Abstract— The incorporation of NeuNAc from CMP‐NeuNAc into endogenous glycolipids and glyco‐proteins, and exogenously added GM1a (monosialoganglioside) and desialylated fetuin (DS‐fetuin) was studied with particulate preparations from 11 to 15 day old rat cerebra. The apparent +K++m values of the enzyme systems for the different substrates, assayed with 0.5 mg enzyme protein, were: CMP‐NeuNAc, 0.13 mm (same with endogenous and exogenous glycolipid and glycoprotein substrates); GM1a, 0.20 mm; DS‐fetuin, 0.15 mm (or 1.2 mm in terms of acceptor sites). The activities, expressed as nmoles NeuNAc incorporated per 0.5 mg enzyme protein per 30 min incubation at 37°C and pH 6.3, were 0.094, 0.039, 0.17 and 0.64 with the endogenous glycolipids, endogenous glycoproteins, exogenous GM1a and exogenous DS‐fetuin, respectively. Incorporation into endogenous glycolipids was mainly in GM3, while exogenously added GM1a was converted to GD1a. Incorporation into endogenous glycoproteins yields about 20 sialoglycopolypeptides on SDS‐polyacrylamide gel electrophoresis. Neura‐minidase pretreatment of the particulate enzyme preparation decreased sialylation of the higher molecule weight polypeptides but increased sialylation of the lower molecule weight species. The sialyltransferase activity with the endogenous glycolipid substrates was more heat resistant than the activities with exogenous GM1a. Since more than 60% of the endogenous glycolipid activity was due to the conversion of lactosylceramide to GM3, the sialyltransferase responsible for this reaction appears to be different from the one that acts on GM1a. This was supported by the observation that exogenously added GM1a did not diminish the incorporation of NeuNAc into endogenous lactosylceramide. These two glycolipid sialyltransferase activities were distinguishable from the glycoprotein sialyltransferase activity since exogenous DS‐fetuin did not compete with either the endogenous or the exogenous glycolipids for CMP‐NeuNAc.

Abstract— Total rat cerebral homogenate, with nuclei removed, yielded sialyltransferase activity peaks that were distinct from the protein distribution profile in a continuous sucrose density gradient. Marker enzyme studies and electron microscopic examinations on the gradient fractions suggested that most of the sialyltransferase activities were not associated with the synaptosomes. The sialyltransferases appeared to be localized in the smooth microsomal membranes and the Golgi complex derivatives. The sialyltransferase activities were stimulated by non‐ionic detergent mixture, Triton CF‐54/Tween 80 (2/1, w/w), the effect being much more pronounced with exogenous substrates. The stimulatory effect was dependent on detergent concentration. With 1 mg detergent mixture per mg enzyme protein, the percent increases in enzyme activities with the different substrates were: endogenous glycolipids, 100; endogenous glycoproteins, 50; exogenous GM1a, 700; exogenous DS‐fetuin, 230. The action of the nonionic detergents appears to be on a hydrophobic segment of the enzyme molecule, bearing the active site, which is buried in the membrane lipid bilayer. This was substantiated by the partial trypsin resistance of the sialyltransferase activities and the abolition of that resistance when trypsiniza‐tion was performed in the presence of nonionic detergents. Furthermore, the sialyltransferase activities were markedly inhibited by organic solvents; and these inhibitory effects were inversely proportional to the solvent dielectric constants.

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