Involvement of dolichol phosphates as intermediates in the mannosyl and galactosyl transferases of rat testicular germ cell Golgi apparatus membranes.
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.
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
D004288:Dolichol Phosphates; D006017:Glycolipids; D006023:Glycoproteins; D011106:Polyisoprenyl Phosphates; D014531:Uridine Diphosphate Galactose; D006155:Guanosine Diphosphate Mannose; D005700:Galactosyltransferases; D006602:Hexosyltransferases; D008364:Mannosyltransferases