e.g. Kobata 1979, Rauvala and Finne 1979, Montreuil 1980). Much indirect evidence has come from cell culture studies, frequently in studies on normal and transformed or malignant cells (Hughes 1976, Harmon 1978, Kottgen et al. 1979). 

Soluble sialyltransferase activity has often been measured in the serum of tumour patients and significant elevation detected (Bernacki and Kim 1977, Ganzinger 1977, Henderson and Kessel 1977, Coombes et al 1978, Ip and Dao... 

Weinstein J, Lee EU, McEntee K, Lai PH, Paulson JC. Primary structure of P-galactoside o2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor. 7. Biol. Chem. 1987 262 17735-17743. 

Bartholomew and Jourdian (1966) and Bartholomew et al. (1973) first reported on this sialyltransferase(s), found in goat, bovine, and human colostrum. This enzyme was different in three principal ways from the previously reported mammary gland enzyme. First, in colostrum the enzyme was soluble and thus could be purified. Second, this sialyltransferase would transfer sialic acid to high-molecular-weight... 

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