Sialyltransferases substrates

It is important to consider the nature of the desialylation experiment in view of the purity of sialidase preparations. The use of highly purified sialidase can provide additional information related to complex carbohydrate structure and the sialidase specificity while effecting desialylation. This approach also generates sialic acid-free complex carbohydrates for use as e.g. sialyltransferase substrates, or, in biological systems, desialylated cell-surface glycoconjugates. Cruder preparations frequently contain proteinase and other glycosidase activities which will also degrade the material. Furthermore, consideration of the buffer system must include those ions necessary for enzyme activity e.g. V. cholerae sialidase requires approx. 4mM Ca for activity). 

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-... 

Substrate-specificity studies on microsomal, frog-liver sialyltrans-ferase revealed the presence of (2—>3) and (2—>6) activities.277 This enzyme system readily sialylates oligosaccharides, but is almost inactive with asialofetuin, which is in contrast to the sialylation of oligosaccharides, as well as asialofetuin, by rat-liver sialyltransferase.278 The conclusion from this observation is that acceptor specificity of sialyl-transferases isolated from liver of evolutionary distant animals is similar for substrates of low molecular weight, but differs for compounds of high molecular weight.279... 

CMP Neu5Ac /f-D-Galp-( I - 4)-t>-GlcpNAc- -(2 —>-6)-sialyltransferase.4 Equimolecular amounts of substrate and coenzyme were used. After these couplings, the recovered enzyme preparation retained full activity. 

A large-scale enzymic synthesis of the trisaccharide a-D-Neu5Ac-(2—>3)-j8-D-Gal-( 1 — 3)-d-G1cNAc (65), the tumor-associated carbohydrate antigen CA 50, has been achieved.113 This is a further illustration of the cross reactivity and efficiency of STB, This sialyltransferase, like STA, was partially purified from porcine liver, according to a modification of Conradt s procedure. 114 The initial rate measured for j8-d-Gal-( 1 - 3)-d-G1cNAc at saturating concentration was 18% of the one measured for the real substrate / -d-... 

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.

It is interesting that all four reactions studied gave the same apparent Km values for CMP-NeuNAc. This observation suggests that the CMP-NeuNAc binding sites of the different species of sialyltransferases are similar in structure and affinity properties, although the binding sites for the acceptors and the catalytic sites may be very different, providing a basis for substrate specificities. 

Another multifunctional bacterial enzyme that has been reported recently is Pasteurella multocida sialyltransferase (PmSTl or tPm0188Ph) (67, 68). It has four different activities, including an a2,3SiaT, a2,6SiaT, a2,3-sialidase, and trans-sialidase activities. PmSTl is a highly active sialyltransferase with broad donor and acceptor substrate specificity therefore it is a powerful tool... 

---