Dolichyl phosphate is known to act as a lipophilic cofactor in mediating the transfer of oligosaccharides to proteins. It has been suggested that the levels of this molecule may be limiting in the glycosylation of nascent polypeptide chains. De novo biosynthesis of dolichyl phosphate may modulate its cellular concentration. This research will focus on the biosynthesis of dolichyl phosphate in two tumor cell lines: Ehrlich ascites tumor cells and cultured mouse L1210 cells. It is proposed that the purification of the key enzyme of dolichyl phosphate biosynthesis in Ehrlich tumor cells, the long-chain prenyl transferase, be undertaken. The enzyme will be purified by conventional means and characterized physically. Cofactor and substrate requirements, as well as the kinetic mechanism, will also be investigated. In addition, the subcellular location and transmembrane topology will be investigated. The induction of long-chain prenyl transferase activity in mouse L1210 cells will be attempted by two means: (1) growing cells at different cell densities; and (2) causing a deficiency in dolichyl phosphate levels by means of the antibiotic compactin, which specifically blocks an early enzyme in the biosynthetic pathway. By correlating changes in enzyme levels with dolichyl phosphate levels, several types of questions may be answered: (1) Is the long-chain prenyl transferase rate-limiting in the synthesis of dolichyl phosphate? (2) What is the rate of decline of dolichyl phosphate levels when de novo synthesis is blocked? and (3) What is the effect of a dolichyl phosphate deficiency on membrane glycoprotein biosynthesis? (B)
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