Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.     

The role of C-4-substituted mannose analogues in protein glycosylation. Effect of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose and 4-deoxy-D-mannose on lipid-linked oligosaccharide assembly.

The effects of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose (GDP-4FMan) and 4-deoxy-D-mannose (GDP-4dMan) on reactions of the dolichol pathway in chick-embryo cell microsomal membranes were investigated by studies with chick-embryo cell microsomal membranes in vitro and in baby-hamster kidney (BHK) cells in vivo. Each nucleotide sugar analogue inhibited lipid-linked oligosaccharide biosynthesis in a concentration-dependent manner. GDP-4FMan blocked in vitro the addition of mannose to Dol-PP-(GlcNAc)2Man from GDP-Man (where Dol represents dolichol), but did not interfere with the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2. Although GDP-4FMan and Dol-P-4FMan were identified as metabolites of 4FMan in BHK cells labelled with [1-14C]4FMan, GDP-4FMan was a very poor substrate for GDP-Man:Dol-P mannosyltransferase and Dol-P-4FMan could only be synthesized in vitro if the chick-embryo cell membranes were primed with Dol-P. It therefore appears that the inhibition of lipid-linked oligosaccharide formation in BHK cells treated with 4FMan [Grier & Rasmussen (1984) J. Biol. Chem. 259, 1027-1030] is due primarily to a blockage in the formation of Dol-PP-(GlcNAc)2Man2 by GDP-4FMan. In contrast, GDP-4dMan was a substrate for those mannosyltransferases that catalyse the transfer of the first five mannose residues to Dol-PP-(GlcNAc)2. In addition, GDP-4dMan was a substrate for GDP-Man:Dol-P mannosyltransferase, which catalysed the formation of Dol-P-4dMan. As a consequence of this, the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2 may be inhibited through competition for Dol-P. In BHK cells treated with 10 mM-4dMan, Dol-PP-(GlcNAc)2Man9 was the major lipid-linked oligosaccharide detected. Nearly normal extents of protein glycosylation were observed, but very little processing to complex oligosaccharides occurred, and the high-mannose structures were smaller than in untreated cells.     

Regulation of the activity of Phosphoenolpyruvate carboxylase isolated from germinating maize (Zea mays L.) seeds by some metabolites

Phosphoenolpyruvate carboxylase (PEPC) was isolated from maize seeds which were germinated for 20 h, using a procedure which included extraction of seed homogenate with Tris-HCl or sodium phosphate buffer, precipitation of the extract with ammonium sulphate, chromatography on DEAE cellulose, and gel filtration on Sephadex G-200. Phosphate buffer was found to be less suitable than Tris-HCl buffer both for maize seed extraction and for further PEPC purification steps. The enzyme preparation obtained was electrophretically homogenous. PEPC activity was inhibited by both phosphate and malate. It values obtained at pH 8.1 which is the pH optimum of the reaction equelled to 42 mmoll^-1 for phosphate and to 13 mmoll^-1 for malate. PEPC isolated from germinating maize seeds was activated by glucose-6-phosphate, glucose-1-phosphate, ribulose-l,5-bisphosphate, fructose-1,6-bisphosphate, and fructose-2,6-bisphosphate. The authors intend to elucidate the mechanism of PEPC activation by sugars by means of the application of a number of derivatives of the sugar phosphates, among which for example 2-deoxy-2-fluoro glucosephosphate also activated PEPC. Sugar phosphates activated PEPC isolated from germinating maize seeds in this order, with increasing effect: fructose-l,6-bisphosphate, glucose-1-phosphate, glucose-6-phosphate, 2-deoxy-2-fluoro glucosephosphate, ribulose-l,5-bisphos-phate, fructose-2-6-bisphosphate.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates; comparison with phosphomannoisomerase

The isotopic discrimination, diastereotopic specificity and intramolecular hydrogen transfer characterizing the reaction catalyzed by phosphomannoisomerase are examined. During the monodirectional conversion of D-[2-3H]mannose 6-phosphate to D-fructose 6-phosphate and D-fructose 1,6-bisphosphate, the reaction velocity is one order of magnitude lower than with D-[U-14C]mannose 6-phosphate and little tritium (less than 6%) is transferred intramolecularly. Inorganic phosphate decreases the reaction velocity but favours the intramolecular transfer of tritium. Likewise, when D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose is exposed solely to phosphomannoisomerase, the generation of tritiated metabolites is virtually restricted to 3H2O and occurs at a much lower rate than the production of D-[U-14C]mannose 6-phosphate from D-[U-14C]fructose 6-phosphate. However, no 3H2O is formed when D-[1-3H]fructose 6-phosphate generated from D-[2-3H]glucose is exposed to phosphomannoisomerase, indicating that the diastereotopic specificity of the latter enzyme represents a mirror image of that of phosphoglucoisomerase. Advantage is taken of such a contrasting enzymic behaviour to assess the back-and-forth flow through the reaction catalyzed by phosphomannoisomerase in intact cells exposed to D-[1-3H]glucose, D-[5-3H]glucose or D-[6-3H]glucose. Relative to the rate of glycolysis, this back-and-forth flow amounted to approx. 4% in human erythrocytes and rat parotid cells, 9% in tumoral cells of the RINm5F line and 47% in rat pancreatic islets.     

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.     

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.     

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.     

Formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone by Zygosaccharomyces rouxii: identification of an intermediate

The formation of the important flavor compound 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDMF; Furaneol) from D-fructose-1,6-bisphosphate by the yeast Zygosaccharomyces rouxii was studied with regard to the identification of intermediates present in the culture medium. Addition of o-phenylenediamine, a trapping reagent for alpha-dicarbonyls, to the culture medium and subsequent analysis by high-pressure liquid chromatography with diode array detection revealed the formation of three quinoxaline derivatives derived from D-fructose-1,6-bisphosphate under the applied growth conditions (30 degrees C; pH 4 to 5). Isolation and characterization of these compounds by tandem mass spectrometry and nuclear magnetic resonance spectroscopy led to the identification of phosphoric acid mono-(2,3,4-trihydroxy-4-quinoxaline-2-yl-butyl) ester (Q1), phosphoric acid mono-[2,3-dihydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q2), and phosphoric acid mono-[2-hydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q3). Q1 and Q2 were formed independently of Z. rouxii cells, whereas Q3 was detected only in incubation systems containing the yeast. Identification of Q2 demonstrated for the first time the chemical formation of 1-deoxy-2,3-hexodiulose-6-phosphate in the culture medium, a generally expected but never identified intermediate in the formation pathway of HDMF. Since HDMF was detected only in the presence of Z. rouxii cells, additional enzymatic steps were presumed. Incubation of periplasmic and cytosolic protein extracts obtained from yeast cells with D-fructose-1,6-bisphosphate led to the formation of HDMF, implying the presence of the required enzymes in both extracts.     

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.     

Amphomycin inhibits the incorporation of mannose and GlcNAc into lipid-linked saccharides by aorta extracts

Amphomycin inhibits the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNac from UDP-[³H]GlcNAc into lipid-linked saccharides by either a particulate or a solubilized enzyme fraction from pig aorta. The solubilized enzyme was much more sensitive to the antibiotic than was the particulate fraction with 50% inhibition being observed at 8–15 μg of amphomycin. Although the antibiotic inhibited mannose transfer from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol, lipid-linked oligosaccharides and glycoprotein, the synthesis of mannosyl-phosphoryl-dolichol was much more sensitive to amphomycin. Amphomycin also inhibited the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryldecaprenol in particulate extracts of $\text{Mycobacterium smegmatis}$.     

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.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

Mannosyltransfer reactions in rabbit liver microsomes

Rabbit liver microsomes catalyzed mannosyltransfer from GDP-[14C]mannose to free $\text{D}$-mannose resulting in the synthesis of α-1,2-, α-1,3-, and α-1,6-mannosyl-mannose. Whereas formation of α-1,2-mannosyl-mannose was stimulated by the addition of manganese chloride or nickel chloride and was inhibited by EDTA, synthesis of α-1,3-mannosyl-mannose was unaffected by manganese or EDTA and was inhibited by nickel. Formation of α-1,6-mannosyl-mannose appeared to be stimulated by manganese and inhibited by nickel. These results suggest that three different mannosyltransferases were involved in the synthesis of mannosyl-mannose glycosidic linkages in rabbit liver.     

Conversion of dolichyl pyrophosphate N,N'-diacetylchitobiose to lipid-tri- to heptasaccharides by liver microsomes from hibernating ground squirrels (Citellus citellus L.)

Incubation of liver microsomes from hibernating ground squirrel with GDP-[14C]mannose and exogenous dolichyl phosphate resulted in the synthesis of dolichyl phosphate [14C]mannose. The mannosyltransferase activity was about 3-fold higher in microsomes from hibernating ground squirrels than in those from active animals. Incubation for 30 min of liver microsomes from hibernating animals with dolichyl pyrophosphate N,N'-diacetyl-[14C]chitobiose and GDP-[14C]mannose led to the synthesis of lipid-[14C]trisaccharide. When liver microsomes were incubated with lipid-[14C]trisaccharide and unlabelled GDP-mannose, lipid-tetra- to heptasaccharides were discovered in the chloroform-methanol (2:1) extract. Since, under the experimental conditions, negligible synthesis of dolichyl phosphate mannose was observed, it was assumed that GDP-mannose was a donor of mannose in the conversion of lipid-trisaccharide into lipid-oligosaccharides containing 2-5 mannose residues.     

alpha-Dihydrodecaprenyl phosphate as a sugar carrier in the membrane of AH 70Btc hepatoma cells

The effect of alpha-dihydrodecaprenyl phosphate, dolichyl phosphate and solanesyl phosphate on the lipid intermediate pathway for protein glycosylation was studied with crude membrane fraction prepared from AH 70Btc hepatoma cells. alpha-Dihydrodecaprenyl phosphate increased the incorporations of [14C]mannose from GDP-[14C]mannose into CHCl3-CH3OH (2:1, v/v) extract, oligosaccharide-lipid and proteins. The above and the other data showed that alpha-dihydrodecaprenyl phosphate may function as a mannose carrier in the lipid intermediate pathway.     

Mechanism and contribution of the pentose phosphate cycle to glucose metabolism in epididymal fat tissue

1. Glucose 6-phosphate, fructose 6-phosphate and altroheptulose 7-phosphate are the major products formed non-oxidatively from ribose 5-phosphate by rat epididymal fat pad enzymes. 2. Arabinose 5-phosphate was detected among the reaction products and significant activity of the new enzyme of the L-type pentose pathway, D-glycero D-ido octulose 1,8-bisphosphate: D-altroheptulose 7-phosphotransferase was found. 3. The glucose moieties of glucose 1-phosphate, glucose 6-phosphate and glucose 1,6-bisphosphate were degraded and showed that epididymal fat pad enzymes relocate 14C from [2-14C]glucose into C-1, C-2, and C-3 of each hexose-phosphate. 4. The 14C-distribution patterns in the hexose-phosphates revealed that these intermediates were not in isotopic equilibrium and the rate of the transaldolase exchange reaction was relatively small. 5. The 14C-distribution data suggest that glucose 1-phosphate, rather than glucose 6-phosphate, is the first intermediate in the path of glycogen synthesis from glucose in this tissue. 6. The data provide the first proof of the mechanism of the pentose pathway in adipose tissue.     

Study of the fructose 6-phosphate/fructose 1,6-bi-phosphate cycle in the liver in vivo.

1. The method proposed by Rognstad & Katz [(1976) Arch, Biochem, Biophys, 177, 337-345] for the determination of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle by the randomization of carbon between C-1 and C-6 of glucose glucose formed from [1-14C] galactose was applied to anaesthetized rats and conscious mice. 2. It was checked that the hydrolysis of fructose 6-phosphate by glucose 6-phosphatase is too weak to invalidate the method. The participation of the Cori cycle in the randomization was negligible within the short experimental period used (2-4 min). 3. No detectable randomization of carbon was observed in starved animals, indicating that phosphofructokinase is inactive in this experimental condition. 4. Randomization of carbon was detected as soon as 1 min after administration of [1-14C] galactose to fed animals and was maximal at about 3-4 min. It was calculated that on average 15% of the glucose formed by the liver to fed rats was recycled through the triose phosphates. The extent of cycling was quite variable. Recycling was also observed in starved rats in which glucose had been administered intravenously 10 min previously. In these animals, recycling was completely inhibited by glucagon. 5. The main factors that appear to be responsible for the very large changes in recycling observed in various experimental conditions are the concentrations of fructose 1,6-bisphosphate and of fructose 6-phosphate and also the affinity of phosphofructokinase for fructose 6-phosphate. The concentration of nucleotides does not seem to play a role.     

Interconversion of D-fructose 1,6-bisphosphate and triose phosphates in human erythrocytes.

Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.     

The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle.

Substrate cycling of fructose 6-phosphate through reactions catalysed by 6-phosphofructokinase and fructose-1,6-bisphosphatase was measured in skeletal muscles of the rat in vitro. The rate of this cycle was calculated from the steady-state values of the 3H/14C ratio in hexose monophosphates and fructose 1,6-bisphosphate after the metabolism of either [5-3H,6-14C]glucose or [3-3H,2-14C] glucose. Two techniques for the separation of hexose phosphates were studied; t.l.c. chromatography on poly(ethyleneimine)-cellulose sheets or ion-exchange chromatography coupled with enzymic conversion. These two methods gave almost identical results, suggesting that either technique could be used for determination of rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling. It was found that more than 50% of the 3H was retained in the fructose 1,6-bisphosphate; it is therefore probable that previous measurement of cycling rates, which have assumed complete loss of 3H, have underestimated the rate of this cycle. The effects of insulin, adrenaline and adrenergic agonists and antagonists on rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling were investigated. In the presence of insulin, adrenaline (1 microM) increased the cycling rate by about 10-fold in epitrochlearis muscle in vitro; the maximum rate under these conditions was about 2.5 mumol/h per g of tissue. The concentration of adrenaline that increased the cycling rate by 50% was about 50 nM. This effect of adrenaline appears to be mediated by the beta-adrenergic receptor, since the rate was increased by beta-adrenergic agonists and blocked by beta-adrenergic antagonists. From the knowledge of the precise rate of this cycle, the possible physiological importance of cycling is discussed.     

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.