Gangliosides are
acidic glycosphingolipids synthesized sequentially by a series of
glycosyltransferases acting in parallel biosynthetic pathways. While most
glycosyltransferases are highly specific, some, however, may catalyze equivalent steps in each pathway using different
gangliosides as substrates (e.g. N-acetylgalactosaminyltransferase,
sialyltransferase-IV). A multi-
enzyme kinetic analysis was developed on the condition that serial enzymatic reactions operate below substrate saturation. A multi-
enzyme kinetic analysis enabled a simultaneous calculation of the Vmax/Km value of each
enzyme derived from the equilibrium concentration of the respective substrate. Substrate concentrations [S] were determined by radioactive labelling of
gangliosides in intact cells with the precursor
sugars [14C]
galactose and [14C]
glucosamine, followed by high-performance thin-layer chromatography and autoradiography of the radiolabelled
glycolipids. On the basis of Michaelis-Menten kinetics, Vmax/Km values were derived from [S] by a system of linear equations. The procedure was used to analyze the development of the
glycolipid composition during differentiation of rat gliomaxmurine
neuroblastoma (NG108-15) cells. The Vmax/Km values calculated by multi-
enzyme kinetic analysis were consistent with the kinetic data obtained with solubilized
enzymes. Application of multi-
enzyme kinetic analysis to published data on the correlation of
enzyme activities with
ganglioside levels in various cell lines and tissues indicated the validity of this method for analysis of the
glycolipid biosynthesis, in particular, of its initial steps. On the basis of the kinetic analysis, it is suggested that the cell lines can be divided into two groups with respect to the substrate pools of GM3 used by
sialyltransferase-II and N-acetylgalactosaminyltransferase-I. The first group encompasses the majority of the
neuroblastoma cell lines and the embryonic rat brain where the two
enzymes share a common pool of GM3. In the second group, the two
enzymes do not compete for the same pool of GM3, indicating a different subcellular localization of
CMP-NeuAc:GM3 alpha2-8-sialyltransferase and
UDP-N-acetylgalactosaminyl:GM3 N-acetylgalactosaminyltransferase. In this study, the theory of a multi-
enzyme kinetic analysis is discussed and its application to analysis of the
glycolipid biosynthesis in
neuroblastoma cells is demonstrated. A multi-
enzyme kinetic analysis can be applied to other biosynthetic pathways and provides the advantage of analyzing kinetic data with intact cells or tissue samples.