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.

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Glucosylation of mannose containing dolichyl diphosphate oligosaccharide.

A maximum rate of dolichyl phosphate [14C]glucose synthesis from 55-day embryos was achieved at 16 nM concentration of exogenous dolichyl phosphate and exceeded about 3 times that without addition of dolichyl phosphate. The highest values of [14C]glucose incorporation from UDP-[14C]glucose into dolichyl phosphate [14C]glucose, dolichyl diphosphate [14C]Glc-oligosaccharides and protein were reached at 5 min time point of incubation of liver microsomes both from embryos and sows. The radioactive incorporation into proteins was about 7-fold higher in liver microsomes from sows compared to that from embryos, probably due to the greater content of acceptor proteins in microsomes from sows. The enzymatic transfer of Glc3-oligosaccharide from a lipid carrier to endogenous protein acceptor in microsomes from pig embryonic and adult livers was considerably faster than the removal of glucose residues during the initial stages of processing of protein-bound oligosaccharides. One labelled compound was discovered in the CHCl3-CH3OH-H2O (1:1:0.3, by vol) extract after incubation of liver microsomes from embryos and sows with UDP-[14C]glucose. On the basis of its mobility on the chromatogram it appears to be GlcNAc2Man9Glc3.

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 lipid-linked oligosaccharides in embryonic liver. Formation of mannose containing derivatives.

In the presence of exogenous dolichyl phosphate mannosyl transferase activity towards dolichyl phosphate was nearly 3-fold higher in microsomes from pig embryonic liver compared to that from adult liver. After incubation of microsomes from embryonic liver with UDP-N-acetylglucosamine and GDP-[14C]mannose lipid-linked tri- to undecasaccharides were discovered in CHCl3-CH3OH (2:1, v/v) and CHCl3-CH3OH-H2O (1:1:0.3, by vol) extracts. The main proportion of the radioactivity was incorporated into penta-, sexta and undecasaccharides. Amphomycin at concentration 500 micrograms/ml inhibited almost completely dolichyl phosphate mannose synthesis in embryonic liver microsomes without inhibition the formation of lipid-linked penta- and sextasaccharides. It was suggested that mannose transferred to lipid-linked tetra- to heptasaccharides comes from GDP-mannose but not from dolichyl phosphate mannose.

Biosynthesis of lipid-linked oligosaccharides by microsomes from virus induced Hepatoma MC-29. Dependence of some glycosyl transferases on dolichyl phosphates of different chain length.

Dolichyl phosphates of various chain length ranging from 7 to 22 isoprene units were tested as lipid acceptors in transglycosylation reactions in chicken liver and Hepatoma MC-29. In the presence of exogenous dolichyl phosphate mixture (18 and 19 isoprene units) the synthesis of dolichyl pyrophosphate N-acetylglucosamine and dolichyl phosphate mannose increased 3 times both in the liver and Hepatoma MC-29, while the formation of dolichyl phosphate glucose was 4 fold higher in the liver and 6-fold higher in Hepatoma MC-29. In liver microsomes the maximum rate of the stimulation of glycosylation was achieved by exogenous dolichyl phosphates, containing 18 and 19 isoprene units, while glycosyl transferases in microsomes from Hepatoma MC-29 did not show any structural requirements to the chain length of dolichyl phosphates.

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.

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Formation of dolichyl pyrophosphate N-acetylglucosaminyl intermediates.

Liver microsomes from pig embryos synthesized dolichyl pyrophosphate N-acetylglucosamine and converted it to dolichyl pyrophosphate N,N'-diacetylchitobiose. N-acetylglucosaminyl transferase activity towards dolichol was about 2-fold greater in microsomes from embryonic liver than in microsomes from adult liver. A maximum level of conversion of dolichyl pyrophosphate N-acetylglucosamine to dolichyl pyrophosphate N,N'-diacetylchitobiose was achieved at 5 mM concentration of unlabelled UDP-N-acetylglucosamine, while this conversion was negligible at lower UDP-N-acetylglucosamine concentrations (0.1 and 0.5 mM). The level of dolichyl phosphate, assessed by the level of dolichyl pyrophosphate N-acetylglucosamine synthesis was 2-fold higher in microsomes from embryonic liver than that in microsomes from adult liver. Tunicamycin (1 microgram/ml) inhibited completely the formation of dolichyl pyrophosphate N-acetyl-glucosamine in embryonic liver microsomes, while the inhibitory effect of UMP (1 mM) was about 70%.

[Effect of ischemia on content of acid-soluble nucleotides in rat liver]. / Vliianie ishemii na soderzhanie kislotorastvorimykh nukleotidov v pecheni krys

A drop in the acid-soluble nucleotide concentrations was studied in the rat liver following different periods of ishemia as well as the resynthesis of these compounds 2 and 24 h after re-establishment of blood circulation. After 5 min ishemia sharp drop was found in the amount of nucleoside triphosphates, UDP-glucose and UDP-glucuronic acid. After 30 to 60 min ischemia the available quantities of acid-soluble nucleotides remained unchanged, then a tendency to a subsequent slight reduction in their amount was observed. All acid-soluble nucleotides the quantity of which dropped noticeably during ischemia were resynthesized to a great extent 2 hours after re-establishment of blood circulation in 30- and 60-min ischemic livers; the synthesizing ability of the 120-min ischemic liver cells was considerably weaker than that of 30 and 60-min ischemic cells, particularly in the later periods after cessation of ischemia.