The formation of a β-(1→4)-d-galactan chain catalysed by a Phaseolus aureus enzyme

With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-alpha-d-[U-(14)C]galactose served as a precursor for a number of products. One of these products was characterized as a beta-(1-->4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of alpha-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The (14)C-labelled beta-(1-->4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [(14)C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [(14)C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive beta-(1-->4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1-->3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [(14)C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [(14)C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.     

Changes in the ribonucleic acid metabolism of aging mouse tissues with particular reference to the prostate gland

1. Mouse mast-cell tumours P815 Y and HC were shown to contain glycoprotein material composed of glucosamine, galactosamine, sialic acid, galactose and mannose. 2. The major amino acids released after acid hydrolysis of Pronase-treated digests of the glycoprotein are aspartic acid, glutamic acid, serine, threonine, proline, glycine and alanine. The Pronase-digested material is not degraded in alkaline solution. 3. On incubation of mast cells with [(35)S]sulphate, heparin is the major radioactive product. However, [1-(14)C]glucosamine and d-[(14)C]glucose are incorporated largely into the glycoprotein. 4. The fate of [(35)S]sulphate-labelled and [1-(14)C]glucosamine-labelled material was studied. In each case high-molecular-weight radioactive material is released from the cells into the culture medium. The t((1/2)) of [(35)S]sulphate-labelled material in cells is 70hr. and that of [1-(14)C]-glucosamine-labelled material in cells is 40hr. 5. About 60% of the [(35)S]sulphate-labelled material is present in the mitochondrial and granular fraction. [1-(14)C]-Glucosamine-labelled material is present in both microsomal and mitochondrial and granular fractions, [(14)C]sialic acid being concentrated in the microsomal fraction.     

The metabolism of glucose 6-phosphate by mammalian cerebral cortex in vitro

1. The metabolism of glucose 6-phosphate in rat cerebral-cortex slices in vitro was compared with that of glucose. It was found that a glucose 6-phosphate concentration of 25mm was required to achieve maximal oxygen uptake rates and ATP concentrations, whereas only 2mm-glucose was required. 2. When 25mm-[U-(14)C]glucose 6-phosphate was used as substrate, the pattern of labelling of metabolites was found to be quantitatively and qualitatively similar to the pattern found with 10mm-[U-(14)C]glucose, except that incorporation into [(14)C]lactate was decreased, and significant amounts of [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate were formed. 3. Unlabelled glucose (10mm) caused a tenfold decrease in the incorporation of 25mm-[U-(14)C]glucose 6-phosphate into all metabolites except [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate. In contrast, unlabelled glucose 6-phosphate (25mm) had no effect on the metabolism of 10mm-[U-(14)C]glucose other than to increase markedly the incorporation into, and amount of, [(14)C]lactate, the specific radioactivity of this compound remaining approximately the same. 4. The effect of glucose 6-phosphate in increasing lactate formation from glucose was found to occur also with a number of other phosphate esters and with inorganic phosphate. Further investigation indicated that the effect was probably due to binding of medium calcium by the phosphate moiety, thereby de-inhibiting glucose uptake. 5. Incubations carried out in a high-phosphate high-potassium medium gave a pattern of metabolism similar to that found when slices were subjected to depolarizing conditions. Tris-buffered medium gave similar results to bicarbonate-buffered saline, except that it allowed much less lactate formation from glucose. 6. Part of the glucose formed from glucose 6-phosphate was extracellular and was produced at a rate of 12mumol/h per g of tissue in Krebs tris medium when glycolysis was blocked. The amount formed was much less when 25mm-P(i) or 26mm-HCO(3) (-) was present, the latter being in the absence of tris. 7. Glucose 6-phosphate also gave rise to an intracellular glucose pool, whereas no intracellular glucose was detectable when glucose was the substrate.     

Cell-free biosynthesis of lipophosphoglycan from Leishmania donovani. Characterization of microsomal galactosyltransferase and mannosyltransferase activities

Incubation of microsomal preparations from Leishmania donovani parasites with UDP-[3H]galactose or GDP-[14C]mannose resulted in incorporation of radiolabel into an endogenous product that exhibited the chemical and chromatographic characteristics of the parasite's major surface glycoconjugate, lipophosphoglycan. The [3H]galactose- or [14C]mannose-labeled product was (i) cleaved by phosphatidylinositol-specific phospholipase C; (ii) deaminated by nitrous acid; and (iii) degraded into radioactive, low molecular weight fragments upon hydrolysis with mild acid. Analysis of the products of mild acid hydrolysis revealed the presence of phosphorylated Gal-beta-Man as the major fragment with lesser amounts of mono-, tri-, and tetrasaccharides. The incorporation of the two isotopic precursors was neither stimulated by the addition of dolichylphosphate nor inhibited by amphomycin, indicating that dolichol-saccharide intermediates are not involved in assembly of the repeating units of lipophosphoglycan. Development of this cell-free glycosylating system will facilitate further studies on the pathway and enzymes involved in lipophosphoglycan biosynthesis.     

Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose is not increased by additions of GDP-alpha-d-mannose. However, final incorporation is increased by addition of GDP-alpha-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is increased by all concentrations of GDP-alpha-d-glucose that are less than that of the GDP-alpha-d-[(14)C]mannose. Maximum stimulation of the initial rate occurs at a GDP-alpha-d-[(14)C]mannose/GDP-alpha-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-alpha-d-[(14)C]mannose is decreased by the addition of GDP-alpha-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2-3 and 50mum. Extrapolated K(m) values for the two linear components are 0.4-1.0 and 700-1500mum. The effect of GDP-alpha-d-glucose on the kinetics of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose also results in biphasic reciprocal plots. One component appears to have K(m) about 2-3mum, the other about 200-400mum. In this reaction, GDP-alpha-d-mannose appears to be a competitive inhibitor with K(i) 20-30mum. With particulate preparations of P. aureus, GDP-alpha-d-[(14)C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-alpha-d-[(14)C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.     

Formation and properties of retinylphosphate galactose.

Crude cell membrane fractions from a number of tissues can form acidic glycolipids. The formation of acidic galactose lipid and mannose lipid was greatly reduced in vitamin A deficiency, primarily in tissues known to be mucus-producing. Mouse mastocytoma tissue was active in forming acidic galactose lipids with UDP-galactose as substrate. One of the products was identified as retinylphosphate galactose. The synthetase reaction producing this compound exhibited an apparent pH optimum at 6.3. The presence of detergent and retinol stimulated the synthetase reaction, which exhibited an absolute requirement for Mn2+ or Mg2+. The synthetase reaction was readily reversible. Incubation of particulate enzyme with retinylphosphate galactose and UDP yielded UDP-galactose and a compound tentatively identified as retinylphosphate. The galactose lipid was isolated by column chromatography on DEAE-cellulose and silica gel. The retinylphosphate galactose was homogeneous when examined by thin layer chromatography. Mild acid hydrolysis of labeled retinylphosphate galactose yields [14C]galactose, whereas alkaline hydrolysis and hydrogenolysis produced [14C]galactose 1-phosphate. Retinylphosphate galactose bound to vitamin A-depleted, retinol-binding protein.     

Radioisotope ratios discriminate between competing pathways of cell wall polysaccharide and RNA biosynthesis in living plant cells

Cell wall polysaccharides are synthesized from sugar-nucleotides, e.g. uridine 5'-diphosphoglucose (UDP-Glc), but the metabolic pathways that produce sugar-nucleotides in plants remain controversial. To help distinguish between potentially 'competing' pathways, we have developed a novel dual-radiolabelling strategy that generates a remarkably wide range of 3H:14C ratios among the various proposed precursors. Arabidopsis cell cultures were fed traces of D-[1-(3)H]galactose and a 14C-labelled hexose (e.g. D-[U-14C]fructose) in the presence of an approximately 10(4)-fold excess of non-radioactive carbon source. Six interconvertible 'core intermediates', galactose 1-phosphate <--> UDP-galactose <--> UDP-glucose <--> glucose 1-phosphate <--> glucose 6-phosphate <--> fructose 6-phosphate, showed a large decrease in 3H:14C ratio along this pathway from left to right. The isotope ratio of a polysaccharide-bound sugar residue indicates from which of the six core intermediates its sugar-nucleotide donor substrate stemmed. Polymer-bound galacturonate, xylose, arabinose and apiose residues (all produced via UDP-glucuronate) stemmed from UDP-glucose, not glucose 6-phosphate; therefore, UDP-glucuronate arose predominantly by the action of UDP-glucose dehydrogenase rather than through the postulated competing pathway leading from glucose 6-phosphate via myo-inositol. The data also indicate that UDP-galacturonate was not formed by a hypothetical UDP-galactose dehydrogenase. Polymer-bound mannose and fucose residues stemmed from fructose 6-phosphate, not glucose 1-phosphate; therefore GDP-mannose (guanosine 5'-diphosphomannose) arose predominantly by a pathway involving phosphomannose isomerase (via mannose phosphates) rather than through a postulated competing pathway involving GDP-glucose epimerization. Curiously, the ribose residues of RNA did not stem directly from hexose 6-phosphates, but predominantly from UDP-glucose; an alternative to the textbook pentose-phosphate pathway therefore predominates in plants.     

Catabolite inhibition: a general phenomenon in the control of carbohydrate utilization

When Escherichia coli is grown in synthetic medium with radioactive galactose or lactose as the carbon source, the addition of glucose rapidly inhibited utilization of the radioactive substrate, whether the formation of (14)CO(2) or acid-insoluble products was measured. The inhibition was reversed after the removal of glucose. Experiments with mutants blocked in subsequent steps of galactose and lactose metabolism demonstrated that the inhibition occurs prior to the formation of the first metabolic product. The utilization of a variety of sugars, including maltose, lactose, mannose, galactose, l-arabinose, xylose, and glycerol was inhibited by glucose. Of a number of carbohydrates tested as potential inhibitors, only glucose and, to a lesser extent, glucose-6-phosphate (G-6-P) were capable of inhibiting the utilization of all of the substrates. Glucose did not inhibit G-6-P utilization but G-6-P inhibited glucose utilization. With all substrates, except glycerol, there was a delay before the onset of inhibition by G-6-P. We conclude that E. coli has a general regulatory mechanism, termed catabolite inhibition, which controls the activity of early reactions in carbohydrate metabolism, allowing certain substrates to be utilized preferentially.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

Polyprenol phosphate as an acceptor of mannose from guanosine diphosphate mannose in Aspergillus niger

Growth of Aspergillus niger in the presence of [2-(14)C]mevalonate and (32)P(i) led to the formation of a lipid, containing (14)C (0.14% of dose) and (32)P (0.009% of dose), that had chromatographic properties identical with those of exo-methylene-hexahydropolyprenol phosphate. When a particulate enzyme preparation from the thallus of A. niger was incubated with GDP-[(14)C]mannose, the main radioactive products were mannose 1-phosphate (57% of products) and mannose (18%). In addition radioactive mannan (8%) and a mannolipid (2%) were formed. The latter was identified as exo-methylene-hexahydropolyprenol phosphate mannose on the basis of its chromatographic properties, acid lability and on the increase in formation of the mannolipid when the phosphate of exo-methylene-hexahydropolyprenols was added to the incubation mixture. The phosphates of ficaprenols and cetyl alcohol caused no corresponding increase. These observations are interpreted as evidence that the thallus of A. niger contains a mannose transferase that uses the phosphate of exo-methylene-hexahydropolyprenols as an acceptor. This situation is discussed in the light of the analogous involvement of a prenol phosphate mannose as an intermediate in the biosynthesis of bacterial wall mannan.     

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.     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.     

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.     

Glucomannan Biosynthesis Catalyzed by Pisum sativum Enzymes

The results of molecular weight studies, structural analysis of the [(14)C]polysaccharides, and enzymic properties indicate that the Pisum sativum guanosine diphosphosphate glucose: glucosyltransferase is an enzymic component involved in the biosynthesis of glucomannan chains. The properties of the Pisum sativum particulate enzyme are essentially identical to the glucomannan synthetase obtained from Phaseolus aureus. Also present in the particulate preparation is an enzyme which catalyzes the formation of a [(14)C]mannolipid, using guanosine diphosphate-[(14)C]mannose as a substrate. The [(14)C]mannolipid is hydrolyzed by treatment with 0.012 m HCl, but is stable to treatment with 0.09 m NaOH. The formation of the [(14)C]mannolipid is apparently reversed by guanosine diphosphate, but not by guanosine monophosphate. The chromatographic mobility of the [(14)C]mannolipid is identical to that of a similar mannolipid synthesized by a Phaseolus aureus enzyme.     

Fatty acid biosynthesis by a particulate preparation from germinating pea

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.     

Functional characterisation of human SGLT-5 as a novel kidney-specific sodium-dependent sugar transporter

Sodium glucose cotransporters (SGLT) actively catalyse carbohydrate transport across cellular membranes. Six of the 12 known SGLT family members have the capacity to bind and/or transport monosaccharides (SGLT-1 to 6); of these, all but SGLT-5 have been characterised. Here we demonstrate that human SGLT-5 is exclusively expressed in the kidney. Four splice variants were detected and the most abundant SGLT-5-mRNA was functionally characterised. SGLT-5 mediates sodium-dependent [(14)C]-α-methyl-D-glucose (AMG) transport that can be inhibited by mannose, fructose, glucose, and galactose. Uptake studies using demonstrated high capacity transport for mannose and fructose and, to a lesser extent, glucose, AMG, and galactose. SGLT-5 mediated mannose, fructose and AMG transport was weakly (μM potency) inhibited by SGLT-2 inhibitors. In summary, we have characterised SGLT-5 as a kidney mannose transporter. Further studies are warranted to explore the physiological role of SGLT-5.     

Glycogen synthesis in the fungus Neurospora crassa.

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [14C]glucose from ADP-[14C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose : glycogen glucosyltransferase as compared with those of the already known UDP glucose : glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-14C]glucose or ADP-[14C]glucose released all the radioactivity as maltose after alpha or beta amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[14C]glucose was the substrate but the stimulation was much greater with ADP-[14C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent &on the presence of a 'primer' of the alpha 1 leads to 4 glucan type.     

Tridecaptin stimulates the formation of lipid-linked saccharides in aorta.

The peptide antibiotic tridecaptin caused a 2--4-fold stimulation in the incorporation of mannose from GDP-[14C]mannose and glucose from UDP-[3H]glucose into lipid-linked monosaccharides by both the particulate and the soluble enzyme fractions from pig aorta. In both cases, the major products and the ones stimulated by antibiotic were dolichyl phosphate mannose and dolichyl phosphate glucose. The stimulation in activity was unaffected by increasing concentrations of dolichyl phosphate, GDP-mannose, UdP-glucose, Mn2+ or the detergent Nonidet P40. Tridecaptin stimulation was apparently not due to protection of sugar nucleotide substrate, since addition of various concentrations of sugar nucleotides did not alter the stimulation. Nor did the addition of tridecaptin result in any increase in the amount of radioactive sugar nucleotide recovered from incubation mixtures. Tridecaptin bound to the particulate enzyme and could not be removed by centrifugation of the particles.     

Differences in the conversion of the polyunsaturated fatty acids [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3) in isolated rat hepatocytes

The reasons why most cellular lipids preferentially accumulate 22:6(n-3) rather than 22:5(n-6) are poorly understood. In the present work the metabolisms of the precursor fatty acids, [1-(14)C]20:4(n-6), [1-(14)C]22:4(n-6) versus [1-(14)C]20:5(n-3), [1-(14)C]22:5(n-3) in isolated rat hepatocytes were compared. The addition of lactate and L-decanoylcarnitine increased the formation of [(14)C]24 fatty acid intermediates and the final products, [(14)C]22:5(n-6) and [(14)C]22:6(n-3). In the absence of lactate and L-decanoylcarnitine, no [(14)C]24 fatty acids and [(14)C]22:5(n-6) were detected when [1-(14)C]22:4(n-6) was the substrate, whereas small amounts of the added [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). Lactate reduced the oxidation of [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) while L-decanoylcarnitine did not. No significant differences between the total oxidation or esterification of the two substrates were observed. By fasting and fructose refeeding the amounts of [(14)C]24:4(n-6) and [(14)C]24:5(n-3) were increased by 2.5- and 4-fold, respectively. However, the levels of [(14)C]22:5(n-6) and [(14)C]22:6(n-3) were similar in hepatocytes from fasted and refed versus fed rats. With hepatocytes from rats fed a fat free diet the levels of [(14)C]24 fatty acid intermediates were low while the further conversion of the n-6 and n-3 substrates was high and more equal, approx. 33% of [1-(14)C]22:4(n-6) was converted to [(14)C]22:5(n-6) and 43% of [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). The moderate differences found in the conversion of [1-(14)C]22:4(n-6) versus [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3), respectively, and the equal rates of oxidation of the two substrates could thus not explain the abundance of 22:6(n-3) versus the near absence of 22:5(n-6) in cellular membranes.     

Glycoproteins in bacterial membranes. In vivo labeling of the sugar portion of energy-transducing ATPase and a low-molecular-weight fraction fromMicrococcus lysodeikticus membranes

The energy-transducing ATPase and a low-molecular-weight fraction ofMicrococcus lysodeikticus membranes incorporated¹⁴C label fromd-[U-¹⁴C]glucose fed to the bacteria in synthetic medium. The specific radioactivity of the sugar portion of the ATPase and low-molecular-weight fraction was, respectively, 2.65 and 2.88 times that of their amino acids. Glucose and mannose in approximately equimolar amounts were identified as the main sugars of the glycoprotein ATPase, thus confirming previous structural studies. Glucose, galactose, and mannose (1:1:2) were identified as the main sugars of the low-molecular-weight glycopeptides. These results confirm and extend the notion that glycoprotein are constituents of prokaryotic membranes.