Multi-enzyme kinetic analysis of glycolipid biosynthesis.
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.
Bieberich
E
E
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA 23298-0614, USA. ebiberi@hsc.vcu.edu
Yu
R K
RK
D000818:Animals; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002460:Cell Line; D005732:Gangliosides / Q000096:biosynthesis; D006017:Glycolipids / Q000096:biosynthesis*; D016695:Glycosyltransferases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008722:Methods; D051379:Mice; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism; D051381:Rats; D012160:Retina / Q000378:metabolism; D012799:Sialyltransferases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D005732:Gangliosides; D006017:Glycolipids; D016695:Glycosyltransferases; D017350:N-Acetylgalactosaminyltransferases; D012799:Sialyltransferases; C043662:alpha-N-acetylneuraminate alpha-2,8-sialyltransferase