Factors affecting glucosyl and mannosyl transfer to dolichyl monophosphate by liver cell-free preparations

GDP-mannose and UDP-mannose (each at less than 1 micrometer) markedly inhibit glucosyl transfer from UDP-glucose (1.6 micrometer( to dolichyl phosphate in liver microsomal preparations. The biphasic response suggests the presence of two glucosyl transferases only one of which is inhibited. The inhibition appears to be a property of the intact nucleotide phosphate sugars and not due to competition for a limited pool of dolichyl phosphate. UDP-galactose and UDP-xylose cause a less marked inhibition of the same enzyme. The failure of UDP-glucose to inhibit mannosyl transfer suggests that the pool of dolichol monophosphate used by mannosyl transferase is not available to the glucosyl transferase. The relationship between the degree to which an exogenous prenol phosphate acts as an acceptor of mannose and the degree to which it inhibits mannosylation of endogenous dolichyl monophosphate varies among different prenyl phosphates. Mannosyl transferase exhibits two pH optima.     

The role of vitamin A in the glycosylation reactions of glycoprotein synthesis in an ''in vitro'' system.

Microsomal membrane preparations from rat livers, when incubated with labelled sugar-nucleotides, were shown to synthesize labelled oligosaccharide-lipids in the presence of excess exogenous dolichyl phosphate. Under the incubation conditions defined in the present study, dolichyl pyrophosphoryl(DolPP)GlcNAc2-Man5, DolPPGlcNAc2Man9 and DolPPGlcNAc2Man9Glc3 were the principal oligosaccharide-lipids formed by both control and vitamin A-deficient membranes. However, deficient membranes synthesized 3.2 +/- 0.8 times as much oligosaccharide-lipids and 2.6 +/- 0.7 times as much dolichyl phosphate mannose (DolPMan) and dolichyl phosphate glucose (DolPGlc) as the controls. The transfer of the oligosaccharide chain from the dolichol carrier to the endogenous protein acceptors in vitamin A-deficient microsomes (microsomal fractions) was only 57.5 +/- 9.5% of that of controls. After endo-beta-N-acetylglucosaminidase treatment, only one oligosaccharide species was isolated from both control and vitamin A-deficient microsomal glycoproteins, and was characterized as GlcNAcMan9Glc3. We conclude that the decreased incorporation of labelled mannose and glucose from sugar-nucleotides into the glycoproteins must be due to decreased transfer of GlcNAc2Man9Glc3 from the dolichol carrier to the protein acceptors. This conclusion was further substantiated by the finding that control membranes transferred 4-6 times as much labelled oligosaccharides from exogenously added dolichol-linked substrate (DolPPGlcNAc2Man9Glc3) to endogenous microsomal protein acceptors as compared with the vitamin A-deficient membranes. Attempts to reverse this defect by addition of retinol or retinyl phosphate (a source of retinyl phosphate mannose) to the incubations were unsuccessful.     

Dolichyl phosphate-dependent glycosyltransferases utilize truncated cofactors.

Synthetic truncated dolichyl phosphates of chain lengths from four to thirteen isoprene units (Jaenicke L. and Siegmund H.-U., Chem. Phys. Lipids 51 (1989) 159-170) were assayed for their cofactor activity in the enzymatic transfer of hexoses and hexosamines. The enzymes were microsomal preparations from the green alga Volvox carteri, baker's yeast, and mammalian liver cells. Under saturating conditions, the acceptor activities of the truncated dolichyl phosphates increased from zero to full strength as compared to the mixture of long-chain dolichyl phosphates from natural sources with growing chain length from five to nine isoprene units. Km determinations confirmed the results. Of the geometric isomers of dolichyl 7-phosphate (35 carbon atoms), the 14-trans compound has unchanged acceptor activity; all-trans dolichyl 7-phosphate, however, was almost inactive. The data suggest that hydrophobicity may be an important, but not the only criterion for the binding of the isoprene moiety to the active sites of the transferase enzyme(s) and that the geometry of more than only one double bond in the dolichols is recognized.     

Inhibition of lipid-linked oligosaccharide synthesis by aryl phosphates.

Our previous work has shown that phenyl phosphate acts as an exogenous substrate for GDP-mannose:dolichyl phosphate mannosyltransferase in rat liver microsomal fractions to give rise to phenyl phosphate beta-D-mannose, a compound which, unlike Dol-P-Man (dolichyl phosphate beta-D-mannose), cannot act as mannose donor for further mannose-adding reactions in microsomal fractions. The study has now been extended to the action of various aryl phosphates and structurally related compounds on several other glycosyltransferase systems in the microsomal fractions. (1) Examination of the ability of these compounds to accept sugars from various sugar nucleotides indicated that the individual compounds have specificity as sugar acceptors. Thus phenyl phosphate acted as an effective acceptor for both mannose and glucose, whereas benzenephosphonic acid was active only in accepting mannose. p-Nitrophenyl phosphate was a more effective glucose acceptor than phenyl phosphate, but had only 8% of the mannose-accepting activity of phenyl phosphate. (2) Phenyl phosphate had an inhibitory effect on the transfer of mannose form GDP-mannose to lipid-linked oligosaccharides and to glycoproteins in rat liver microsomal fractions. The inhibition depended on the concentration of phenyl phosphate and on the extent of inhibition of Dol-P-Man synthesis. It is proposed that phenyl phosphate has a direct effect on the synthesis of Dol-P-Man and that its inhibition of synthesis of lipid-linked oligosaccharides and glycoproteins could be a consequence of this effect.     

Voltage-dependent behaviour of dolichyl phosphate-phosphatidylcholine bilayer lipid membranes

The current-voltage steady-state characteristics, cyclic voltammograms and capacitance-voltage steady-state relationships of bilayer lipid membranes made from dioleoylphosphatidylcholine or its mixtures with dolichyl-12 phosphate have been studied. Sustained fluctuations of the capacitance of dolichyl phosphate modified bilayers under applied voltage were observed. The results suggest that the dynamics of dolichyl phosphate molecules in membranes can be regulated by transmembrane electrical potential.     

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.     

Mannosyltransferase activity in calf pancreas microsomes. Formation of 14C-labeled lipid-linked oligosaccharides from GDP-D-[14C]mannose and pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate.

Calf pancreas microsomes incorporated radioactive D-mannose from GDP-D-[14C]mannose into lipid-bound oligosaccharides extracted with chloroform/methanol/water (10/10/2.5, v/v). Several products, which probably differed in the size of the oligosaccharide moiety, were labeled. These could be partially resolved by thin layer chromatography and DEAE-cellulose chromatography. The labeled lipid-bound oligosaccharides were retained on DEAE-cellulose more strongly than synthetic dolichyl alpha-D-[14C]mannopyranosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a neutral 14C-labeled oligosaccharide fraction which was estimated by gel filtration to have a minimum of 8 monosaccharide residues. Hot alkali treatment yielded a mixture of neutral and acidic 14C-labeled oligosaccharides which could be transformed into neutral products by alkaline phosphatase. The D-[14C]mannose residues were alpha-linked at the nonreducing terminus of the oligosaccharides since they could be removed completely with alpha-mannosidase. Most of the D-[14C]mannose-labeled oligosaccharides were retained on concanavalin A Sepharose and eluted with methyl alpha-D-mannopyranoside. Pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate incubated with calf pancreas microsomes in the presence of sodium taurocholate was efficiently utilized as donor of alpha-D-mannosyl residues in lipid-bound oligosaccharides. The products formed from dolichyl beta-D-[14C]mannopyranosyl phosphate were identical with those formed from GDP-D-[14C]mannose, and evidence was obtained to show that the dolichyl beta-D-[14C]mannopyranosyl phosphate was serving as donor without prior conversion to GDP-D-[14C]mannose. Transfer of mannose from dolichyl beta-D-[14C]mannopyranosyl phosphate to lipid-bound oligosaccharides took place at a pH optimum of 7.3, whereas transfer to the precipitate containing glycoproteins was greatest at pH 6.0 in Tris/maleate buffer. The addition of divalent cation was not required, but low concentrations of EDTA were extremely inhibitory. The carbohydrate composition of the lipid-bound oligosaccharides of microsomal membranes was investigated by gas-liquid chromatography and by reduction with sodium borotritide. A heterogeneous mixture of oligosaccharides containing N-acetyl-D-glucosamine, D-mannose, and D-glucose varying in proportions from approximately 1/2.5/0.5 to 1/5/1.5 was obtained with glucosamine at the reducing end. Acid treatment of the lipid-bound oligosaccharide fraction yielded dolichyl pyrophosphate, suggesting that at least some of the oligosaccharides were linked to dolichol through a pyrophosphate group.     

Dolichyl sulphate and H-phosphonate: enzymatic reactions with activated sugars.

Two phosphate-modified analogues of dolichyl phosphate were evaluated as substrates or inhibitors of the reactions catalyzed by mammalian microsomal enzymes. Dolichyl H-phosphonate could serve as an efficient acceptor for mannosyl and glucosyl transfer. The reaction products were chromatographically different from those formed from dolichyl phosphate. Lower activity of the H-phosphonate was observed for the reaction of N-acetylglucosaminyl phosphate transfer from UDP-GlcNAc. Dolichyl sulphate was shown not to serve as a substrate for the transfer of mannosyl (from GDP-Man), glucosyl (from UDP-Glc) or N-acetylglucosaminyl phosphate (from UDP-GlcNAc) residues in the presence of rat liver microsomes. Weak inhibitory properties of this analogue were demonstrated.     

Dolichol pathway in lymphocytes from rat spleen. Influence of the glucosylation on the cleavage of dolichyl diphosphate oligosaccharides into phosphooligosaccharides

Incubation of rat-spleen lymphocytes with UDP-glucose together with GDP-mannose and UDP-N-acetylglucosamine leads to the formation of glucosylated lipid intermediates characterized as dolichyl phosphate glucose and dolichyl diphosphate oligosaccharides. This latter can be either transferred onto endogenous protein acceptors or cleaved into phosphooligosaccharides. The striking fact is that phosphooligosaccharide populations contain far less glucosylated products than the dolichyl diphosphate oligosaccharide ones from which they are derived. Two hypotheses have been investigated: either a rapid action of glucosidases on the liberated phosphooligosaccharides or a preferential splitting of the non-glucosylated population of dolichyl diphosphate oligosaccharides. Addition of p-nitrophenyl-alpha-D-glucoside inhibits glucosidase activities and allows the production of a major population of dolichyl diphosphate oligosaccharides containing three glucose residues. Using these conditions, it is shown that the amount of phosphooligosaccharides generated from the splitting of dolichyl diphosphate oligosaccharides is greatly decreased and that the major part of these remaining phosphooligosaccharides do not contain glucose. These results show that the presence of glucosyl units prevent dolichyl diphosphate oligosaccharides from further degradation into phosphooligosaccharides.     

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.     

Rat liver microsomes catalyse mannosyl transfer from GDP-d-mannose to retinyl phosphate with high efficiency in the absence of detergents

In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose 6-phosphatase activity was latent. The transfer process was linear with time up to 5min and with protein concentration up to 1.5mg/0.2ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15mum giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent K(m) of 18mum. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[(3)H]galactose, UDP- or GDP-[(14)C]glucose, UDP-N-acetyl[(14)C]-glucosamine and UDP-N-acetyl[(14)C]galactosamine, all at 24mum. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the beta-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is about 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl(2) concentrations between 5 and 10mm and could not be detected below 0.6mm-MnCl(2), when synthesis of dolichyl phosphate mannose from endogenous dolichyl phosphate was about 80% of optimal synthesis. Under optimal conditions (5mm-MnCl(2)) endogenous retinyl phosphate mannose represented about 20% of dolichyl phosphate mannose at 15min of incubation at 37 degrees C.     

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of β-1,4-glucans in vivo.     

Formation of alpha-1,2- and alpha-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-alpha-D-mannose, p-nitrophenyl-alpha-D-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-alpha-D-mannose or with mannose were alpha-linked. The disaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-alpha-1,2-mannose and mannose-alpha-1,3-mannose. synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-alpha-1,3-mannose was severely inhibited by Triton X-100.     

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.     

Effect of ethionine on dolichyl phosphate-dependent transglycosylation reactions in rat liver

Dolichyl phosphates of different chain length (C35, C55 , C75 , Dol-mixture of C90 , 95, 100, 105 and C110 ) were tested as lipid acceptors in transglycosylation reactions. In the absence of exogenously added dolichyl phosphates there were no differences in the rate of synthesis in liver of dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate N-acetylglucosamine between normal and ethionine-treated animals. Addition of exogenous dolichyl phosphates of different chain length stimulated the synthesis of dolichyl phosphate mannose and dolichyl pyrophosphate N-acetyl-glucosamine 2 to 4 times depending on the chain length of dolichols , both in normal and ethionine-treated animals. In liver of ethionine-treated rats the formation of dolichyl phosphate glucose was not stimulated. Following ethionine treatment the concentration of free and esterified with fatty acids dolichols was increased.     

Formation of α-1,2- and α-1,3-linked mannose disaccharides from dolichyl mannosyl phosphate by rat liver membrane enzymes

Dolichyl mannosyl phosphate and GDPmannose were active substrates for the transfer of mannose to methyl-α-d-mannose, p-nitrophenyl-α-d-mannose, and free mannose with rat liver microsomal membranes. The products formed during dolichyl mannosyl phosphate incubation with methyl-α-d-mannose or with mannose were α-linked. The dissaccharides formed by incubation of dolichyl mannosyl phosphate or GDPmannose with mannose were identified by paper chromatography and electrophoresis as mannose-α-1,2-mannose and mannose-α-1,3-mannose. Synthesis of each product was dependent on the assay conditions used and was most markedly affected by the presence of detergent. Transfer of mannose from either substrate to form mannose-α-1,3-mannose was severely inhibited by Triton X-100.     

Lipid-bound sugars in malignant human breast tumors

Microsomal preparations from malignant human breast tumors catalyzed the transfer of mannose and glucose from GDP-[14C]-Man and UDP-[14C]-Glc into lipid-linked sugars and glycoprotein-like substances. As judged by several criteria the obtained lipid-linked monosaccharides behaved as dolichyl phosphate mannose and dolichyl phosphate glucose whereas lipid-linked oligosaccharides behaved as polyprenyl diphosphate derivatives. The optimum conditions for mannosyl- and glucosyl-transfer reactions and the effect of dolichyl phosphate, detergent and EDTA on incubation mixture were described.     

Dihydroheptaprenyl and dihydrodecaprenyl monophosphates induce apoptosis mediated by activation of caspase-3-like protease

Dolichyl phosphate, an essential carrier lipid in the biosynthesis of N-linked glycoprotein, has been found to induce apoptosis in rat glioma C6 cells and human monoblastic leukemia U937 cells. In the present study, dolichyl phosphate and structurally related compounds were examined regarding their apoptosis-inducing activities in U937 cells. Dihydroheptaprenyl and dihydrodecaprenyl phosphates, of which isoprene units are shorter than that of dolichyl phosphate, induced apoptosis in U937 cells. This phenomenon occurred in a dose- and time-dependent manner, as seen with dolichyl phosphate-induced apoptosis. Derivatives of the same isoprene units of dolichyl phosphate, such as dolichol, dolichal or dolichoic acid, did not induce DNA fragmentation. Farnesyl phosphate and geranylgeranyl phosphate also failed to induce apoptosis. During apoptosis, the caspase family of cysteine proteases play important roles. We observed that apoptosis induced by dihydroprenyl phosphate was mediated by caspase-3-like (CPP32-like) activation but not by caspase-1-like (ICE-like) activation. This caspase-3-like activation was inhibited by a specific inhibitor of caspase-3, DEVD-CHO, but not by an caspase-1 inhibitor YVAD-CHO. We interpret these results to mean that dihydroprenyl phosphates with more than seven isoprene units have apoptosis-inducing activity and that their signal is mediated by caspase-3-like activation.     

The effect of flavomycin on the synthesis and transfer of lipid-linked saccharides in pig brain

Particulate membrane fractions from pig brain catalyse the synthesis of lipid-linked sugar derivatives of the dolichyl phosphate pathway. Flavomycin, a phosphoglycolipid antibiotic produced by various species of streptomycetes, interferes with the formation of these glycolipids to a different extent. The formation of dolichyl phosphate glucose was shown to be most susceptible to the antibiotic, being blocked by about 50% in the presence of 0.2mm-flavomycin, whereas the synthesis of dolichyl diphosphate N-acetylglucosamine, dolichyl diphosphate chitobiose and dolichyl diphosphate chitobiosyl mannose required higher concentrations to achieve a comparable inhibition. Although the formation of dolichyl phosphate mannose was hardly affected, the accumulation of oligosaccharides with five to seven sugar units was observed, when dolichyl diphosphate oligosaccharides were synthesized with GDP-[(14)C]mannose in the presence of 1mm-flavomycin. This indicates that the inhibition of the synthesis of larger-sized oligosaccharides, known to be mediated by lipid-bound mannose, was not caused by an actual deficiency in dolichyl phosphate mannose. At flavomycin concentrations that inhibited the formation of dolichyl phosphate glucose by 50%, the transfer of lipid-linked saccharides to either the hexapeptide Tyr-Asn-Gly-Thr-Ser-Val or endogenous protein acceptors was hardly influenced. The mode of action of flavomycin is still obscure, but seems not to be of a competitive nature, since the inhibition was unaffected by increasing concentrations of dolichyl phosphate. Some evidence indicates that, besides a direct interaction of the antibiotic with some transferases, a non-specific incorporation into the membrane and alteration of its properties might be responsible for those inhibitory effects on all enzymes which were observed at high concentrations of flavomycin.     

A high-performance liquid chromatographic method for the determination of dolichyl phosphates in tissues

A sensitive high-performance liquid chromatographic method has been developed for the determination of dolichyl phosphate homologs in tissues. Dolichyl phosphates were methylated and derivatized with 3-(9-anthryl)-diazo-2-propene to yield methyl 3-(9-anthryl)-2-propenyl dolichyl phosphates. As an internal standard, 2,2-didecaprenylethyl phosphate was used. The derivatives were separated on a reversed-phase column using isocratic elution, and detected fluorometrically. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichyl phosphate homologs in tissues.