Properties of mannosyl- and N-acetylglucosamine-1-phosphate transferases towards dolichol phosphate in liver microsomes from pig embryos.

1. The activity of mannosyl- and N-acetylglucosamine-1-phosphate transferases in microsomes from pig embryonic liver was linear to 1 min of incubation at 37 degrees C. 2. The activity of both enzymes was higher in the presence of Mg2+ as compared to Mn2+. A maximal stimulatory effect of Mn2+ was obtained at 2 mM concentration and greater concentrations of it inhibited the activities of both enzymes. 3. The activity of mannosyl transferase was found to be highest after treatment of microsomes with Nonidet P-40 while the activity of N-acetylglucosamine-1-phosphate transferase was greatest in the presence of sodium deoxycholate. 4. The Km for acceptor substrate was 1.6 x 10(-5)M in the reaction for dolichol phosphate mannose synthesis and 2.2 x 10(-5)M in the reaction for dolichol pyrophosphate N-acetylglucosamine formation. 5. The Km for GDP-mannose was 1.4 x 10(-5)M and for UDP-N-acetylglucosamine-6.2 x 10(-5)M. At saturating concentrations of donor substrates V values (pmol/min/mg) were 1330 and 150, respectively.

Conversion of dolichyl pyrophosphate N,N'-diacetylchitobiose to lipid-tri- to heptasaccharides by liver microsomes from hibernating ground squirrels (Citellus citellus L.).

Incubation of liver microsomes from hibernating ground squirrel with GDP-[14C]mannose and exogenous dolichyl phosphate resulted in the synthesis of dolichyl phosphate [14C]mannose. The mannosyltransferase activity was about 3-fold higher in microsomes from hibernating ground squirrels than in those from active animals. Incubation for 30 min of liver microsomes from hibernating animals with dolichyl pyrophosphate N,N'-diacetyl-[14C]chitobiose and GDP-[14C]mannose led to the synthesis of lipid-[14C]trisaccharide. When liver microsomes were incubated with lipid-[14C]trisaccharide and unlabelled GDP-mannose, lipid-tetra- to heptasaccharides were discovered in the chloroform-methanol (2:1) extract. Since, under the experimental conditions, negligible synthesis of dolichyl phosphate mannose was observed, it was assumed that GDP-mannose was a donor of mannose in the conversion of lipid-trisaccharide into lipid-oligosaccharides containing 2-5 mannose residues.

Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intermediates in liver microsomes from hibernating ground squirrels (Citellus citellus L.).

Dolichyl pyrophosphate N-acetyl[14C]glucosamine was synthesized after incubation of liver microsomes from hibernating ground squirrels with UDP-N-acetyl[14C )glucosamine. The radioactivity of glycolipid formed by liver microsomes from hibernating ground squirrels was about 2-fold greater than by liver microsomes from active animals. Addition of exogenous dolichyl phosphate to the incubation mixture increased the formation of dolichyl pyrophosphate N-acetyl[14C]glucosamine by microsomes from both active and hibernating ground squirrels about 6 times. Liver microsomes from hibernating ground squirrels converted dolichyl pyrophosphate N-acetyl[14C]glucosamine into dolichyl pyrophosphate N,N'-diacetyl[14C]chitobiose in the presence of unlabelled UDP-N-acetylglucosamine. This conversion was maximal at 1.0 M concentration of unlabelled UDP-N-acetylglucosamine. The level of dolichyl phosphate assessed by the level of dolichyl pyrophosphate N-acetylglucosamine formation was nearly 2 times greater in liver microsomes from hibernating ground squirrels than from active animals.