Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of light and auxin

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-¹⁴C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH₃ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-¹⁴C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of ¹⁴CO₂ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inhibitory effect of ethanol on growth and solute accumulation by Saccharomyces cerevisiae as affected by plasma-membrane lipid composition

Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-³H] glucose, d-[1-¹⁴C] glucosamine, l-[U-¹⁴C] lysine or arginine, or KH₂ ³²PO₄ lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.     

Effects of salt stress on the growth,ion content,stomatal behaviour and photosynthetic capacity of a salt-sensitive species,Phaseolus vulgaris L.

Phaseolus vulgaris (cv. Hawkesbury Wonder) was grown over a range of NaCl concentrations (0–150 mM), and the effects on growth, ion relations and photosynthetic performance were examined. Dry and fresh weight decreased with increasing external NaCl concentration while the root/shoot ratio increased. The Cl^- concentration of leaf tissue increased linearly with increasing external NaCl concentration, as did K⁺ concentration, although to a lesser degree. Increases in leaf Na⁺ concentration occurred only at the higher external NaCl concentrations (100 mM). Increases in leaf Cl^- were primarily balanced by increases in K⁺ and Na⁺. X-ray microanalysis of leaf cells from salinized plants showed that Cl^- concentration was high in both the cell vacuole and chloroplast-cytoplasm (250–300 mM in both compartments for the most stressed plants), indicating a lack of effective intracellular ion compartmentation in this species. Salinity had little effect on the total nitrogen and ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) content per unit leaf area. Chlorophyll per unit leaf area was reduced considerably by salt stress, however. Stomatal conductance declined substantially with salt stress such that the intercellular CO₂ concentration (C _i) was reduced by up to 30%. Salinization of plants was found to alter the ¹³C value of leaves of Phaseolus by up to 5 and this change agreed quantitatively with that predicted by the theory relating carbon-isotope fractionation to the corresponding measured intercellular CO₂ concentration. Salt stress also brought about a reduction in photosynthetic CO₂ fixation independent of altered diffusional limitations. The initial slope of the photosynthesis versus C _i response declined with salinity stress, indicating that the apparent in-vivo activity of RuBP carboxylase was decreased by up to 40% at high leaf Cl^- concentrations. The quantum yield for net CO₂ uptake was also reduced by salt stress.Abbreviations and symbols A net CO₂ assimilation rate - C _a ambient CO₂ concentration - C _i intercellular CO₂ concentration - RuBP ribulose-1,5-bisphosphate - ¹³C ratio of ¹³C to ¹²C relative to standard limestone     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

Production of pyruvic acid from 1,2-propanediol byPseudomonas sp. strain TB-135 which does not require thiamine

Summary Pseudomonas sp. strain TB-135 produces D-lactic acid from 1,2-propanediol (1,2-PD) and requires Ca²⁺, Mg²⁺ and Fe²⁺ for growth but does not require thiamine. The strain produced pyruvic acid only under Fe²⁺-deficient conditions and the addition of Cu²⁺ increased pyruvic acid production. Under optimal conditions (0.03 ppm of FeSO₄ and 0.5ppm of CuSO₄), the strain accumulated 14 mg pyruvic acid par ml after 3 days of cultivation. The thiamine concentration in the cells grown on Fe²⁺-deficient medium was about 6% of that in the cells grown on Fe²⁺-sufficient medium, though pyruvate dehydrogenase (EC 1.2.4.1) activities of both types of cells were the same. We conclude that the low thiamine content of the cells is responsible for the acid production.     

Effect of 4-Pentenoic Acid on Intermediate Metabolism of Tetrahymena*

SYNOPSIS. The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium with acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-pentenoic acid on the incorporation of label from [1-¹⁴C]acetate, [2-¹⁴C]glycerol, [1-³⁴]ribose, [U-¹⁴C]fructose, or [1-¹⁴C]glucose into CO₂, but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest (～ 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-¹⁴C]acetyl CoA into glycogen, lipids, CO₂, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit β-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments. At least 2 mechanisms have been proposed for the action of 4-pentenoic acid: (a) reduction of the levels of acetyl CoA or free CoA and (b) direct inhibition of enzymes by 4-pentenoyl CoA or its metabolites. Although 4-pentenoic acid has little effect on acetyl-CoA metabolism in the inner mitochondrial compartment, the present data suggest that the flux through the outer mitochondrial compartment of acetyl-CoA derived from pyruvate is inhibited largely by the first, and that the glyconeogenic flux of acetyl-CoA is inhibited largely by the 2nd mechanism.     

Effect of tolbutamide on the metabolism of Tetrahymena

Tolbutamide partially inhibited the growth but increased the glycogen content of Tetrahymena pyriformis in logarithmically growing cultures. Tolbutamide slightly increased ¹⁴CO² production from [1-¹⁴C] and [6-¹⁴HC] glucose and [2-¹⁴C] pyruvate, but had little effect on the oxidation of [1-¹⁴C] acetate when any of these substrates were added to the proteose-peptone medium in which the cells had been grown. Measurement of ¹⁴CO₂ production from [1-¹⁴C] and [2-^I4C]-glyoxylate showed that this substrate was primarily oxidized via the glyoxylate cycle, with little if any oxidation occurring via the peroxisomal glyoxylate oxidase. Addition of tolbutamide inhibited the glyoxylate cycle as indicated by a marked reduction in label appearing in CO₂ and in glycogen from labeled acetate. In control cells, addition of acetate strongly inhibited the oxidation of [2-¹⁴C]-pyruvate whereas addition of pyruvate had little effect on the oxidation of [1-¹⁴C]-acetate. Acetate was more effective than pyruvate in preventing the growth inhibitory and glycogen-increasing effects of tolbutamide. The data suggest that one effect of tolbutamide may be to interfere with the transfer of isocitrate and acetyl CoA across mitochondrial membranes.     

Growth response of barley and tomato to nitrogen stress and its control by abscisic acid,water relations and photosynthesis

Barley (Hordeum vulgare L.) and tomato Lycopersicon esculentum Mill.) were grown hydroponically and examined 2, 5, and 10 d after being deprived of nitrogen (N) supply. Leaf elongation rate declined in both species in response to N stress before there was any reduction in rate of dryweight accumulation. Changes in water transport to the shoot could not explain reduced leaf elongation in tomato because leaf water content and water potential were unaffected by N stress at the time leaf elongation began to decline. Tomato maintained its shoot water status in N-stressed plants, despite reduced water absorption per gram root, because the decline in root hydraulic conductance with N stress was matched by a decline in stomatal conductance. In barley the decline in leaf elongation coincided with a small (8%) decline in water content per unit area of young leaves; this decline occurred because root hydraulic conductance was reduced more strongly by N stress than was stomatal conductance. Nitrogen stress caused a rapid decline in tissue NO ₃ ^- pools and in NO ₃ ^- flux to the xylem, particularly in tomato which had smaller tissue NO ₃ ^- reserves. Even in barley, tissue NO ₃ ^- reserves were too small and were mobilized too slowly (60% in 2 d) to support maximal growth for more than a few hours. Organic N mobilized from old leaves provided an additional N source to support continued growth of N-stressed plants. Abscisic acid (ABA) levels increased in leaves of both species within 2 d in response to N stress. Addition of ABA to roots caused an increase in volume of xylem exudate but had no effect upon NO ₃ ^- flux to the xylem. After leaf-elongation rate had been reduced by N stress, photosynthesis declined in both barley and tomato. This decline was associated with increased leaf ABA content, reduced stomatal conductance and a decrease in organic N content. We suggest that N stress reduces growth by several mechanisms operating on different time scales: (1) increased leaf ABA content causing reduced cell-wall extensibility and leaf elongation and (2) a more gradual decline in photosynthesis caused by ABA-induced stomatal closure and by a decrease in leaf organic N.Abbreviation and symbols ABA abscisic acid - c_i leaf internal CO₂ concentration - L_p root hydraulic conductance     

Effect of Chlorpromazine on Active Transport of Amino Acids in Tetrahymena*

SYNOPSIS. Low concentrations of chlorpromazine (～0.01 mM) inhibit growth and nucleic acid synthesis in the ciliate Tetrahymena pyriformis. Brief exposure of the cells to, e.g. 0.018 mM chlorpromazine, had very little effect on ¹⁴CO₂ production or on label incorporation into glycogen from [1-¹⁴C]glucetate, [6–14C]glucose, or [1-¹⁴C]leucine, but 17-h exposure of stationary phase cultures to this drug caused marked alterations in metabolism, including an almost complete loss of ability to decarboxylate L-[1-¹⁴C]leucine and L-[1-¹⁴C]tyrosine. It was shown that loss of ability to decarboxylate these amino acids results from loss of ability to transport them.     

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.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

盐分胁迫对啤酒大麦幼苗生长、离子平衡和根际pH变化的影响

盐分胁迫不仅影响植物的生长,而且会影响植物根际微域环境。根际pH的改变对土壤养分的有效性和微生物群落组成的变化有重要影响。为了探究啤酒大麦幼苗对不同类型盐分胁迫的生理生态响应机制和根际pH变化影响的生理机制,采用水培法,通过不同类型盐分(对照、混合Na盐、混合Cl盐和NaCl)胁迫处理啤酒大麦幼苗,对其生长、离子平衡和根际pH变化进行了研究。结果表明,1)在3种不同类型盐分胁迫下,啤酒大麦幼苗地上部干重、含水量均有所降低,而根冠比增加,尤其在NaCl胁迫下啤酒大麦幼苗地上部干重较对照显著降低了17.88%,而根干重和根冠比则分别增加了19.12%和43.86%。不同类型盐分胁迫抑制了啤酒大麦幼苗根长的生长,尤其在混合Na盐胁迫下根长降低明显(P0.05),但促进了根表面积和根体积的增加,尤其在混合Cl盐胁迫下,根表面积和根体积分别增加了41.76%和84.38%。2)不同类型盐分胁迫下啤酒大麦幼苗地上部离子平衡发生改变,在混合Na盐和NaCl胁迫下啤酒大麦幼苗主要吸收Na~+,地上部K~+/Na~+、Ca~(2+)/Na~+和Mg~(2+)/Na~+显著降低;混合Cl盐和NaCl胁迫下则过量吸收Cl~-,抑制了H_2PO_4~-、NO_3~-和SO_4~(2-)的吸收。3)在混合Na盐、混合Cl盐和NaCl盐分胁迫下,啤酒大麦幼苗对阴离子的吸收总量高于对阳离子的吸收总量,离子平衡计算结果表明根际呈碱化现象,与原位显色结果一致,且在混合Cl盐胁迫下根际碱化程度最大。     

In vivo incorporation of radioactive metabolites by Golgi apparatus and other cell fractions of onion stem

Incorporation in vivo of various ¹⁴C-labeled substrates into dictyosomes of onion (Allium cepa) stem was determined, and comparisons were made with other cell fractions on a nitrogen basis. Tissue explants were incubated for varying times in the presence of the radioactive metabolites supplied in the external medium. Fractions were then obtained from homogenates stabilized with glutaraldehyde. Purified fractions containing dictyosomes (individual stacks of cisternae) of the Golgi apparatus were obtained by centrifugation in a sucrose gradient also yielding a smooth membrane fraction free of dictyosomes. Dictyosomes were preferentially labeled with choline-1,2-¹⁴C and acetate-2-¹⁴C, suggesting that plant Golgi apparatus participate in the synthesis or modification of membrane lipids. Dictyosomes were also labeled with glucose-U-¹⁴C and leucine-U-¹⁴C, but on a molar basis incorporation was less than with choline or acetate.     

Effect of vitamin B6 deficiency on pancreatic acpinar cell function

The present study was designed to determine the effect of vitamin B₆ deficiency on pancreatic acinar cell function. Rats were either fed $\text{ad}$$\text{lib}$ or rendered B₆-deficient by a purified B₆-deficient diet; half of the latter being replenished with IP pyridoxine before sacrifice. Body weight, pancreatic weight, RNA and DNA content were decreased in B₆-deficient animals. These changes were considered to be due to inanition resulting from decreased food intake. Amylase content of pancreas in B₆-deficient animals was less compared with B₆-replenished animals. Although slightly higher in B₆-deficient animals, the incorporation of L-phenylalanine¹⁴C into total tissue proteins was not significantly different in the three groups of animals. On B₆-replenishment, incorporation of L-phenylalanine¹⁴C into nascent proteins was diminished in spite of higher tissue amylase and protein content. Vitamin B₆ deficiency decreased total RNA content and adenine-8-¹⁴C incorporation into RNA. DNA content was diminished but incorporation of thymidine-2-¹⁴C into DNA was increased. On replenishment with B₆, thymidine-2-¹⁴C incorporation decreased significantly compared to control animals. Secretion of amylase was diminished commensurate with decreased content. It is concluded from these studies that B₆-deficiency induced DNA injury, decreased RNA turnover and increased protein turnover resulting in diminished amylase content. These data indicate that B₆-deficiency so frequently encountered in alcoholism may contribute to the pancreatic injury in this clinical condition.     

Synthesis of phenylalanine ammonia-lyase in anthocyanin-containing and anthocyanin-free callus cells of Daucus carota L.

When callus cells of Daucus carota are grown on a medium containing gibberellic acid (GA₃) in a physiological concentration of 3x10^-6 M the cells cease to accumulate anthocyanins. This anthocyanin-free cell line has a very low activity of phenylalanine ammonia-lyase. After density labelling with D₂O an intensive de novo synthesis of the phenylalanine ammonia-lyase (E.C. 4.3.1.5; PAL) in the anthocyanin-containing cells does occur. 58% of the C-bound H-atoms are replaced by deuterium. The anthocyanin-free cells show only a very low enzyme synthesis which is difficult to detect with density labelling experiments. To ascertain that de novo synthesis occurs in the anthocyanin-free cells, the incorporation of ¹⁴C-labelled amino acids into the partially purified enzyme protein was measured after separation of the protein a) in CsCl gradients and b) on polyacrylamide gels. In both cases the enzyme bears ¹⁴C-label. These results suggest that in the anthocyanin-free cells de novo synthesis of PAL is still occuring but the synthesis is reduced in comparison to the anthocyanin-containing cells.Abbreviations GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase (E.C.4.3.1.5) - DCb anthocyanin-containing cells - DCw anthocyanin-free cells     

Synthesis of Sterols In Herpetomonas Samuelpessoai: Influence of Growth Conditions*

Ergosterol was the only sterol detected in Herpetomonas samuelpessoai grown in a defined, lipid-free medium. When cultivated in a complex medium, this flagellate was found to contain 6 additional sterols. As measured by incorporation of L-[Me-¹⁴C]methionine, in the absence of acetate, the sterol synthesis was greater at 28 C than at 37 C; in the presence of acetate, however, this synthesis was greater at 37 C. When [2-¹⁴C]acetate was used as the sterol precursor, the synthesis level at 37 C exceeded that at 28 C.     

Control of starch and exocellular polysaccharides biosynthesis by gibberellic acid with cells of sweet potato cultured in vitro

The regulation of starch synthesis and exocellular polysaccharide synthesis by GA₃ was studied with cells of sweet potato grown as suspension in glycerol medium. In the presence of GA₃, and under normal cell growth, starch formation was inhibited. The incorporation activity (starch synthesis) from ADP-[¹⁴C] glucose or UDP-[¹⁴C] glucose with GA₃ treated cells was reduced. On the other hand, the synthesis of exocellular polysaccharides composed of glucose, galactose, mannose and arabinose etc., was stimulated and a clear increase of the Man/Ara ratio was observed in the presence of GA₃. These results may indicate that GA₃ affects the regulation of starch synthesis and exocellular polysaccharide synthesis.     

In vivo 13C NMR analysis of acetate metabolism in Thiobacillus versutus under denitrifying conditions

The disappearance of 2-¹³C-acetate and the subsequent incorporation of label into cellular metabolites were followed in denitrifying cells of Thiobacillus versutus by ¹³C NMR spectroscopy. In cells grown under acetate-limitation, the specific rate of consumption was idependent of the density of the cell suspension. An isotopic steady state was reached within 30 min if sufficient substrate was added to the cell suspension. In cells grown under nitrate-limitation, the consumption of 2-¹³C-acetate proceeded at a significantly lower rate. The decrease and final disappearance of 2-¹³C-acetate were accompanied by incorporation of ¹³C into glutamate, glutamine, and by the release of labeled HCO ₃ ^– and CO₂. The appearance of a broad resonance being the methyl endgroup of poly-3-hydroxybutyrate (PHB) was indicative for PHB mobilization during the incubation. The sequence of label incorporation and the distribution among the various carbon nuclei were consistent with the operation of the tricarboxylic acid cycle.