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.     

HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.     

Measurement of mouse brain glucose utilization in vivo using [U-14C]glucose

The rate of [2-¹⁴C]glucose uptake has been used as an indication of the status of energy consumption by the rat brain, but the cost of this radiolabel can be prohibitive and the surgical manipulation involved in published methods is extensive. A method for measuring glucose utilization in vivo in mouse brain with [U-¹⁴C]glucose is described in this article. Glucose consumption in whole mouse brain obtained with [U-¹⁴C]glucose or [2-¹⁴C]glucose was 0.650±0.022 and 0.716±0.36 nmol/mg/min, respectively. In all instances the rate obtained with the uniformly labeled isotope was somewhat lower than that found with [2-¹⁴C]glucose. The rate of glucose utilization measured with either isotope was significantly depressed in sodium pentobarbital anesthetized mice. The method described here is advantageous because [U-¹⁴C]glucose is substantially less expensive than [2-¹⁴C]glucose and surgical intervention is avoided.     

Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

A simple method for determining total glucose utilisation by isolated adipocytes using (5-3H)-glucose

Glucose utilisation by adipocytes incubated with and without insulin and at two concentrations of extracellular glucose has been estimated by three different procedures. Glucose disappearance from the medium was calculated by using glucose oxidase to determine the glucose concentration remaining after incubation and comparing this with the glucose concentration in standard solutions made up by appropriate dilution of the original medium. [U-¹⁴C]-glucose utilisation was calculated by summing the ¹⁴C found in CO₂, triglycerides, and anions. [³H]-H₂O formation from [5-³H]-glucose was the third measure of glucose utilisation. All three methods gave similar answers, but the [5-³H]-glucose is simpler to use than [U-¹⁴C]-glucose and gives substantially more reproducible results than glucose oxidase.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

Effects of galactose on direct and indirect pathway estimates of hepatic glycogen synthesis

Hepatic glycogen is formed by direct and indirect pathways whose activities reflect altered nutrition or disease. Direct/indirect pathway measurements often involve test meals where ～10% of carbohydrate is galactose, but its effects on direct/indirect pathway estimates are unknown. Therefore, direct/indirect pathway contributions in 24-h fasted rats given 2 g/kg 100% glucose (GLU, n=6) or 90% glucose–10% galactose (GLU+GAL, n=6) were measured by [U-¹³C]glucose dilution and by position-5/position-2 glycogen enrichment (H5/H2) from 2H₂O. For GLU+GAL, galactose glycogenesis was independently measured with [1-¹³C]galactose. Glycogenesis was equivalent in both groups but for GLU+GAL, 23±4% of glycogen was derived from galactose. [U-¹³C]glucose reported a 30±3% direct pathway contribution to glycogenesis for GLU but only 20±3% for GLU+GAL (p=0.012 vs. GLU). H5/H2 yielded identical direct pathway estimates (32±3% GLU, 29±6% GLU+GAL). Thus, galactose glycogenesis was undetected by H5/H2 while [U-¹³C]glucose reported a reduced direct/indirect pathway ratio. With [1-¹³C]galactose also present, correct glycogenic source contributions were obtained.     

Metabolism of 3H- and 14C-labelled lactate in starved rats

1. [2-³H,U-¹⁴C]- or [3-³H,U-¹⁴C]-Lactate was administered by infusion or bolus injection to overnight-starved rats. Tracer lactate was injected or infused through indwelling cannulas into the aorta and blood was sampled from the vena cava (A–VC mode), or it was administered into the vena cava and sampled from the aorta (V–A mode). Sampling was continued after infusion was terminated to obtain the wash-out curves for the tracer. The activities of lactate, glucose, amino acids and water were followed. 2. The kinetics of labelled lactate in the two modes differed markedly, but the kinetics of labelled glucose were much the same irrespective of mode. 3. The kinetics of ³H-labelled lactate differed markedly from those for [U-¹⁴C]lactate. Isotopic steady state was attained in less than 1h of infusion of [³H]lactate but required over 6h for [U-¹⁴C]lactate. 4. ³H from [2-³H]lactate labels glucose more extensive than does that from [3-³H]lactate. [3-³H]Lactate also labels plasma amino acids. The distribution of ³H in glucose was determined. 5. Maximal radioactivity in ³HOH in plasma is attained in less than 1min after injection. Near-maximal radioactivity in [¹⁴C]glucose and [³H]glucose is attained within 2–3min after injection. 6. The apparent replacement rates for lactate were calculated from the areas under the specific-radioactivity curves or plateau specific radioactivities after primed infusion. Results calculated from bolus injection and infusion agreed closely. The apparent replacement rate for [³H]lactate from the A–VC mode averaged about 16mg/min per kg body wt. and that in the V–A mode about 8.5mg/min per kg body wt. The apparent rates for [¹⁴C]lactate (`rate of irreversible disposal') were 8mg/min per kg body wt. for the A–VC mode and 5.5mg/min per kg body wt. for the V–A mode. Apparent recycling of lactate carbon was 55–60% according to the A–VC mode and 35% according to the V–A mode. 7. The specific radioactivities of [U-¹⁴C]glucose at isotopic steady state were 55% and 45% that of [U-¹⁴C]lactate in the A–VC and V–A modes respectively. We calculated, correcting for the dilution of ¹⁴C in gluconeogenesis via oxaloacetate, that over 70% of newly synthesized glucose was derived from circulating lactate. 8. Recycling of ³H between lactate and glucose was evaluated. It has no significant effect on the calculation of the replacement rate, but affects considerably the areas under the wash-out curves for both [2-³H]- and [3-³H]-lactate, and calculation of mean transit time and total lactate mass in the body. Corrected for recycling, in the A–VC mode the mean transit time is about 3min, the lactate mass about 50mg/kg body wt. and the lactate space about 65% of body space. The V–A mode yields a mass and lactate space about half those with the A–VC mode. 9. The area under the wash-out curve for [¹⁴C]lactate is some 20–30 times that for [³H]lactate, and apparent carbon mass is 400–500mg/kg body wt. and presumably includes the carbon of glucose, pyruvate and amino acids, which are exchanging rapidly with that of lactate.     

Labelling of lipids by D-[1-14C]glucose, D-[6-14C]glucose and D-[3-3H]glucose in pancreatic islets from normal and GK rats

In pancreatic islets prepared from either normal or GK rats and incubated at either low (2.8 mM) or high (16.7 mM) D-glucose concentration, the labelling of both lipids and their glycerol moiety is higher in the presence of D-[1-¹⁴C]glucose than D-[6-¹⁴C]glucose. The rise in D-glucose concentration augments the labelling of lipids, the paired ¹⁴C/³H ratio found in islets exposed to both D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose and D-[3-³H]glucose being even slightly higher at 16.7 mM D-glucose than that found, under otherwise identical conditions, at 2.8 mM D-glucose. Such a paired ratio exceeds unity in islets exposed to D-[1-¹⁴C]glucose. The labelling of islet lipids by D-[6-¹⁴C]glucose is about 30 times lower than the generation of acidic metabolites from the same tracer. These findings indicate (i) that the labelling of islet lipids accounts for only a minor fraction of D-glucose catabolism in pancreatic islets, (ii) a greater escape to L-glycerol-3-phosphate of glycerone-3-phosphate generated from the C₁-C₂-C₃ moiety of D-glucose than D-glyceraldehyde-3-phosphate produced from the C₄-C₅-C₆ moiety of the hexose, (iii) that only a limited amount of [3-³H]glycerone 3-phosphate generated from D-[3-³H]glucose is detritiated at the triose phosphate isomerase level before being converted to L-glycerol-3-phosphate, and (iv) that a rise in D-glucose concentration results in an increased labelling of islet lipids, this phenomenon being somewhat more pronounced in the case of D-[1-¹⁴C]glucose or D-[6-¹⁴C]glucose rather than D-[3-³H]glucose.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: isotopic discrimination between hydrogen and deuterium

Summary The discrimination between the isotopes of hydrogen in the reaction catalyzed by yeast phosphoglucoisomerase is examined by NMR, as well as by spectrofluorometric or radioisotopic methods. The monodirectional conversion of D-glucose 6-phosphate to D-fructose 6-phosphate displays a lower maximal velocity with D-[2-²H]glucose 6-phosphate than unlabelled D-glucose 6-phosphate, with little difference in the affinity of the enzyme for these two substrates. About 72% of the deuterium located on the C₂ of D-[1-¹³C,2-²H]glucose 6-phosphate is transferred intramolecularly to the C₁ of D-[1-¹³C,1-²H]fructose 6-phosphate. The velocity of the monodirectional conversion of D-[U-¹⁴C]glucose 6-phosphate (or D-[2-³H]glucose 6-phosphate) to D-fructose 6-phosphate is virtually identical in H₂O and D₂O, respectively, but is four times lower with the tritiated than ¹⁴C-labelled ester. In the monodirectional reaction, the intramolecular transfer from the C₂ of D-[2-³H]glucose 6-phosphate is higher in the presence of D₂O than H₂O. Whereas prolonged exposure of D-[1-¹³C]glucose 6-phosphate to D₂O, in the presence of phosphoglucoisomerase, leads to the formation of both D-[1-¹³C,2-²H]glucose 6-phosphate and D-[1-¹³C,1-²H]fructose 6-phosphate, no sizeable incorporation of deuterium from D₂O on the C₁ of D-[1-¹³C]fructose 1,6-bisphosphate is observed when the monodirectional conversion of D-[1-¹³C]glucose 6-phosphate occurs in the concomitant presence of phosphoglucoisomerase and phosphofructokinase. The latter finding contrasts with the incorporation of hydrogen from ¹H₂O or tritium from ³H₂O in the monodirectional conversion of D-[2-³H]glucose 6-phosphate and unlabelled D-glucose 6-phosphate, respectively, to their corresponding ketohexose esters.     

A model study of the fructose diphosphatase-phosphofructokinase substrate cycle

A fructose diphosphatase–phosphofructokinase substrate cycle has been reconstructed in vitro to provide a system that recycles fructose 6-phosphate and hydrolyses ATP to ADP and P_i. The concerted actions of glucose phosphate isomerase, phosphofructokinase, aldolase and triose phosphate isomerase catalysed the loss of ³H from [5-³H,U-¹⁴C]glucose 6-phosphate. This was used as the basis of a method for the estimation of the fructose diphosphatase–phosphofructokinase substrate cycle. For the reconstructed cycle, the rate of decrease of the ³H/¹⁴C ratio in [5-³H,U-¹⁴C]hexose 6-phosphate was proportional to the rate of fructose 6-phosphate substrate cycling. A detailed theoretical treatment of this relationship is developed, which enables the rate of substrate cycling to be determined in vivo.     

TURNOVER OF PHOSPHATIDYLCHOLINE IN MICROSOMES AND MYELIN IN BRAINS OF YOUNG AND ADULT RATS

We have tested the hypothesis that the turnover of phosphatidylcholine in subcellular fractions of rat brain is a function of the age at which this lipid is deposited. Rats, 60 days of age, were injected intracranially with [2-³H]glycerol and either [methyl-¹⁴C]choline (to label the base moiety) or [U-¹⁴C]glucose (to label acyl moieties). Littermates were killed up to 90 days after injection and brain microsomes and myelin isolated. Lipids were extracted and the phosphatidylcholine was isolated by 2-dimensional TLC and hydrolyzed to its constituent moieties. The ³H in the glycerol backbone and ¹⁴C in the choline or acyl residues was quantitated. The microsomal and myelin ³H/¹⁴C ratios decreased with time with either set of precursors, indicating that labeled choline and acyl moieties were reutilized more efficiently than the glycerol backbone. The various precursors exhibited first order decay curves with half-lives for the glycerol backbone of 6 and 11 days for the microsomal and myelin fractions respectively. These results contrast with those previously obtained with identical experimental procedures when 17-day-old animals were injected. In that study, although much of the phosphatidylcholine turned over rapidly as for the older animals, by 2 weeks after injection most of the remaining phosphatidylcholine was turning over more slowly with a half-life of 13 and 25 days for microsomes and myelin respectively (Miller et al., 1977). The base and acyl moieties also had a corresponding shorter half-life in older animals relative to the slow turnover phase in younger rats.     

Evidence for two different herbicide-binding proteins at the reducing side of Photosystem II

The binding behaviour at the thylakoid membrane of the radioactively labelled phenolic inhibitors 2-iodo-4-nitro-6-[2′,3′-³H]isobutylphenol and 3,5-diiodo-4-hydroxy[U-¹⁴C]benzonitrile (ioxynil) has been studied. As judged from displacement experiments with other herbicides, phenolic herbicides and herbicides as represented best by 3-(3,4-dichlorophenyl)-1,1-dimethylurea have different binding sites at the reducing side of Photosystem II. The binding parameters of phenolic herbicides are not, or only slightly, affected by trypsin treatment of chloroplasts. In chloroplasts, besides free pigments, lipids, and the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex, a protein of molecular weight 41 000 is radioactively labelled by the photoaffinity label 4-nitro-2-azido-6-[2′,3′-³H]isobutylphenol. The amount of radioactivity bound to the 41 kDa protein is diminished if chloroplasts are incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea prior to addition of the photoaffinity label, but not if the 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol is used instead. These two compounds are characteristic representatives of inhibitiors acting at the reducing or the oxidizing site of plastoquinone, respectively. Based on these results, a model for two different herbicide-binding proteins at the reducing side of Photosystem II is presented.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.