Effects of training duration on substrate turnover and oxidation during exercise

Phillips, S. M., H. J. Green, M. A. Tarnopolsky, G. J. F. Heigenhauser, R. E. Hill, and S. M. Grant. Effects of training duration on substrate turnover and oxidation during exercise. J. Appl. Physiol. 81(5):2182-2191, 1996.Adaptations in fat and carbohydrate metabolismafter a prolonged endurance training program were examined using stableisotope tracers of glucose([6,6-²H₂]glucose),glycerol([²H₅]glycerol),and palmitate([²H₂]palmitate).Active, but untrained, males exercised on a cycle for 2 h/day[60% pretraining peak O₂consumption (O_{2 peak}) = 44.3 ± 2.4 ml ^· kg^{1 ·} min¹]for a total of 31 days. Three cycle tests (90 min at 60% pretraining O_{2 peak}) wereadministered before training (PRE) and after 5 (5D) and 31 (31D) daysof training. Exercise increased the rate of glucose production(R_a) and utilization(R_d) as well as the rate oflipolysis (glycerol R_a) and freefatty acid turnover (FFA R_a/R_d).At 5D, training induced a 10% (P < 0.05) increase in total fat oxidation because of an increase inintramuscular triglyceride oxidation (+63%,P < 0.05) and a decreased glycogenoxidation (16%, P < 0.05).At 31D, total fat oxidation during exercise increased a further 58%(P < 0.01). The pattern of fatutilization during exercise at 31D showed a reduced reliance on plasmaFFA oxidation (FFA R_d) and agreater dependence on oxidation of intramuscular triglyceride, whichincreased more than twofold (P < 0.001). In addition, glucose R_aand R_d were reduced at all timepoints during exercise at 31D compared with PRE and 5D. We concludethat long-term training induces a progressive increase in fatutilization mediated by a greater oxidation of fats from intramuscularsources and a reduction in glucose oxidation. Initial changes arepresent as early as 5D and occur before increases in muscle maximalmitochondrial enzyme activity [S. M. Phillips, H. J. Green, M. A. Tarnopolsky, G. J. F. Heigenhauser, and S. M. Grant.Am. J. Physiol. 270 (Endocrinol. Metab. 33):E265-E272, 1996].

     

Specific rates of substrate oxidation and product formation in autotrophically growingChromatium vinosum cultures

Eighty-six species of fungi belonging to sixty-four genera were examined for their ability to metabolize naphthalene. Analysis by thin-layer and high pressure liquid chromatography revealed that naphthalene metabolism occurred in forty-seven species belonging to thirty-four genera from the major fungal taxa. All organisms tested from the order Mucorales oxidized naphthalene with species of Cunninghamella, Syncephalastrum and Mucor showing the greatest activity. Significant metabolism was also observed with Neurospora crassa, Claviceps paspali and four species of Psilocybe. The predominant metabolite formed by most organisms was 1-naphthol. Other products identified were, 4-hydroxy-1-tetralone, trans-1,2-dihydroxy-1,2-dihydronaphthalene, 2-naphthol, 1,2-and 1,4-naphthoquinone.     

Actual and potential rates of substrate oxidation and product formation in continuous cultures ofChromatium vinosum

Kinetics of electron-donor oxidation, storage-polymer formation and growth were studied in continuous cultures ofChromatium under conditions of balanced growth as well as during transient states.Under steady-state conditions, glycogen was accumulated at all dilution rates. This observation is consistent with previously postulated ideas about an ineffective glycogen-synthesis regulation.Upon perturbing the steady states, brought about by injection of extra sulfide into steady-state cultures, the following phenomena were observed immediately, irrespective of the dilution rate: the specific rate of sulfide oxidation increased to the value found in batch cultures, the sulfur-oxidation rate was decreased, the specific glycogen-synthesis rate increased, the increment being higher the lower the dilution rate, but an increase in the specific growth rate, if any, was below the limit of detection. The inverse relationship between the specific rates of glycogen synthesis and growth after removing the substrate limitation is to be explained by a shortage of intermediates, rather than by a growth-rate dependent intrinsic glycogen-synthesis limitation, because upon complete inhibition of growth a further increase in the rate of glycogen synthesis was observed. Essayed in this way, identical glycogen-synthesis rates were found at all dilution rates.Competitive advantages of such an apparently not adapted metabolism in environments with diurnal fluctuations in substrate concentrations are discussed.Non-Standard Abbreviations N_c cell nitrogen - TS total sugar - PHB poly--hydroxybutyrate - D dilution rate - S_R reservoir concentration of the growth-limiting substrate - CAP chloramphenicol     

Prolonged exercise after diuretic-induced hypohydration: effects on substrate turnover and oxidation

To determine the influence of a diuretic-induced reduction in plasma volume (PV) on substrate turnover and oxidation, 10 healthy young males were studied during 60 min of cycling exercise at 61% peak oxygen uptake on two separate occasions > or =1 wk apart. Exercise was performed under control conditions (CON; placebo), and after 4 days of diuretic administration (DIU; Novotriamazide; 100 mg triamterene and 50 mg hydrochlorothiazide). DIU resulted in a calculated reduction of PV by 14.6 +/- 3.3% (P < 0.05). Rates of glucose appearance (R(a)) and disappearance (R(d)) and glycerol R(a) were determined by using primed constant infusions of [6,6-(2)H]glucose and [(2)H(5)]glycerol, respectively. No differences in oxygen uptake during exercise were observed between trials. Main effects for condition (P < 0.05) were observed for plasma glucose and glycerol, such that the values observed for DIU were higher than for CON. No differences were observed in plasma lactate and serum free fatty acid concentrations either at rest or during exercise. Hypohydration led to lower (P < 0.05) glucose R(a) and R(d) at rest and at 15 and 30 min of exercise, but by 60 min, the effects were reversed (P < 0. 05). Hypohydration had no effect on rates of whole body lipolysis or total carbohydrate or fat oxidation. A main effect for condition (P < 0.05) was observed for plasma glucagon concentrations such that larger values were observed for DIU than for CON. A similar decline in plasma insulin occurred with exercise in both conditions. These results indicate that diuretic-induced reductions in PV decreases glucose kinetics during moderate-intensity dynamic exercise in the absence of changes in total carbohydrate and fat oxidation. The specific effect on glucose kinetics depends on the duration of the exercise.     

Functional characterization of the promiscuous prenyltransferase responsible for furaquinocin biosynthesis: identification of a physiological polyketide substrate and its prenylated reaction products

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) that exhibits antitumor activity and is produced by the Streptomyces sp. strain KO-3988. Bioinformatic analysis of furaquinocin biosynthesis has identified Fur7 as a possible prenyltransferase that attaches a geranyl group to an unidentified polyketide scaffold. Here, we report the identification of a physiological polyketide substrate for Fur7, as well as its reaction product and the biochemical characterization of Fur7. A Streptomyces albus transformant (S. albus/pWHM-Fur2_del7) harboring the furaquinocin biosynthetic gene cluster lacking the fur7 gene did not produce furaquinocin but synthesized the novel intermediate 2-methoxy-3-methyl-flaviolin. After expression and purification from Escherichia coli, the recombinant Fur7 enzyme catalyzed the transfer of a geranyl group to 2-methoxy-3-methyl-flaviolin to yield 6-prenyl-2-methoxy-3-methyl-flaviolin and 7-O-geranyl-2-methoxy-3-methyl-flaviolin in a 10:1 ratio. The reaction proceeded independently of divalent cations. When 6-prenyl-2-methoxy-3-methyl-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del7, furaquinocin production was restored. The promiscuous substrate specificity of Fur7 was demonstrated with respect to prenyl acceptor substrates and prenyl donor substrates. The steady-state kinetic constants of Fur7 with each prenyl acceptor substrate were also calculated.     

Heterocycle formation in vibriobactin biosynthesis: alternative substrate utilization and identification of a condensed intermediate

The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin. When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step. This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases. Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate. We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

NovJ/NovK catalyze benzylic oxidation of a beta-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis

The bicyclic coumarin ring in the aminocoumarin natural product antibiotics that target bacterial DNA gyrase is assembled from tyrosine by nonribosomal peptide synthetase logic. Tyrosine has previously been shown to be activated and installed as a phosphopantetheinyl thioester on the thiolation domain of NovH and then hydroxylated on the benzylic carbon by the heme protein NovI, generating beta-OH-Tyr-S-NovH. This aminoacyl-S-protein is the substrate for the next two orfs, Streptomyces sphaeroides NovJ and NovK, that have now been expressed in and purified from Escherichia coli as a J2K2 heterotetramer. NovJ/NovK use NADP as an electron acceptor to oxidize the beta-OH of the tyrosyl moiety to yield the tethered beta-ketotyrosyl-S-NovH. The enol tautomer is the form that predominates in the subsequently cyclized aminocoumarin scaffold. The labile beta-ketotyrosyl thioester moiety was identified by hydrolytic release from NovH, analysis by mass spectroscopy, and comparison with a synthetic sample. We also have identified a residue in NovJ that when mutated results in a 50-fold reduction in catalytic activity.     

The peroxidase-catalysed oxidation of a chalcone and its possible physiological significance

1. Crystalline horseradish peroxidase catalysed the oxidation of 2',4,4'-trihydroxychalcone (isoliquiritigenin) in the presence of trace amounts of hydrogen peroxide under aerobic conditions. One atom of oxygen was consumed for each molecule of substrate. 2. The reaction course comprised a lag phase and a linear phase. The optimum pH for the linear phase of the reaction was about 7.5. The length of the lag phase decreased with increasing pH. It is suggested that the chalcone anion is the actual substrate for the reaction. 3. No evidence for the production of reducing free radicals or perhydroxyl radicals during the reaction could be found. 4. 4',7-Dihydroxyflavonol and 4',6-dihydroxyaurone were isolated from the reaction mixture. The immediate products of the reaction may have included 3,4',7-trihydroxyflavanone and 4',6-dihydroxy-2-(alpha-hydroxybenzyl)coumaran-one, which can be readily converted non-enzymically into the flavonol and aurone respectively. 5. A similar reaction was catalysed by cell-free extracts of hypocotyls of Phaseolus vulgaris. 6. The physiological significance of the reaction is discussed in terms of a possible free-radical mechanism. An analogy may exist between flavonoid biosynthesis and lignin formation.     

Single turnover of substrate-bound ferric cysteine dioxygenase with superoxide anion: enzymatic reactivation, product formation, and a transient intermediate

Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O(2)-dependent oxidation of L-cysteine (Cys) to produce cysteine sulfinic acid (CSA). In this study we demonstrate that the catalytic cycle of CDO can be "primed" by one electron through chemical oxidation to produce CDO with ferric iron in the active site (Fe(III)-CDO, termed 2). While catalytically inactive, the substrate-bound form of Fe(III)-CDO (2a) is more amenable to interrogation by UV-vis and EPR spectroscopy than the 'as-isolated' Fe(II)-CDO enzyme (1). Chemical-rescue experiments were performed in which superoxide (O(2)(?-)) anions were introduced to 2a to explore the possibility that a Fe(III)-superoxide species represents the first intermediate within the catalytic pathway of CDO. In principle, O(2)(?-) can serve as a suitable acceptor for the remaining 3-electrons necessary for CSA formation and regeneration of the active Fe(II)-CDO enzyme (1). Indeed, addition of O(2)(?-) to 2a resulted in the rapid formation of a transient species (termed 3a) observable at 565 nm by UV-vis spectroscopy. The subsequent decay of 3a is kinetically matched to CSA formation. Moreover, a signal attributed to 3a was also identified using parallel mode X-band EPR spectroscopy (g ~ 11). Spectroscopic simulations, observed temperature dependence, and the microwave power saturation behavior of 3a are consistent with a ground state S = 3 from a ferromagnetically coupled (J ~ -8 cm(-1)) high-spin ferric iron (S(A) = 5/2) with a bound radical (S(B) = 1/2), presumably O(2)(?-). Following treatment with O(2)(?-), the specific activity of recovered CDO increased to ~60% relative to untreated enzyme.     

Modeling of microbial substrate conversion,growth and product formation in a recycling fermentor

Paracoccus denitrificans and Bacillus licheniformis were grown in a carbon- and energy source-limited recycling fermentor with 100% biomass feedback. Experimental data for biomass accumulation and product formation as well as rates of carbon dioxide evolution and oxygen consumption were used in a parameter optimization procedure. This procedure was applied on a model which describes biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of the biomass growth rate. The fitting procedure yielded two growth domains for P. denitrificans. In the first domain the values for the maximal growth yield and the maintenance coefficient were identical to those found in a series of chemostat experiments. The second domain could be described best with linear biomass increase, which is equal to a constant growth yield. Experimental data of a protease producing B. licheniformis also yielded two growth domains via the fitting procedure. Again, in the first domain, maximal growth yield and maintenance requirements were not significantly different from those derived from a series of chemostat experiments. Domain 2 behaviour was different from that observed with P. denitrificans. Product formation halts and more glucose becomes available for biomass formation, and consequently the specific growth rate increases in the shift from domain 1 to 2. It is concluded that for many industrial production processes, it is important to select organisms on the basis of a low maintenance coefficient and a high basic production of the desired product. It seems less important that the maximal production becomes optimized, which is the basis of most selection procedures.     

Simulation of cell growth,substrate consumption and product formation with recombinant Escherichia coli

Summary The growth of recombinant Escherichia coli, consumption of different amino acids and glucose as well as fusion protein formation are simulated by a structured mathematical model and compared with measurements carried out in a stirred tank reactor under well-controlled conditions. The model parameters were identified based on the variation in cell concentration, particular amino acids, and glucose as well as intracellular products during cultivation. The dependence of the model parameters on cultivation time and culture conditions (temperature, medium composition, dissolved oxygen concentration) are discussed.Offprint requests to: K. Schügerl     

Enzymatic protein carboxyl methylation at physiological pH: cyclic imide formation explains rapid methyl turnover

At pH 7.4, 37 degrees C, bovine brain protein carboxyl methyltransferase transiently methylates deamidated adrenocorticotropin. The methylation occurs at the alpha-carboxyl group of an atypical beta-carboxyl-linked isoaspartyl residue (position 25). Several lines of evidence indicate that the immediate product of demethylation is an aspartyl cyclic imide involving positions 25 and 26. The evidence includes (1) the rapid rate of methyl ester hydrolysis, which is consistent with intramolecular catalysis, (2) the inability of the demethylated product to be remethylated, (3) the charge of this product, and (4) its rate of breakdown. The eventual hydrolysis of the cyclic imide produces a 30/70 mixture of peptides containing either alpha- or beta-carboxyl-linked aspartyl residues, respectively. Cyclic imide formation is nonenzymatic and can explain the unusual lability of mammalian protein methyl esters in general. These findings suggest that protein carboxyl methylation in mammalian tissues is not a simple on/off reversible modification as it apparently is in chemotactic bacteria. Carboxyl methylation may serve to activate selected protein carboxyl groups for subsequent longer lasting modifications, possibly subserving a role in protein repair, degradation, cross-linking, or some other as yet undiscovered alteration of protein structure.     

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.