Cell surface glycoproteins have been shown to undergo qualitative and quantitative changes during cellular transformation and during the growth phases of cells in culture. They have been implicated as being involved in cellular adhesion, cellular migration and aggregation, and in cell-cell communication. During the coming year, we will continue our studies on the biosynthesis of cell surface and soluble glycoproteins. Our research involves the solubilization, purification, and characterization of enzymes that catalyze the incorporation of sugars into the carbohydrate side chains of glycoproteins. We will also continue to isolate, purify, and characterize oligosaccharide-lipids that are intermediates in the biosynthetic pathway. The oligosaccharide-lipids will be isolated in sufficient quantities for use as acceptors during studies on the glycosyltransferases. Our major emphasis, however, will be to continue studies on the reconstitution of the purified enzymes with phospholipids. Results obtained during the last year demonstrate that at least two of the enzymes in the biosynthetic pathway, mannosyltransferase II and dolichol-p-mannose synthetase, are optimally active in the presence of nonbilayer phospholipid phases. We have also shown that dolichol induces phase changes in membranes composed of phosphatidylethanolamine and phosphatidylcholine. Our goal is to determine whether other enzymes in the pathway require nonbilayer phospholipid phases for activity and if these unique phases are involved in vitro in glycoprotein biosynthesis. (A)

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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University of Alabama Birmingham
School of Medicine & Dentistry
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Schutzbach, John S (2006) Assay of dolichyl-phospho-mannose synthase reconstituted in a lipid matrix. Methods Mol Biol 347:31-41
Schutzbach, J S; Zimmerman, J W (1992) Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids. Biochem Cell Biol 70:460-5
Schutzbach, J S; Forsee, W T (1990) Calcium ion activation of rabbit liver alpha 1,2-mannosidase. J Biol Chem 265:2546-9
Schutzbach, J S; Jensen, J W (1989) Bilayer membrane destabilization induced by dolichylphosphate. Chem Phys Lipids 51:213-8
Forsee, W T; Palmer, C F; Schutzbach, J S (1989) Purification and characterization of an alpha-1,2-mannosidase involved in processing asparagine-linked oligosaccharides. J Biol Chem 264:3869-76
Jensen, J W; Schutzbach, J S (1989) Phospholipase-induced modulation of dolichyl-phosphomannose synthase activity. Biochemistry 28:851-5
Jensen, J W; Schutzbach, J S (1988) Modulation of dolichyl-phosphomannose synthase activity by changes in the lipid environment of the enzyme. Biochemistry 27:6315-20
Monti, J A; Christian, S T; Schutzbach, J S (1987) Effects of dolichol on membrane permeability. Biochim Biophys Acta 905:133-42
Lai, C S; Schutzbach, J S (1986) Localization of dolichols in phospholipid membranes. An ESR spin label study. FEBS Lett 203:153-6
Jensen, J W; Schutzbach, J S (1985) Activation of dolichyl-phospho-mannose synthase by phospholipids. Eur J Biochem 153:41-8