Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii

When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.     

Use of 3H and 14C double-labeled glucose to assess in vivo pathways of amino acid biosynthesis in Escherichia coli.

3H and 14C tracing data concerning amino acid biosynthetic pathways in Escherichia coli K12 are presented. Thirteen acidic and neutral amino acids were isolated from protein hydrolysates of wild type E. coli K12 grown aerobically or anaerobically in the presence of [U-14C]glucose together with [1-3H]glucose, [3-3H]glucose, [4-3H]glucose, or [6-3H]glucose. The observed 3H/14C counts of the amino acids were compared with the ratios expected on the basis of the input substrate specific activities and present understanding of biosynthetic pathways. For nine amino acids, serine, valine, leucine, threonine, isoleucine, glycine, glutamate, proline, and phenylalanine, the agreement between anticipated and observed specific activities was satisfactory. For the remaining four, methionine, alanine, aspartate, and (in cells labeled with [3-3H]glucose) tyrosine, the anticipated and observed specific activities differed markedly. For alanine, aspartate, and tyrosine, the differences are probably due to exchange of tritium in the course of biosynthesis; for methionine, it may be that there is a principle source of the methyl group other than carbon 3 of serine.     

Chemical Composition of Exosporium from Spores of Bacillus cereus

Homogeneous fragments of exosporium were extricated in centigram amounts from dormant spores of Bacillus cereus and analyzed for intrinsic constituents. The membrane proved to be chemically complex but not unique, consisting mainly of protein (52%), amino and neutral polysaccharides (20%), lipids (18%), and ash (4%). Seventeen common amino acids were identified by chromatography, and were present in usual proportions except for low levels of cystine-cysteine, methionine, tyrosine, and histidine. Glucosamine was the only amino sugar, and glucose and rhamnose were the principal neutral sugars. The lipid fraction contained 5.5% cardiolipin as the only phospholipid, 12.5% neutral lipids, and at least 19 fatty acids, among which normal C(16) and C(18) ones predominated. Calcium and phosphorus occurred in the ash. Small amounts of teichoic, ribonucleic, and dipicolinic acids were believed to represent contamination.     

Some aspects of metabolic adaptations in lipid metabolism during starvation are mimicked by epinephrine in rat adipocytes

1. The effects of fasting on the neutral lipid synthesis to insulin and/or epinephrine in isolated fat cells have been examined using [1-14C]glucose. 2. The ability of adipocytes from starved rats to synthesize fatty acids from both labeled substrates was markedly diminished compared to adipocytes from control rats. 3. The response of lipogenic stimulation to insulin at all concentrations tested was greatly diminished in adipocytes from 24 hr starved rats. 4. [1-14C]glucose utilization rates in the absence or in the presence of insulin were not significantly different in adipocytes from 24 hr starved rats as compared with control adipocytes, although basal and insulin stimulated glyceride-glycerol synthesis were significantly higher in starved adipocytes. 5. Epinephrine acutely inhibited [1-14C]acetate incorporation into fatty acids for insulin-stimulated lipogenesis in control adipocytes, in contrast, this lipolytic agent strongly increased [1-14C]glucose conversion to triacylglycerols. 6. In both cases, the differences in lipid synthesis capacities found in both nutritional states were abolished by epinephrine.     

Lipid metabolism in various regions of squid giant nerve fiber

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.     

Distribution and composition of lipopolysaccharides from mycoplasmas.

Polymeric carbohydrates containing glycerol and fatty acids were isolated from whole cells and membranes of mycoplasmas by hot aqueous phenol extraction and gel filtration. Lipopolysaccharides were found to occur in four species of Acholeplasma, two of Anaeroplasma, and in Mycoplasma neurolyticum. None were detected in Spiroplasma citri or in five species of Mycoplasma. All lipopolysaccharides contained both neutral and N-acylated amino sugars in ratios varying from 1:1 to 3:1. The neutral sugars found in varying distribution were glucose, galactose, and mannose. The amino sugars included fucosamine, an unidentified deoxyhexosamine, galactosamine, and glucosamine. Fucosamine and glucose were the only sugars common to all lipopolysaccharides. The fatty acids were similar to those found in the lipids of each organism.     

Carbohydrate Metabolism in Photosynthetic and Nonphotosynthetic Tissues of Variegated Leaves of Coleus blumei Benth

Mature, variegated leaves of Coleus blumei Benth. contained stachyose and other raffinose series sugars in both green, photosynthetic and white, nonphotosynthetic tissues. However, unlike the green tissues, white tissues had no detectable level of galactinol synthase activity and a low level of sucrose phosphate synthase indicating that stachyose and possibly sucrose present in white tissues may have originated in green tissues. Uptake of exogenously supplied [¹⁴C]stachyose or [¹⁴C]sucrose into either tissue type showed conventional kinetic profiles indicating combined operation of linear first-order and saturable systems. Autoradiographs of white discs showed no detectable minor vein labelling with [¹⁴C]stachyose, but some degree of vein labeling with [¹⁴C]sucrose. Autoradiographs of green discs showed substantial vein loading with either sugar. In both tissues, p-chloromercuribenzenesulfonic acid had no effect on the linear component of sucrose or stachyose uptake but inhibited the saturable component. Both tissues contained high levels of invertase, sucrose synthase and α-galactosidase and extensively metabolized exogenously supplied ¹⁴C-sugars. In green tissues, label from exogenous sugars was recovered as raffinose-series sugars. In white tissues, exogenous sugars were hydrolysed and converted to amino acids and organic acids. The results indicate that variegated Coleus leaves may be useful for studies on both phloem loading and phloem unloading processes in stachyose-transporting species.     

Long-term Metabolism of [14C]Sugars in Stored Potatoes

[¹⁴C]Sucrose, [¹⁴C]glucose and [¹⁴C]fructose were introducedinto potato tubers held at 10 °C and the redistributionof label chased over a 65 d period in storage. Respiratory losseswere identical in all treatments, as was the partitioning of¹⁴C between soluble and insoluble forms. Sucrose was the predominantlabelled sugar in the tubers after 20 h, regardless of the original[¹⁴C]sugar introduced, and was loaded and distributed throughoutthe tubers by the internal phloem system. After 20 h the proportionsof labelled sugars bore no relationship to those of the unlabelledendogenous sugars. However, with time the percentage of ¹⁴Cin sucrose fell while that in glucose increased and by 65 dthe proportions of the labelled sugars more closely resembledthe endogenous pools. Fructose represented a consistently lowproportion of both the labelled and unlabelled sugars. By 21d a considerable proportion of the soluble ¹⁴C had been convertedto starch (approx. 25% of the total tuber 14C), this value remainingrelatively constant for the remainder of the storage period.Sprouts which formed on the tubers contained up to 6% of thetotal tuber ¹⁴C but less than 0.2% of the tuber dry matter.It is suggested that the bulk of the translocated [¹⁴C]sucroseentered the symplast and exchanged slowly with the bulk of thesugars in the storage cell vacuoles. [¹⁴C]sugars, phloem loading, starch, potato tuber, Solunum tuberosum, cold storage     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Sugar metabolism and partitioning in cytosol and bacteroid fractions of chickpea nodules

The contents of free sugars in nodules of chickpea (Cicer arietinum) were maximum around flowering. In stem and root tissues, the relative incorporation of ¹⁴C from [¹⁴C]-labelled sucrose or glucose into extracted sucrose was over 70 %. In the former tissue, the relative incorporation of ¹⁴C from glutamate into sucrose was about 50 % at 50 d after sowing (DAS) but the same decreased to about 25 % at 80 DAS. However, from glutamate, 63–68 % of ¹⁴C from extracted sugars of root tissue appeared in invert sugars. Feeding via stem [¹⁴C]-glutamate to intact nodules led to intense labelling of sucrose and invert sugars in nodule cytosol. Upon injecting labelled sugars or glutamate into isolated nodules, maximum ¹⁴C appeared in glucose of this nodule fraction. In bacteroids, incorporation of ¹⁴C from glutamate was much higher in amino acids. In the cytosol of younger (50 DAS) nodules, sucrose was cleaved largely by soluble alkaline invertase (EC 3.2.1.26). However, sucrose cleavage in this fraction of older (80 DAS) nodules was catalysed by this enzyme as well as sucrose synthase (reversal, EC 2.4.1.13) and such nodules also contained higher activity of nitrogenase. The bacteroid fraction, which contained 10–17 % of nodule sugars, lacked the activities of sucrose-cleaving enzymes. The activities of ATP-dependent phosphofructokinase (EC 2.7.1.11), glyceraldehyde-3-phosphate dehydrogenase (EC 1.1.1.12), NADP⁺-dependent isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) were higher in cytosol than bacteroids. However, the reverse was true for glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The results suggest that in chickpea nodules sugar metabolism occurs largely via the glycolytic pathway in cytosol and the pentose phosphate pathway in bacteroids and there is some transport of glutamate from cytosol to bacteroids.     

Biosynthesis of glycosylated glycerolphosphate polymers in Streptococcus sanguis.

Two types of glycosylated glycerolphosphates were synthesized when a particulate enzyme prepared from Streptococcus sanguis was incubated with [3H]-phosphatidylglycerol and uridine diphosphate-[14C]glucose in the presence of MgCl2. The first type was extractable with saline and contained no fatty acid. The second type was pellet bound and could be extracted with 0.1% sodium dodecyl sulfate. Both types of polymers were purified and partially characterized. The first type of polymer was fractionated into five polymers, peaks 2a, 2b, 2c, 3a, and 3b. All except peak 2a, which contained only [3H]glycerol, contained both [3H]glycerol and [14C]glucose. [3H]NaBH4 reduction of acid hydrolysates of the polymers revealed that all of the polymers contained glucose as the major sugar componenta nd xylose as the minor sugar component. The second type of polymer was fractionated into three polymers, P-1, P-2, and P-3. All contained [3H]-glycerol, [14C]glucose, and fatty acids. P-1 appeared to be pure, whereas P-2 and P-3 contained two polymers each, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Patterns of polysaccharide biosynthesis in differentiating cells of maize root-tips

1. The patterns of incorporation of radioactivity from d-[6-(3)H]-, d-[1-(14)C]-, d-[U-(14)C]- and d-[6-(14)C]-glucose and [U-(14)C]myoinositol into the neutral sugars and uronic acids of the polysaccharides synthesized in different regions of the root-tip of maize were determined. 2. The root-cap tissue synthesized a slime in which a polysaccharide that contained a high proportion of fucose (32%) and galactose (21%) was found. This polysaccharide is synthesized only by the root-cap cells, and very little polysaccharide containing fucose is synthesized in adjacent tissues. Part of the meristematic tissue of the root is surrounded by the cap cells. A section of the root that contains both these tissues can be analysed, and the polysaccharide synthesized by the meristematic region can be obtained since the contribution of the root-cap cells can be found by the amount of fucose formed. 3. It was shown that there is very little difference in the polysaccharide synthesis of the meristematic region from that of the cells immediately behind it. In the more mature cells, however, the amount of xylose synthesized relative to that of arabinose is increased, and the proportion of xylose and arabinose formed in the matrix polysaccharides is increased whereas that of galactose is decreased. 4. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on polysaccharide synthesis was to bring about a decrease in the relative amount of galactose synthesized in the matrix polysaccharides of cells immediately adjacent to the meristematic region and also in the more mature tissue. The growth factor also increased the amount of xylose synthesized relative to that of arabinose in the more mature tissue. These metabolic effects were related to a very obvious change in the morphological appearance of the root-tips. 5. Radioactivity from [U-(14)C]myoinositol was incorporated mainly into xylose, arabinose and galacturonic acid rather than into the hexoses, although small amounts of these sugars were formed.     

Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of [2-3H]glucose and [U-14C]glucose utilization is due to contamination of precursor pool with 14C-labeled products and incomplete recovery of 14C-labeled metabolites

Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported (Huang, M., and Veech, R.L. (1982) J. Biol. Chem. 257, 11358-11363). The evidence was an apparent more rapid 3H than 14C loss from the glucose pool and faster [2-3H]glucose than [U-14C]glucose utilization following pulse labeling of the brain with [2-3H,U-14C]glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, 14C-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from [14C]glutamine; 2) [14C]glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, 14C-labeled derivatives of [2-3H,U-14C]glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found.     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

Glucose metabolism in the testis of the normal and streptozotocin diabetic rat

In the present work, the metabolism of [U-14C]glucose was studied in the testicular tissue of normal and streptozotocin diabetic rats. The results show that diabetes alters 14CO2 production and 14C incorporation into lipids from [U-14C]glucose. No differences were found in the [14C]proteins and [14C]nucleic acids between experimental groups. Results are discussed in relation to an insulin deficiency and/or an alteration in the hypothalamic-hypophyseal-gonadal axis.     

Intracellular phosphorylation of glucose analogs via the phosphoenolpyruvate: mannose-phosphotransferase system in Streptococcus lactis.

The bacterial phosphoenolpyruvate:sugar-phosphotransferase system (PTS) mediates the vectorial translocation and concomitant phosphorylation of sugars. The question arises of whether the PTS can also mediate the phosphorylation of intracellular sugars. To investigate this possibility in Streptococcus lactis 133, lactose derivatives have been prepared containing 14C-labeled 2-deoxy-glucose (2DG), 2-deoxy-2-fluoro-D-glucose (2FG), or alpha-methylglucoside as the aglycon substituent of the disaccharide. Two of the compounds, beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-D-glucopyranose (2'D-lactose) and beta-O-D-galactopyranosyl-(1,4')-2'-deoxy-2'-fluoro-D-glucopyranose (2'F-lactose), were high-affinity substrates of the lactose-PTS. After translocation, the radiolabeled 2'F-lactose 6-phosphate (2'F-lactose-6P) and 2'D-lactose-6P derivatives were hydrolyzed by P-beta-galactoside-galactohydrolase to galactose-6P and either [14C]2FG or [14C]2DG, respectively. Thereafter, the glucose analogs appeared in the medium, but the rates of sugar exit from mannose-PTS-defective mutants were greater than those determined in the parent strain. Unexpectedly, the results of kinetic studies and quantitative analyses of intracellular products in S. lactis 133 showed that initially (and before exit) the glucose analogs existed primarily in phosphorylated form. Furthermore, the production of intracellular [14C]2FG-6P and [14C]2DG-6P (during uptake of the lactose analogs) continued when the possibility for reentry of [14C]2FG and 2DG was precluded by addition of mannose-PTS inhibitors (N-acetylglucosamine or N-acetylmannosamine) to the medium. By contrast, (i) only [14C]2DG, [14C]2FG, and trace amounts of [14C]2FG-6P were found in cells of a mannose-PTS-defective mutant, and (ii) only [14C]2FG and [14C]2DG were present in cells of a double mutant lacking both mannose-PTS and glucokinase activities. We conclude from these data that the mannose-PTS can effect the intracellular phosphorylation of glucose and its analogs in S. lactis 133.     

Cell wall carbohydrates in Phycomyces blakesleeanus burgeff

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fucose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosugars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-¹⁴C]-glucose into wall materials.Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but not by 5-fluorouracil.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of acetate, pyruvate, lactate, palmitate, electron-acceptors, uncoupling agents and oligomycin

1. The incorporation of 5mm-[U-(14)C]glucose into glyceride fatty acids by fat cells from normal rats incubated in the presence of 20munits of insulin/ml was increased by acetate, pyruvate, palmitate, NNN'N'-tetramethyl-p-phenylenediamine, phenazine methosulphate, dinitrophenol, tetrachlorotrifluoromethyl benzimidazole and oligomycin. Lactate did not stimulate glucose incorporation into fatty acids. The effects of these agents were concentration-dependent. 2. In the presence of 5mm-glucose+insulin, [U-(14)C]acetate, [U-(14)C]pyruvate and [U-(14)C]lactate were incorporated into fatty acids in a concentration-dependent manner, thereby further increasing the total rate of fatty acid synthesis. 3. NNN'N'-tetramethyl-p-phenylenediamine decreased the incorporation of [U-(14)C]pyruvate into fatty acids in normal cells and increased the incorporation of [U-(14)C]lactate into fatty acids. 4. In fact cells from 72h-starved rats the stimulatory effects of NNN'N'-tetramethyl-p-phenylenediamine upon glucose and lactate incorporation into fatty acids were totally and partially abolished respectively whereas the stimulatory effects of acetate upon glucose incorporation were retained. 5. Combinations of the optimum concentrations of the substances that stimulate glucose incorporation into fatty acids were tested and compared. The effects of acetate+NNN'N'-tetramethyl-p-phenylenediamine and acetate+palmitate upon normal cells were additive. The effects of NNN'N'-tetramethyl-p-phenylenediamine+palmitate were not additive. It was found that total fatty acid synthesis in the presence of glucose was most effectively increased by raising the concentration of pyruvate in the incubation system. 6. The significance of these results in supporting the proposal that fatty acid synthesis from glucose in adipose tissue is a ;self-limiting process' is discussed.