Annette Herscovics

Effect of bacitracin on the biosynthesis of dolichol derivatives in calf pancreas microsomes.

Publisher Summary bacitracin is a cyclic peptide antibiotic that forms a complex with polyisoprenyl pyrophosphates in the presence of a divalent cation. In bacteria, the formation of this complex prevents the regeneration of undecaprenyl phosphate, which is essential for the biosynthesis of peptidoglycan in the cell wall. This chapter discusses an experiment in which the effect of bacitracin on the some enzymic reactions was investigated. In the experiment, the calf pancreas microsomes were incubated with labeled nucleotide sugars. The radioactivity incorporated into glycolipids extracted with chloroform–methanol (2:1) (CM extract) and chloroform–methanol–water (10:10:2.5) (CMW extract) and into the precipitate (Ppt) containing glycoproteins was measured. It was found from the experiment that bacitracin inhibited the formation of Dol-PP-GlcNAc and Dol-PP-(GlcNAc) 2 from UDP-N-acetyl-D-[ 14 C]lglucosamine and endogenous dolichyl phosphate and the incorporation of D-[14-4C]mannose into dolichyl pyrophosphate oligosaccharides. It also inhibited the incorporation of N-acetyl-D-[14C] glucosamine and D-[14C] mannose into the precipitate.

Occurrence of a β-D-mannopyranosyl phosphate residue in the polyprenyl mannosyl phosphate formed in calf pancreas microsomes and in human lymphocytes

Polyprenyl mannosyl phosphates, which have been implicated as intermediates in the biosynthesis of glycoproteins [2-53, are formed from GDP-D[14C]mannose in a variety of animal tissues [2131, including calf pancreas microsomes [ 121 and human lymphocyte homogenates [ 131. Although in some cases the polyprenyl mannosyl phosphates were shown to have properties similar to those of synthetic Dol-P-a-Man [3,9,12,13], the configuration of the

Biosynthesis of dolichyl pyrophosphate trisaccharide from synthetic dolichyl pyrophosphate di-N-acetylchitobiose and GDP-D[14C]mannose in calf pancreas microsomes

The biosynthesis of asparagine-linked oligosaccharides in glycoproteins begins with the transfer of an oligosaccharide from dolichyl pyrophosphate to protein [ 1,2]. The major oligosaccharide thus transferred has a composition of GlcsMangGlcNAcs [3,4], and a scheme for its biosynthesis in vivo has been proposed [5]. However, details of the complete enzymic synthesis of the oligosaccharide moiety remain to be elucidated. We have shown that calf pancreas microsomes are very active in the synthesis of dolichyl pyrophosphate oligosaccharides [6,7]. Using synthetic dolichol derivatives as substrates we have characterized the first two enzymic reactions involved in the synthesis of the oligosaccharide moiety [8,9] :

Oxonolytic cleavage of authentic and pancreatic dolichyl mannopyranosyl phosphates: Determination of sugar configuration in the fragments with α- and β-mannosidases☆

Abstract Exposure of authentic dolichyl α-D-[14C]mannopyranosyl phosphate ( I ) or calf pancreas dolichyl [14C]mannopyranosyl phosphate ( II ) to ozone at −70° in pentane followed by treatment with triphenylphosphine gave water-soluble fragments in 65–95% yield. The radioactive products obtained were similar; the major fragment had a mobility on tlc greater than that of mannose but lower than that of citronellyl β-D-mannopyranosyl phosphate. The electrophoretic behavior of the fragments indicated that they possessed intact phosphodiester linkages. α-Mannosidase released [14C]mannose from the fragments of I but not from the fragments of II ; however, the latter were susceptible to β-mannosidase indicating that the pancreatic mannolipid contains a β-linked mannosyl residue.

Glycoprotein biosynthesis in intestinal epithelial cells during differentiation incorporation of [14C]mannose from GDP-[14C]mannose into dolichol derivatives

Epithelial cells of the rat small intestine were collected as a gradient of villus to crypt cells. Homogenates of these cells incubated with GDP-D-[14C]mannose in the presence of MnCl2 incorporated radioactivity into dolichyl mannosyl phosphate and a mixutre of dolichyl pyrophosphate oligosaccharides varying in the size of their oligosaccharide moiety. The labeled oligosaccharides formed in villus cell homogenates appeared shorter than those formed in crypt cell homogenates. The addition of dolichyl phosphate greatly stimulated the synthesis of dolichyl mannosyl phosphate. The initial rate of synthesis of dolichyl mannosyl phosphate from GDP-D-[14C]mannose and exogenous dolichyl phosphate was highest in an intermediate cell fraction having a low specific activity of sucrase and alkaline phosphatase and an intermediate specific activity of thymidine kinase. To compare the rates of dolichyl mannosyl phosphate synthesis in the different cell fractions, it was essential to control degradation of GDP-D-[14]mannose by the addition of AMP to the incubation, since villus cells degraded GDP-D-[14C]mannose much faster than crypt cells.

Biosynthesis of a D-glucosyl polyisoprenyl diphosphate in particulate preparations of Micrococcus lysodeikticus

Particulate fractions of Micrococcus lysodeikticus incubated with UDP-D-[14C]glucose incorporated radioactivity into a chloroform - methanol-soluble, low-mol. wt. compound, and into a polymer. The low-mol. wt. compound consisted of a glucolipid that was extremely labile to mild acid hydrolysis with the formation of D-[14C]glucose, and to mild alkali, yielding 14C-labeled alpha-D-glucopyranose 1,2-phosphate and D-glucose 2-phosphate. The labeled glucolipid was eluted from a DEAE-cellulose column at a salt concentration higher than that required by synthetic ficaprenyl (D-glucopyranosyl phosphate), and it migrated more slowly than the latter compound in t.l.c. Formation of the glucolipid was stimulated by exogenous ficaprenyl phosphate, but not by C55-dolichyl phosphate. These results suggest that the [14C]glucolipid has the characteristic properties of a polyisoprenyl glucosyl diphosphate.