The long term objective of this research program is to develop a thorough understanding of the multi-enzyme systems that are involved in the biosynthesis of the glycosaminoglycan chains of proteoglycans. Since a large number of different enzymes are involved in the assembly and modification of these oligosaccharides, studies must be directed to investigations on the kinetics, properties, and mechanisms of the individual enzymes, with the final aim of reconstituting the complete systems. Recent results indicate that is will be important to characterize the enzymes in a phospholipid matrix and to investigate the properties of the enzyme/phospholipid complexes. The work proposed in this application will therefore involve a comprehensive investigation of a number of the glycosyltransferases catalyzing synthesis of the glycosidic linkages found in glycosaminoglycan chains. The transferases will be solubilized, at least partially purified, and characterized. Investigations will then be directed at understanding how the glycosyltransferases act in concert to direct the synthesis of specific glycosaminoglycan structures that are found on proteoglycans. More specifically, the objectives of the proposed research are: a) To reconstitute purified glycosyltransferases in a phospholipid matrix, b) To establish the optimal parameters of enzyme activity for the reconstituted enzymes, c) To investigate the effects of the phospholipid environment on the activities of the enzymes including specificity for phospholipid in regard to head group and acyl chains, phospholipid phase properties, and membrane surface charge density, d) To identify the nature of the enzyme/phospholipid complexes as to whether they are hydrophobic, ionic, or combination of both, e) To investigate factors that might serve to control or regulate enzyme activities in a phospholipid environment, and f) Since the enzymes under consideration are organized in a membrane environment in vivo, purified enzymes that catalyze sequential reactions in the biosynthetic pathway will be reconstituted in a common phospholipid matrix and the properties of the multi-enzyme """"""""complexes"""""""" will be thoroughly characterized. Results obtained from the proposed research will provide significant new insight about the activities of glycosyltransferses in a phospholipid matrix.
