title: Involvement of dolichol phosphates as intermediates in the mannosyl and galactosyl transferases of rat testicular germ cell Golgi apparatus membranes.
abstract: Isolated Golgi apparatus membranes from the germinal elements (spermatocytes and early spermatids) of rat testis were examined for their ability to incorporate [14C]mannose and [14C]galactose into glycolipid and glycoprotein fractions. Transfer of mannose from GDP-[14C]mannose into a Lipid I fractions (GPD:MPP mannosyl transferase activity), identified as mannosyl phosphoryl dolichol, showed optimal activity at 1.5 mM manganese and at pH 7.5. Low concentrations of Triton X-100 (0.1%) stimulated transferase activity in the presence of exogenous dolichol phosphate (Dol-P); however, inhibition occurred at Triton X-100 concentrations greater than 0.1%. Maximal activity of this GDP:MPP mannosyl transferase occurred at 25 microM Dol-P. Activity using endogenous acceptor was 2.34 pmole/min/mg, whereas in the presence of 25 microM Dol-P the specific activity was 284 pmole/min/mg, a stimulation of 125-fold. Incorporation of mannose into a Lipid II (oligosaccharide pyrophosphoryl dolichol) and a glycoprotein fraction was also examined. In the absence of exogenous Dol-P, rapid incorporation into Lipid I occurred with a subsequent rise in Lipid II and glycoprotein fractions suggesting precursor-product relationships. Addition of exogenous Dol-P to galactosyl transferase assays showed only a minor stimulation, less than twofold, in all fractions. Over the concentration range of 9.4 to 62.5 micrograms/ml Dol-P, only 1% of radioactive product accumulated in the combined lipid fractions. These observations suggest that the mannose transfer involves Dol-P intermediates and also that spermatocyte Golgi membranes may be involved in formation of the oligosaccharide core as well as in terminal glycosylations.
mesh_terms: D000818:Animals; D004288:Dolichol Phosphates / Q000494:pharmacology*; D005700:Galactosyltransferases / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006602:Hexosyltransferases / Q000378:metabolism*; D008297:Male; D008364:Mannosyltransferases / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013087:Spermatids / Q000648:ultrastructure; D013090:Spermatocytes / Q000648:ultrastructure; D013094:Spermatozoa / Q000648:ultrastructure*; D013737:Testis / Q000166:cytology*; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
chemical_list: D004288:Dolichol Phosphates; D006017:Glycolipids; D006023:Glycoproteins; D011106:Polyisoprenyl Phosphates; D014531:Uridine Diphosphate Galactose; D006155:Guanosine Diphosphate Mannose; D005700:Galactosyltransferases; D006602:Hexosyltransferases; D008364:Mannosyltransferases