The biosynthesis of glycogen from UDPG 5 with a livcr enzyme has been reported previously ( l ). The reaction has been investigatcd further using a partially purified preparation form rat muscle with which the •• general properties of thc system have been studied. l\ f ETHODS AnalyticnlCl ycoge n wa s es timated by lhe phe.;ol-sulfuric acid method (2), after digest.i.D.Jl with KOH and ethlanol precipilat ion (3) . A sampJ.e of glywgrn prepared as described by Somogyi (4) wa s used a:; standard. lts co ncentral ion was checked against gl ucose using the k l nthrone method (5). U DP was estima ted as describcd by Cabib and L eloir (6) , but h alvwg the amounts o[ reagcnls. UDPG W\lS measured spectrophotometrica ll y with a partially purifiecl UDPG dehydrogenase (7). Phosphory lase was cstimratecl as dt scribed by Cori el al. (8). Protein was measurcd by the m ethods of Kunitz a ncl \1 cDonalcl (9) and of W arb urg ancl Christian ( lO). Amylase ac tivity was d e term ined under the ~a m e co nditions as the glycogen-formil.g enzyme but withoul U DPG or G-6-P. After clepn:!c inization with Ba (OH) , a nd ZnSO,, lh c reclucing substances were lltC'ast wcd according to Park and Johnson (11). G-6-P was cstim a ted spectrophotometricall y (12) . Radioacti,•ity was meas urecl with a gas-Elow co unter. Radioactive sugars in paper chrolll'atogra ms were locatecl wilh a silver reagent (13) and th en eluted, platee! , ancl co unted. App roximately halE oE thc added counts werc dctcctecl afte r this treatment.

Incubation of GDP-['4C]mannose with liver microsomes and magnesium ions led to the formation of dolichol monophosphate mannose and of other acidlabile compounds that contain oligosaccharides. Formation of these compounds was enhanced by addition of an acceptor lipid in the same fractions of DEAE-cellulose chromatography where bound dolichol is found. Alkaline treatment of the oligosaccharides, obtained by acid methanolysis, followed by paper electrophoresis, gave rise to the formation of two positively charged substances believed to be formed by deacetylation of hexosamine residues. Incubation of the oligosaccharide-containing compounds with liver microsomes and manganese ions resulted in a transfer to endogenous protein. The role of dolichol derivatives in glycoprotein synthesis is discussed.Incubation of liver microsomes with UDP-Glc and dolichol-P leads to the formation of dolichol-P-Glc and a substance believed to be dolichol-PP-oligosaccharide (1, 2). This substance, which has been referred to as glucosylated endogenous acceptor, can also be obtained by direct transfer of glucose from dolichol-P-Glc and acts as donor in oligosaccharide transfer to protein (3).The structure of the oligosaccharide portion of glucosylated endogenous acceptor is not known, but studies with the substance labeled in the glucose moiety have given some information. Measurements of molecular weight by ion exclusion gave values of 3500, and the rate of migration on paper chromatography corresponds to that of a maltooligosaccharide of about 17 units (4). The oligosaccharide, therefore, has around 20 monosaccharide units.When the oligosaccharide prepared by acid methanolysis was subjected to electrophoresis, it behaved as an uncharged substance and on treatment with alkali, it gave rise to two positively charged substances (5). These became neutral after treatment with acetic anhydride under conditions that led to N-acetylation. From these facts it was tentatively concluded that the oligosaccharide of glucosylated endogenous acceptor contains two hexosamine residues.In experiments where UDP-['4C]GlcNAc was used as donor and dolichol-P as acceptor, it was found that substances that appear to be dolichol-PP-GlcNAc (6) and dolichol-PP-N,N'-diacetylchitobiose are formed (7). The transfer of both the sugar residue and phosphate from UDP-GlcNAc to a lipid had been described by Molnar et al. (8).These findings can be related to the fact that two N-acetylglucosamine residues are present in the inner portion of the oligosaccharide of many glycoproteins (9). In these, an N,N'-diacetylchitobiose residue is linked to the amide N of asparagine and chains of mannose residues are linked to it.In experiments with GDP-Man as donor, lipid-bound mannose has been detected (10-12). Its formation was enhanced by addition of dolichol-P (6, 13), and the structure of the product has been unambiguously proved to be dolichol-PMIan by Hemming's group (14).In previous experiments it was found that, besides dolichol-P-Man, other substances are formed which,...

It has been reported that liver microsomes catalyze the transfer of glucose from uridine diphosphate glucose to dolichol monophosphate so as to produce dolichol monophosphate glucose. Dolichol is a polyprenol containing about 20 isoprene units. The glucosyl residue of dolichol monophosphate glucose is transferred to an endogenous acceptor on further incubation with liver microsomes. The glucosylated endogenous acceptor appears to be an oligosaccharide of about 20 monosaccharide units bound to dolichol through a phosphate or pyrophosphate bridge. In this paper it is reported that liver microsomes catalyze the transfer of the oligosaccharide from the glucosylated endogenous acceptor to an endogenous protein. This transfer reaction requires the presence of bivalent cations,-manganese being more effective than magnesium. The presence of deoxycholate is also required. Besides the glycoprotein, several watersoluble products are also formed. Preliminary evidence indicates that they are glucose, oligosaccharides of different size, and possibly oligosaccbarides bound to amino acids.In the first paper on the formation of dolichol monophosphate glucose (dolichol-P-Glc) it was reported that the glucosyl residue of this substance was transferred to an endogenous acceptor on further incubation with liver microsomes (1). The product thus formed was believed to be a glycoprotein because of its insolubility in certain organic solvents and in trichloroacetic acid. However, further work (2, 3) showed that most of the radioactivity transferred from glucoselabeled dolichol-P-Glc under the conditions described was recovered in an amphipatic substance, the hydrophilic residue of which appears to be an' oligosaccharide of about 20 monosaccharide units. The lipophilic residue had some properties in common with dolichol-P, and evidence was obtained indicating that the bridge between the lipid moiety and the oligosaccharide is a pyrophosphate. The substance was referred to as glucosylated endogenous acceptor (Glc-acceptor). On the basis of present evidence its structure can be written as dolichol-P-P-glycoseg-Glc. Dolichol (4) is a polyprenol containing about 20 isoprene units and glycosel9 stands for an oligosaccharide of about 19 unidentified monosaccharide residues. It should be emphasized that this is a tentative formula for the Glc-acceptor. Several aspects of its structure have not been fully clarified, especially the number and identity of the monosaccharide residues and the number of phosphates in the bond between the lipophilic and hydrophilic moieties.In this paper we report that under appropriate conditions, liver microsomes catalyze the transfer of the hydrophilic moiety of the Glc-acceptor to an endogenous protein. The reactions of glucose transfer under study can be schematically written as follows:UDP-Glc -dolichol-P-Glc dolichol-P-P-glycoseis-Glc protein-glycoseig-Glc MATERIALS AND METHODSRadioactive Glc-acceptor labeled in the glucose residue was obtained directly from UDP-Glc as described (5).Enzymes. Papain 2 X ...

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