Role of mannosyl lipid intermediate in the synthesis of Neurospora crassa glycoproteins.

Particulate membrane preparations from Neurospora crassa incorporated mannose from GDP-[14C] mannose into endogenous lipid and particulate protein acceptors. Synthesis of the mannosyl lipid is reversible in the presence of GDP. Chemical and chromatographic characterization of the mannosyl lipid suggest that it is a mannosylphosphorylpolyisoprenol. The other endogenous acceptor was precipitated by trichloracetic acid. Gel filtration and electrophoresis studies before and after treatment with proteolytic enzymes indicate that the second acceptor is a glycoprotein(s). beta Elimination studies on the mannosyl protein formed from GDP-[14C] mannose with Mg2+ in the reaction mixture or formed from mannosyl lipid indicate thad with the peptide chain. Several lines of evidence indicate that in Neurospora crassa the mannosyl lipid is an obligatory intermediate in the in vitro mannosylation of the protein. (a) At 15 degrees C the initial formation of the mannosyl lipid is faster than the initial formation of the mannosyl protein. (b) Exogenous partially purified mannosyl lipid can function as a mannosyl donor for the synthesis of the mannosyl protein. This reaction was also dependent on a divalent metal. The rate of this reaction was optimal at a concentration of Triton X-100 which effectively inhibited the transfer of mannose from GDP-[14C] mannose to lipid and protein, indicating that GDP-mannose was not an intermediate in the transfer of mannose from lipid to protein. The mannosyl protein formed in this reaction was indistinguishable by several criteria from the mannosyl protein formed from GDP-[14C] mannose and Mg2+. (c) The effect of a chase with an excess of unlabeled GDP-mannose on the incorporation of mannose into endogenous acceptors was immediate cessation of the synthesis and subsequent turnover of the mannosyl lipid; in contrast, however, incorporation of mannose into protein continued and was proportional to the loss of mannose from the mannosyl lipid.     

Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain alpha-saturated polyprenyl monophosphate

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.     

Dietary effects on the formation of dolichyl monophosphate mannose by microsomal preparations of rat adipose tissue

Microsomal preparations from rat adipose tissue catalyse the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to an endogenous acceptor forming a [¹⁴C]mannosyl lipid. The mannosyl lipid co-chromatographs with hen oviduct dolichyl monophosphate β-mannose on three solvent systems. It is stable to mild alkaline hydrolysis, but strong alkaline treatment yields a compound that co-migrates with mannose 1-phosphate. The mannosyl lipid is labile to mild acid hydrolysis, yielding [¹⁴C]mannose. Formation of the compound is reversible by GDP, but not GMP, and is stimulated by exogenous dolichyl phosphate.

The kinetics of transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose to form dolichyl monophosphate mannose were studied by using preparations derived from rats fed on one of four diets: G (high glucose), L (high lard), F (fructose) or GC (high glucose, 0.9% cholesterol). The K_m and V_max. values for transfer from GDP-mannose were virtually indistinguishable in the four preparations.

In the absence of exogenous dolichyl phosphate, the largest amount of transfer of [¹⁴C]mannose into the mannosyl lipid was observed with preparations from fructose-fed animals. Preparations from glucose-fed animals showed about 60% as much transfer, whereas membranes from rats fed the other diets showed intermediate values between the fructose- and glucose-fed animals. The inclusion of cholesterol in the glucose diet elicited an increase in transfer of mannose.

Under conditions of saturating exogenous dolichyl phosphate, preparations from lard-fed animals have 1.5 times as much enzyme activity as do preparations from animals fed the other three diets.

     

Synthesis of a mannosyl phosphoryl polyprenol by the cellular slime mold Dictyostelium discoideum

A membrane fraction isolated from the cellular slime mold Dictyostelium discoideum was incubated with GDP-[14C]mannose and found to catalyze the incorporation of [14C]mannose into an endogenous acceptor to yield a product with the chemical and chromatographic properties of a polyprenol phosphate sugar derivative. These results suggest that D. discoideum can synthesize a mannosyl phosphoryl polyprenol.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus.

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated. Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [14C]mannose from GDP-[14C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [14C]mannosyl-1-phosphorylundecaprenol. In contrast, this is the major mannolipid synthesized from GDP-[14C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents. Both membrane preparations possessed the ability to catalyse the transfer of [14C]mannose from purified [14C]mannosyl-1-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [14C]mannosyl units from 14C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

Glycosylation in vitro of Semliki-Forest-virus and influenza-virus glycoproteins and its suppression by nucleotide-2-deoxy-hexose

Cell-free enzyme preparations from cultured fibroblasts infected with Semliki forest virus or fowl plague virus (an influenza A virus) incorporate [14C]-mannose into dolichol-phosphate-mannose, lipid-linked oligosaccharides and into endogenous virus-specific glycoproteins. When GDP-2-deoxy-D-[14C]glucose serves as substrate 2-deoxy-D-[14C]glucose is transferred to dolichol phosphate yielding dolichol-monophosphate-2-deoxy-D-[14C]glucose. UDP-2-deoxy-D-[14C]glucose gives rise also to a lipid which, however, is not a polyprenol derivative. The transfer of [14C]mannose to lipid-extractable fractions and glycoproteins in vitro is blocked by GDP-2-deoxy-D-glucose. It can be restored by exogenous dolichol monophosphate only with regard to the formation of dolichol-monophosphate-[14C]mannose-labelled oligosaccharides into glycoproteins. UDP-2-deoxy-D-glucose has no inhibitory effect on transfer reactions of [14C]mannose from GDP-[14C]mannose into various lipid fractions or into glycoprotein. It is concluded therefore, that the inhibition of glycosylation brought about by 2-deoxyglucose in vivo is caused by an interference of its GDP derivative with the formation of a correct lipid-oligosaccharide.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated.Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [¹⁴C]mannosyl-l-phosphorylundecaprenol. In contrast, this is the major manno-lipid synthesized from GDP-[¹⁴C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents.Both membrane preparations possessed the ability to catalyse the transfer of [¹⁴C]mannose from purified [¹⁴C]mannosyl-l-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [¹⁴C]mannosyl units from ¹⁴C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

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.     

Schistosoma mansoni: glycosyl transferase activity and the carbohydrate composition of the tegument.

Homogenates of adult Schistosoma mansoni contain enzymes which transferred [¹⁴C]mannose, [¹⁴C]glucose, and [¹⁴C]galactose from GDP-[U-¹⁴C]mannose, UDP-[U-¹⁴C]glucose, and UDP-[U-¹⁴C]galactose respectively to a lipid acceptor; in comparison, free [¹⁴C]mannose, GDP-[U-¹⁴C]fucose, and UDP-[U-¹⁴C]acetyl-glucosamine were poorly transferred. The lipid acceptor is believed to be an intermediate in the glycosylation of the worm's glycoproteins and in the biosynthesis of oligosaccharides and glycolipids. The tegument of adult worms was isolated by the freeze-thaw procedure and sugars associated with macromolecules in this fraction were analyzed; the major monosaccharide components were glucose, galactose, and mannose. These results suggest that the mechanism of glycosylation of the adult schistosome's tegumental macromolecules may occur through the glycosyl transferase system. The schistosome mannosyl transferase (EC 2.4.1), which is membrane bound was solubilized with 0.1% Triton X-100 without loss of activity; after density gradient centrifugation there was a peak of enzymic activity in a region of density 1.08, which could not be associated with any particular organelle.     

Mannosylation of endogenous and exogenous phosphatidic acid by liver microsomal membranes. Formation of phosphatidylmannose

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.     

Lipid-linked oligosaccharides containing glucose in lactating rabbit mammary gland.

1. Microsomal fractions of lactating rabbit mammary gland incubated with UDP-glucose formed lipid-linked mono- and oligo-saccharides. The lipid-linked monosaccharide had chromatographic properties similar to those of dolichol phosphate mannose and yielded glucose on acid hydrolysis. 2. Incubation of the microsomal fraction with GDP-[U14C]-mannose yielded an oligosaccharide lipid of approximately seven monosaccharide units. Further incubation with UDP-glucose increased the size of the oligosaccharide by approximately two units. 3. Explants of lactating rabbit mammary gland incorporated [U-14C]glucose into both lipid-linked mono- and oligo-saccharides. The oligosaccharide lipid was of approx. 11 monosaccharide units. 4. Considerable redistribution of radioactive label occurred in the explant system, and radioactively labelled glucosamine and mannose, as well as glucose, were detected on acid hydrolysis of the oligosaccharide lipid. 5. Glucose was also detected in the acid hydrolysate of explant proteins. Radioactive glucosamine, galactosamine, galactose and mannose were also found in this fraction.     

Solubilization and properties of mannose and N-acetylglucosamine transferases involved in formation of polyprenyl-sugar intermediates.

A particulate fraction from porcine aorta catalyzed the incorporation of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc into both GlcNAc-pyrophosphorylpolyprenol and GlcNAc-GlcNAc-pyrophosphorylpolyprenol. This transfer utilized endogenous lipid and required a divalent cation. Mn2+ was the best metal ion and was optimum at 2.3 mM. This same particulate fraction was previously shown to transfer mannose from GDP-[14C]mannose to endogenous lipid to form mannosylphosphorylpolyprenol (Chambers, J., and Elbein, A.D. (1975) J. Biol. Chem. 250, 6904-6915). Both the GlcNAc activities and the mannose activity were solubilized by treatment of the particulate fraction with the detergent Nonidet P-40. The enzymes were partially purified by chromatography on DEAE-cellulose and on Sephadex G-200. These soluble enzymes required the addition of acceptor lipid for activity. An acidic lipid fraction, isolated from pig liver and having the properties of dolichyl phosphate, was active with either the GlcNAc or the mannose transferase. Chemically synthesized dolichyl phosphate was also active with either of these enzymes. The products formed from either GlcNAc or mannose by the soluble transferases were similar to those formed by the particulate enzyme. Thus the major product formed from UDP-[3H]GlcNAc was GlcNAc-pyrophosphoryldolichol with small amounts of the disaccharide-lipid while the product formed from GDP-[14C]mannose was mannosylphosphoryldolichol.     

Enzymatic synthesis of mannosyl retinyl phosphate from retinyl phosphate and guanosine diphosphate mannose.

A study was conducted to determine whether retinyl phosphate would act as substrate for the enzymatic synthesis of mannosyl retinyl phosphate. Retinyl phosphate, prepared chemically, supported the growth of vitamin A-deficient rats at the same rate as retinol. It also stimulated the uptake of [14C]mannose from GDP-[14C]mannose into total chloroform-methanol extractable lipid. This reaction occurred in the presence of ATP, Mn2+, detergent (Zonyl A), and a membrane-rich enzyme preparation from the livers of vitamin A-deficient rats, provided that a lipid extract of the membrane preparation of alpha-L-lecithin was also added. Total chloroform-methanol-extractable, labeled mannolipid was separated into two principal labeled mannolipids by thin-layer or column chromatography or by differential solvent extraction. The properties of these mannolipids identified them as glycophospholipids: one was identical with authentic synthetic dolichyl mannosyl phosphate, and the other was concluded to be mannosyl retinyl phosphate because of its incorporation of radioactivity from [3H]retinyl phosphate, its rapid hydrolysis by dilute acid, and the formation of substance that cochromatographed with retinol upon its acid hydrolysis. The presence of ATP or GTP was essential for the stimulation of mannolipid synthesis, probably because of their protective action on the substrates against phosphatases present in the crude enzyme fraction. A pH of 6.0-6.2 favored the formation of dolichyl mannosyl phosphate; a higher pH (6.7-7.0) that of mannosyl retinyl phosphate.     

Synthesis of a mannosyl phosphoryl polyprenol by the cellular slime mold Dictyostelium discoideum

A membrane fraction isolated from the cellular slime mold Dictyostelium discoideum was incubated with GDP-[¹⁴C]mannose and found to catalyze the incorporation of [¹⁴C]mannose into an endogenous acceptor to yield a product with the chemical and chromatographic properties of a polyprenol phosphate sugar derivative. These result suggest that D. discoideum can synthesize a mannosyl phosphoryl polyprenol.     

Role of mannosyl phosphoryl polyisoprenol in biosynthesis of mammary glycoproteins

When a membrane preparation from the lactating bovine mammary gland is incubated with GDP-[¹⁴C] mannose, mannose is incorporated into a [¹⁴C] mannolipid, a [Man-¹⁴C] oligosaccharide-lipid, and metabolically stable endogenous acceptor(s). The rate of mannosyl incorporation is the fastest into [¹⁴C] mannolipid, intermediate in [Man-¹⁴C] oligosaccharide-lipid, and least into [Man-¹⁴C] endogenous acceptor(s). The [¹⁴C] mannolipid has been partially purified and characterized. Mild acid hydrolysis of this compound gives [¹⁴C] mannose, whereas alkaline hydrolysis yielded [¹⁴C] mannose phosphate as the labeled product. The t_½ of hydrolysis of the mannolipid under the acidic and basic conditions are comparable to values obtained for mannosyl phosphoryl dolichol in other systems. The mannolipid is chromatographically indistinguishable from calf brain mannosyl phosphoryl polyisoprenol and chemically synthesized β-mannosyl phosphoryl dolichol. Exogenous dolichol phosphate stimulates the synthesis of mannolipid in mammary particulate preparations 8.5-fold. Synthesis of mannolipid is freely reversible; in the presence of GDP, the transfer of mannosyl moiety from endogenously labeled mannolipid to GDP-mannose is obtained. All of these results indicate that the structure of mannolipid is mannosyl phosphoryl polyisoprenol. Even though the precise chain length of the polyisoprenol portion has not been established, it is tentatively suggested to be dolichol. Partially purified [¹⁴C] mannolipid can directly serve as a mannosyl donor in the synthesis of [Man-¹⁴C] oligosaccharide-lipid and [Man-¹⁴C] endogenous acceptor(s). Pulse and chase kinetics utilizing GDP-mannose to chase the mannosyl transfer from GDP-[¹⁴C] mannose in the mammary membrane incubations caused an immediate and rapid turnover of [¹⁴C] mannose from [¹⁴C] mannolipid while the incorporation of label in [Man-¹⁴C] oligosaccharide-lipid and radioactive endogenous acceptor(s) continued for a short period before coming to a halt. Both gel filtration and electrophoresis indicate that the endogenous acceptor(s) are a mixture of 2 or more glycoproteins since incubation with proteases releases all of the radioactivity into water soluble low-molecular-weight components, perhaps glycopeptides. All of the above evidence is consistent with the following precursor-product relationship: GDP-mannose ? mannosyl phosphoryl polyisoprenol → mannosyl-oligosaccharide-lipid → mannosyl-proteins. The exact structure of the oligosaccharide-lipid and the endogenous glycoproteins is unknown.     

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates

Pea membranes supplied with GDP-[14C]mannose, UDP-N-[14C]acetylglucosamine or UDP-[14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogenous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl-P-dolichol was formed most rapidly in the presence of long-chained dolichyl-P while mannosyl-PP-, glucosyl-PP- and GlcNAc-PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl-PP- and mannosyl-PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc-PP-dolichol was lengthened by addition of a second GlcNAc plus several [14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glycosylated in the presence of UDP-[14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.     

Biosynthesis of the O9 antigen of Escherichia coli. Synthetic glycosyldiphosphomoraprenols as probes for requirement of mannose acceptors

Synthetic monosaccharide derivatives (alpha-glucosyl, beta-glucosyl, alpha-mannosyl) and disaccharide derivatives (alpha-mannosyl-1,2-alpha-glucosyl, alpha-mannosyl-1,3-alpha-glucosyl, alpha-mannosyl-1,4-alpha-glucosyl, alpha-mannosyl-1,6-alpha-glucosyl) of diphosphomoraprenol were used as putative mannose acceptors in the biosynthesis of Escherichia coli O9 antigen. Membranes of E. coli O9 derived from the rfe mutant F 1357 were reconstituted with these compounds and then incubated with different concentrations of GDP-[14C]mannose. Of the monosaccharide derivatives tested, only alpha-glucodiphosphomoraprenol was a mannose acceptor and the only disaccharide derivative which accepted mannose was alpha-mannosyl-1,3-alpha-glucosyldiphosphomoraprenol. The alpha-glucosyl derivative accepted only one mannose unit at 4 microM GDP-[14C]mannose, and above 50 microM GDP-[14C]mannose about 25% of the product had a minimum size of about 30 mannose units. The alpha-mannosyl-1,3-alpha-glucosyl derivative was only a mannose acceptor at a GDP-[14C]mannose concentration of 50 microM and higher, and the product had a minimum size of about 30 mannose units. The results are discussed with respect to requirement of mannose acceptors.     

Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate.

The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.     

Purification and properties of beta-mannosyltransferase that synthesizes Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol

The beta-mannosyltransferase that adds mannose, from GDP-mannose, to GlcNAc-GlcNAc-pyrophosphoryl-dolichol, to form Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol was solubilized from pig aorta microsomal preparations, using 0.5% NP-40, and was purified about 116-fold using conventional methods. The purified enzyme was mostly free of alpha 1,3- or alpha 1,6-mannosyltransferase activities, since Man beta-GlcNAc-GlcNAc-PP-dolichol (PP = pyrophosphoryl) accounted for more than 95% of the product when enzyme was incubated with GDP-[14C]mannose and GlcNAc-GlcNAc-PP-dolichol. Very little Man-beta-GlcNAc-GlcNAc-PP-dolichol was formed when GDP-[14C]mannose was replaced by dolichol-phosphoryl-[14C]mannose, indicating that GDP-mannose was the mannosyl donor. The oligosaccharide portion of this lipid was released by mild acid hydrolysis and was characterized by gel filtration as well as by susceptibility to beta-mannosidase and resistance to alpha-mannosidase. The partially purified enzyme could be stabilized by the addition of 20% glycerol and 0.5 mM dithiothreitol to the buffer, and could be kept in this solution for 5 or 6 days in ice. The enzyme was greatly stimulated by the addition of detergent (NP-40) with optimum activity being observed at 0.1%. However, no stimulation was seen with any phospholipid. The partially purified enzyme had a pH optimum of about 7.0, and showed an almost absolute requirement for Mg2+ with optimal activity occurring at about 5 mM Mg2+. Mn2+ and Ca2+ were only slightly active. The Km for GDP-mannose was about 5 X 10(-7) M and that for GlcNAc-GlcNAc-PP-dolichol about 1 X 10(-6) M. Beta-Mannosyltransferase activity was inhibited competitively by a variety of guanosine nucleotides with GDP and GDP-glucose being most active, but GTP, GMP, guanosine, and periodate-oxidized guanosine were also effective. The enzyme was strongly inhibited by p-chloromercuribenzenesulfonic acid and this inhibition was partially prevented by the addition of dithiothreitol.     

The involvement of endogenous dolichol in the formation of lipid-linked precursors of glycoprotein in rat liver

Endogenous dolichol was shown to function as a natural acceptor of mannose residues by using regenerating rat liver containing [(3)H]dolichol. When subcellular fractions from this liver were incubated with GDP-[(14)C]mannose a double-labelled lipid, which represented 30% of the total [(14)C]mannolipid, could be isolated. This lipid was shown to be identical with the dolichol phosphate mannose formed from exogenous dolichol phosphate, by chromatography, stability to alkali and by chemical cleavage to mannose and dolichol derivatives. It was formed by the rough endoplasmic reticulum and mitochondria. If it is concerned in glycoprotein synthesis this would suggest that it functions in the formation of both secreted and mitochondrial glycoproteins. When both the dolichol and retinol of rat tissue were radioactive they made similar contributions to the synthesis of the lipid by liver microsomal fractions and intestinal epithelial cells.