Nicolas H. Behrens

The role of dolichol monophosphate in sugar transfer.

Abstract The specificity of the transfer of monosaccharides from sugar nucleotides to dolichol monophosphate catalyzed by liver microsomes was studied. Besides uridine diphosphate glucose, uridine diphosphate-N-acetylglucosamine and guanosine diphosphate mannose were found to act as donors for the formation of the respective dolichol monophosphate sugars. Uridine diphosphate galactose and uridine diphosphate- N -acetylgalactosamine gave negative results. The optimal conditions for transfer from dolichol monophosphate glucose to endogenous acceptor was determined. Studies were carried out on the glucosylation of ceramide by brain extracts and of collagen by skin enzymes in order to find out if dolichol monophosphate glucose is an intermediate in these reactions. The results, while not definite, were not in favor of this assumption.

Mannose transfer to lipid linked di-N-acetylchitobiose

Abstract Mannose was found to be transferred from guanosine diphosphate mannose to dolichol-P-P-di-N-acetylchitobiose when these substrates were incubated with hen oviduct or rat liver microsomes. The oligosaccharide moiety of the product appears to be the trisaccharide β-mannosyl-β-N-acetylglucosaminyl-(1→4)-N-acetylglucosamine as judged by the action of glycosydases. Under certain conditions further transfer of mannose occurs and larger oligosaccharides bound to lipid are formed.

Dolicholmonophosphates: Mannosyl acceptors in a particulate in vitro system of S. cerevisiae.

Undecaprenols have been shown to play a major role in the synthesis of cell wall polysaccharides in bacteria [l-4] . For eucaryotic cells the role of “lipids” as intermediates in the biosynthesis of polysaccharides and possibly glycoproteins has also been established [&lo]. Evidence that in liver a dolichol-monophosphate is identical to the “lipid” component of the intermediate has been presented by Behrens and Leloir [9]. Since yeast cells also contain polyprenols of the dolichol family 11 I] it has been considered possible that the lipid intermediate in yeast mannan biosynthesis described previously [S j is a mannosyldolicholphosphate. To test this hypothesis dolicholphosphates from yeast as well as from liver were tested as possible mannosyl acceptors in the yeast system. It will be shown that both can serve as acceptors for the mannosyl residue of GDP-mannose.

Further studies on a glycolipid formed from dolichyl-d-glucosyl monophosphate

Abstract Incubation of liver microsomes with dolichyl- d -glucosyl- 14 C monophosphate led to the labelling of an endogenous acceptor. This compound seems to be also a dolichol derivative. It contains a high-molecular weight oligosaccharide bound to dolichol through a phosphate or pyrophosphate bond. Various treatments of the labelled oligosaccharide afforded further information on its structure: Reduction with sodium borohydride, followed by acid hydrolysis gave only radioactive d -glucose indicating that the labelled d -glucose is not incorporated at the reducing end of the oligosaccharide. The percentage of radioactivity, liberated as formic acid after periodate oxidation, indicates that more than one molecule of d -glucose is incorporated and that at least one of them is inside the oligosaccharide chain. Alkaline treatment of the otherwise neutral oligosaccharide gave two positively charged derivatives which could be neutralized by N -acetylation, indicating the presence of two hexosamine residues. The oligosaccharides isolated from different tissues by the same method as that used for rat liver, were similar as judged by their migration in paper chromatography and by the pattern of products liberated by acetolysis.

Subcellular distribution of dolichol phosphate.

A crude preparation of liver microsomes was shown to catalyze the transfer of glucose from UDP-Glc to Dol-P** to form Dol-P-Glc [ 1 ] . ~ub~quently it was observed that microsomes also transferred the glucose from Dol-P-Glc to an endogenous acceptor. This latter compound is probably another dolichol derivative containing about 20 monosaccharide units bound to dolichol through a phosphate or pyrophosphate bridge [2,3]. The abbreviation Glc-acceptor will be used for this dolichol derivative until the details of its structure are known with certainty, Liver microsomes also catalyze the transfer of mannose to Dol-P from GDP-mannose and of N-acetyl ~ucos~ine from UDP-GlcNAc to form what seems to be dolichol monophosphate mannose and dolichol monophosphate N-aeetyl glucosamine [4, S] . It is possible that the dolichol derivatives are intermediates involved in the synthesis of the sugar portions of the various glycoproteins and glycolipids occurring in smooth microsomes, the Golgi system and other cellular membranes. It is therefore necessary to establish the subcellular loc~ization of these derivatives.

Glucose transfer from dolichol monophosphate glucose The lipid moiety of the endogenous microsomal acceptor

Abstract Further studies on the structure of the microsomal endogenous substance which accepts labeled glucose from dolichol monophosphate glucose are reported. In order to obtain sufficient amounts of substance for measuring bound dolichol monophosphate, a concentrate was prepared by treating liver with mixtures of chloroform-methanol-water which lead to purification of the radioactive compound. Estimation of dolichol monophosphate, by its accelerating action on dolichol monophosphate glucose formation, showed that it is liberated by mild acid treatment of the concentrate. The dolichol monophosphate-yielding substance behaved like the labeled glucosylated acceptor when tested by its solubility properties in some solvent mixtures, thin-layer and DEAE-cellulose chromatographies and acid or alkaline treatments. Therefore the results provide further evidence that the lipid moiety of the endogenous acceptor of microsomes is dolichol phosphate.

[71] Mannan synthetase from yeast

Publisher Summary This chapter discusses the synthesis of mannan synthetase from yeast. A particulate preparation from disrupted yeast protoplasts catalyzes the incorporation of mannose from galactokinase diphosphate (GDP)-l4C-mannose into a mannose polysaccharide that precipitates with a copper reagent, as does authentic yeast mannan. Two assay methods are described in the chapter. In the first, the reaction mixture is submitted to paper electrophoresis; the radioactive mannan formed remains at the starting line, while the substrate and decomposition products travel toward the anode. In the other, more specific but also much more laborious method, the radioactive polysaccharide is isolated before counting. A blank, prepared by adding GDP-mannose to the reaction mixture just before transfer to the paper, is counted in the same way and subtracted. The alcohol precipitate contains glycogen and other substances besides mannan. The reagents used, procedure followed, and the enzyme preparation is also described in the chapter. All operations are carried out at 0–2°, unless otherwise stated.

Galactose transfer from dolichyl monophosphate galactose in animal tissues.

The involvement of the dolichyl phosphate derivatives of N-acetylglucosamine, mannose and glucose in the biosynthesis of glycoproteins has been well established [l-3]. A similar role for galactose derivatives is not clear. The formation of an acid-labile galactolipid has been reported [4,5] and an increase in galactose incorporation into protein in cell cultures by the addition of dolichyl phosphate has been described [6]. Studies have also been carried out on the biosynthesis of retinol phosphate galactose [7]. This communication describes experiments carried out using Dol-P-Gal prepared with a bacterial system. Incubation of this compound with liver microsomes was found to lead to galactose transfer to an endogenous acceptor having the properties of a dolichyl diphosphate oligosaccharide.

A lipid linked oligosaccharide which contains hexosamine, mannose and glucose.

SummaryPrevious work showed that liver microsomes catalyze the transfer of glucose from dolichol monophosphate glucose to an endogenous acceptor believed to be a dolichol pyrophosphate derivative of an oligosaccharide. This oligosaccharide has now been prepared on a larger scale so as to permit the determination of its sugars. The purification procedure includes, as a last step, a thin layer chromatography on kieselguhr-silica gel to obviate glucose-containing contaminants. After complete hydrolysis mannose, glucose and a small amount of hexosamine were detected.