Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Differential effects of insulin, epinephrine, and glucagon on rat hepatocyte mitochondrial activity

Oxidation of [2,3-14C]succinate carbons in the mitochondrial Krebs cycle was used as a probe to investigate the effects of insulin, epinephrine, glucagon, and 2,4-dinitrophenol (2,4-DNP) on isolated rat hepatocytes. Epinephrine, glucagon, and 2,4-DNP had a far greater stimulatory effect on 14CO2 formation from [2,3-14C]succinate than insulin. Unlike insulin, epinephrine and glucagon had no significant effect on the anabolic utilization of succinate carbons for protein synthesis. Our results suggest that although epinephrine, glucagon, and 2,4-DNP enhance the movement of tracer carbons through the Krebs cycle, only insulin is capable of enhancing amphibolite utilization for protein synthesis.     

Insulin as a probe of mitochondrial metabolism in situ

Our previous studies of insulin action have led us to the finding that insulin acts specifically on the mitochondrial Krebs cycle to stimulate, by 30%, the oxidation of carbons 2 and 3 of pyruvate to CO₂. Insulin also stimulates the oxidation of both carbons of acetate. These carbons can be converted to CO₂ only after passing through all of the reactions of the Krebs cycle more than once. Carboxyl groups, such as number 1 of pyruvate, are oxidized to CO₂ without any effect of insulin, and can be converted to CO₂ by extramitochondrial enzyme. We conclude that insulin must act on the complete intramitochondrial cycle and not on the four enzymes of the Krebs cycle which are present in the cytoplasm. The path taken by those carbons affected by insulin is traced through the complete Krebs cycle, and the necessity for this effect to be mitochondrial has been verified by demonstration of the same specific effect of insulin on the oxidation of the 2 and 3 carbons of succinate. The use of this phenomenon is proposed for the study not only of human diabetes, but of all mitochondrial disorders, by using ¹⁴C specifically labeled tracers in culture or biopsy material, or ¹³C labeled tracer material in vivo. (Mol Cell Biochem 174: 91–96, 1997)     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

The biogenesis of ethylene in Penicillium digitatum

The origin of ethylene in Penicillium digitatum has been shown to be intimately associated with the Krebs cycle. 2-Ketoglutaric acid and glutamic acid are the most efficient precursors of ethylene, which is derived from carbons 3 and 4 of these substrates as a unit. However, which of these is the immediate precursor has not been established. Since 2-ketoglutaric acid is a very efficient precursor and succinic acid is an inefficient one, 2-ketoglutaric acid must be the branching point at which the pathway of ethylene biosynthesis leads away from the Krebs cycle. This conclusion is in full agreement with the following observations: Three of the four hydrogen atoms of the ethylene molecule were derived from protons of the medium; C-2 but not C-1 of acetate was incorporated into ethylene; and [2,3-¹⁴C]succinic acid but not [2,3-³H]succinic acid was incorporated.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Preparation of 1-acyl-2-succinyl glycero-3-phosphorylcholine and evidence against its involvement in succinate dehydrogenase action

1-Acyl-2-succinyl glycero-3-phosphorylcholine (GPC) was synthesized and its properties described. Although 1-acyl-2-succinyl GPC is a good substrate for succinate dehydrogenase, experiments on the incorporation of [2,3-¹⁴C]succinate into mitochondrial lipids gave no evidence to indicate that it is an intermediate in the enzymic oxidation of succinate to fumarate, as has been suggested earlier.     

Role of transamination in the mobilization of respiratory substrates in germinating seeds of castor oil plants

The metabolism of 1,4-¹⁴C-succinate and 2,3-¹⁴C-succinate and the activity of succinic semialdehyde dehydrogenase (EC 1.2.1.16) were studied in germinating seeds of castor oil plants (Ricinus communis L.). Succinate metabolism involved succinate dehydrogenase and was sensitive to metabolites of the γ-aminobutyric acid shunt. Considerable accumulation of the label in amino acids reflected the progression of transamination reactions. Succinic semialdehyde dehydrogenase was purified from the endosperm of castor oil plants. Kinetic characteristics of the enzyme were evaluated. Our study indicates that the mobilization of respiratory substrates during germination of castor oil plants is related to active transamination of ketoacids in the Krebs cycle and involves the γ-aminobutyric acid shunt.     

The role of the Krebs cycle in the generation of intramitochondrial reducing equivalents for the 11 beta-hydroxylation of deoxycorticosterone in isolated rat adrenal cells

Isolated rat adrenal cells were used to study the possible pathways of intramitochondrial NADPH generation for 11β-hydroxylation of 11-deoxycorticosterone. Pyruvate was efficiently utilized by the mitochondria as shown by evolution of ¹⁴CO₂ from [1-¹⁴C]- and [2-¹⁴C]pyruvate. Citrate, isocitrate, succinate, and malate were not utilized by intact cells due to their inability to permeate the plasma membrane. For every mole of corticosterone formed, 1.9 and 0.8 moles of ¹⁴CO₂ were formed from [1-¹⁴C]- and [2-¹⁴C]pyruvate, respectively, indicating that pyruvate dehydrogenase was quite active and supplied acetyl C?oA to the Krebs cycle. Fluorocitrate and 2,4-dinitrophenol inhibited 11β-hydroxylation of 11-deoxycorticosterone as well as the production of ¹⁴CO₂ from [2-¹⁴C]pyruvate. Comparison of data with the two inhibitors showed that for the same percentage of inhibition of ¹⁴CO₂ production, the inhibition of 11β-hydroxylation was greater with 2,4-dinitrophenol than with fluorocitrate. It is concluded that operation of the Krebs cycle may be essential for 11β-hydroxylation to occur primarily because NADH generated by the cycle provides ATP, via the respiratory chain, as well as the substrate for the energy-linked transhydrogenase that forms NADPH. The NADPH required for 11β-hydroxylation seems to be derived to a large extent via the energy-linked transhydrogenase.     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.     

The Mechanism of Acetate and Pyruvate Oxidation by Trypanosoma cruzi

The epimastigote or culture form of Trypanosoma cruzi oxidizes [3-¹⁴C] pyruvate and [2-¹⁴C] acetate to ¹⁴CO₂ without an apparent increase in overall respiration. This oxidation takes place through the tricarboxylic acid cycle as shown by (a) the incorporation of substrate ¹⁴C into cycle intermediates; (b) the earlier liberation of acetate carboxyl carbon as CO₂; and (c) the characteristic intramolecular distribution of pyruvate and acetate carbon atoms in the skeletal carbon of aspartic and glutamic acids. Upon oxidation of [3-¹⁴C] pyruvate and [2-¹⁴C] acetate, two of the products, alanine and glutamic acid, are found to account for more than 50% of incorporated ¹⁴C; labeling of alanine predominates with [3-¹⁴C] pyruvate while labeling of glutamic acid predominates with [2-¹⁴C] acetate. Using [1- or 6-¹⁴C] glucose as substrate, the pattern of ¹⁴C distribution in soluble metabolites closely resembles that obtained with [3-¹⁴C] pyruvate, in accordance with the joint operation of the Embden-Meyerhof pathway and Krebs cycle. The cycle operation depends on electron transport through the mitochondrial respiratory chain, since antimycin A, at a relatively low concentration, inhibits the oxidation of [2-¹⁴C] acetate to ¹⁴CO₂, to the same extent as the parasite respiration. Though functional in T. cruzi epimastigotes, the oxidative role of the Krebs’ cycle is apparently limited by the absence of an efficient oxidative apparatus. The cycle operation does, however, constitute an important source of skeletal carbon for the biosynthesis of amino acids and can contribute to the process of glycogenesis.     

Extracellular phosphate requirement for insulin action on isolated rat hepatocytes

Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30 degrees in the presence of tracer [2,3-14C]succinate and a 0.5 mM concentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3-14C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of 32P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state.     

Partial characterization of the pathway from propionate to acetate in the cabbage looper,Trichoplusia ni

Experiments were performed to characterize the metabolism of propionate to acetate in the cabbage looper Trichoplusia ni and correlate the results with vitamin B₁₂ levels. Fourth and fifth instar larvae contain 2–4 pg vitamin B₁₂/mg dry wt whereas pupae and adults do not contain detectable amounts. In vivo studies as a function of time in larvae, pupae and adults gave evidence that [2-¹⁴C]propionate was converted to 3-hydroxypropionate and then to acetate, which subsequently labeled Krebs cycle intermediates. Radioactivity from [1-¹⁴C]propionate was recovered only in the propionate and 3-hydroxypropionate fractions, and not in acetate or Krebs cycle intermediates, suggesting that carbon 1 of propionate was lost as carbon dioxide and that carbons 2 and 3 of propionate were retained during conversion to acetate. The enzymes of this pathway were located entirely in the mitochondrial fraction. Cyanide inhibited the metabolism of propionate to 3-hydroxypropionate and acetate in mitochondrial preparations, whereas carbon monoxide did not. [2,3-¹⁴C]Acrylic acid was metabolized to 3-hydroxypropionate, which is consistent with a dehydrogenase converting propionate to acrylate which is then hydrated to 3-hydroxypropionate and then oxidized and decarboxylated to acetate.     

Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate

It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.     

Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-¹³C]- and [1,4-¹³C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-¹³C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber. Correspondence to: A. J. Anderson     

Oxidation of Alanine, Acetate, Glutamate, and Succinate by Digitonin-Permeabilized Leishmania major Promastigotes

Leishmania major promastigotes were treated with digitonin and the rates at which [1 -¹⁴C]acetate, [1,4-¹⁴C]succinate, [1-¹⁴C]glutamate, and [U-¹⁴C]alanine are oxidized were measured in the presence of suitable cofactors. Acetate was oxidized at the lowest rate of the four substrates examined, even in the presence of added NAD, CoA, ADP and acetyl-CoA synthase. Its rate of oxidation was negligible if the permeabilized cells were washed before the cofactors were added, indicating the requirement for an as yet unknown factor. Succinate was oxidized at a rate much higher than the very slow rate at which it is oxidized by intact cells. Its rate of oxidation was strongly inhibited by antimycin A, but that of glutamate was scarcely affected. Fumarate inhibited the rate of oxidation of acetate, glutamate, and succinate, but increased that of alanine, Ca⁺⁺ inhibited the rates of oxidation of alanine and succinate, but not of acetate or glutamate. Increasing the osmolality by addition of mannitol partially inhibited the rate of oxidation of alanine but had little effect on that of glutamate. These results show that appreciable transaminase activity remains in the permeabilized cells and support earlier data indicating the presence of a branched NAD-to-cytochrome oxidase system. These results also provide preliminary information on the sensitivity of the two branches to Ca⁺⁺, hyperosmolality, and Krebs cycle intermediates     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Incorporation of [2,3-13C]succinate into methyl-branched alkanes in a termite

Carbon-13 nuclear magnetic resonance was used to examine the incorporation of [2,3-¹³C]succinate into methylalkanes in the termite Zootermopsis angusticollis. Carbons 2 and 3 of succinate were preferentially incorporated into the branching methyl group(s) and the tertiary carbon(s) of monomethyl- and dimethylalkanes. These data support a pathway in which succinate is metabolized to methylmalonyl-coenzyme A, and is then incorporated in place of malonyl-coenzyme A at specific points during chain elongation.