TY - JOUR
T1 - Multi-enzyme kinetic analysis of glycolipid biosynthesis
AU - Bieberich, Erhard
AU - Yu, Robert K.
N1 - Funding Information:
We thank Dr. Darrell L. Peterson for his helpful comments during the preparation of this manuscript. This study was supported by USPHS Grant NS 11853-24 (to Dr. R.K. Yu) and an A.D. Williams grant (to Dr. E. Bieberich).
PY - 1999/6/15
Y1 - 1999/6/15
N2 - 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 V(max)/K(m) 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, V(max)/K(m) 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 V(max)/K(m) 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 α2-8-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. Copyright (C) 1999 Elsevier Science B.V.
AB - 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 V(max)/K(m) 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, V(max)/K(m) 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 V(max)/K(m) 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 α2-8-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. Copyright (C) 1999 Elsevier Science B.V.
KW - Enzyme kinetic
KW - Ganglioside
KW - Glycolipid
KW - Glycosyltransferase
KW - Metabolic control
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U2 - 10.1016/S0167-4838(99)00085-0
DO - 10.1016/S0167-4838(99)00085-0
M3 - Article
C2 - 10366734
AN - SCOPUS:0033040971
SN - 0167-4838
VL - 1432
SP - 113
EP - 124
JO - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
JF - Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology
IS - 1
ER -