The long term objective of this research is to develop a thorough understanding of the biosyntheis of the asparagine-linked saccharide side chains of mammalian glycoproteins. Since a large number of different enzymes are involved in the assembly of these oligosaccharides, studies must be directed to investigations on the kinetics, properties, and mechanisms of the individual glycosyltransferases. Recent results indicate that it will also be important to reconstitue some of the enzymes in a phospholipid matrix and investigate the properties of the enzyme/phospholipid complexes. The work proposed will therefor involve a comprehensive investigation of a number of the glycosyltranferases catalyzing synthesis of the glycosidic linkages found in glycoproteins. Specific assays will be developed for the enzymes, and the transferases will be solubilized, at least partialyy purified, and characterized. Investigations will them be directed at understanding how the glycoytransferases act in concert to direct the synthesis of specific oligosaccharide structures that are found on mammalian glycoproteins. In addition, this research will also be directed to studies of the effects of dolichols on the physical properties of membranes. More specifically, the objectives of th proposed research are to: a) Solubilize and purify membrane-bound glycosyltransferases present in microsomes prepared from rabbit liver and other mammalian tissues. b) Characterize the purified glycosyltranferases in regard to mechanism, specificity, and physical glycosyltransferases in regard to mechanism, specificity, and physical and kinetic properties. c) Reconstitue the enzymes in a phospholipid matrix and investigate the effects of the phospholipids enzyme properties. d) Determine the phospholipid specificity and the physical properties of the phospholipid matrix required for optimal enzyme activity, and e) Investigate the effects of dolichols on the properties of membranes including bilayer stability, permeability, and membrane fusion. Although information currently available supports a common pathway for the synthesis of all asparagine-linked oligosaccarides, additinal research is required to fully understand glycoprotein biosynthesis.
