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1.
When a metabolic system undergoes a transition between steady states, the lag or transition time of the system is determined by the aggregated lifetimes of the metabolite pools. This allows the transition time, and hence the temporal responsiveness of the system, to be estimated from a knowledge of the starting and finishing steady states and obviates the need for dynamic measurements. The analysis of temporal response in metabolic systems may be integrated with the general field of metabolic control analysis by the definition of a temporal control coefficient ( C) in terms of flux and concentration control coefficients. The temporal control coefficient exhibits summation and other properties analogous to the flux and concentration control coefficients. For systems in which static metabolite channels exits, the major kinetic advantage of channelling is a reduction in pool sizes and, as a result, a more rapid system response reflected in a reduced transition time. The extent of the channelling advantage may therefore be assessed from a knowledge of the system transition time. This reveals that no channelling advantage is achieved at high enzyme concentration (i.e., comparable to Km) or, in the case of ‘leaky’ channels, where rapid equilibrium kinetic mechanisms obtain. In the case of a perfect channel with no leakage and direct transfer of metabolite between adjacent enzyme active sites, the transition time is minimized and equal to the lifetime of the enzyme–substrate complex. 相似文献
2.
A bacterium degrading pentachlorophenol (PCP) as the only source of carbon and energy was grown in a nutristat, i.e., a continuous culture with on-line measurement and control of the substrate concentration. We improved the PCP nutristat by incorporation of a personal computer with a proportional integral derivative (PID) algorithm for controlling the medium feed pump. The controlled value deviated from the average (set-point) value by 1% maximally. In the PCP nutristat (30°C), the steadystate dilution rate, and hence, specific growth rate, showed a maximum value of 0.142±0.004 h -1 at set-point PCP concentrations between 37 and 168 M. At PCP concentrations above 168 M, the steady-state growth rate decreased because of inhibition. The growth yield coefficient was not seriously affected by the PCP concentration, suggesting that uncoupling was not the inhibitory mechanism. It was concluded that the PCP nutristat is very useful for establishing steady-state conditions that maintain growth-inhibitory PCP concentrations and high cell concentrations, conditions for which the chemostat is not suitable.Abbreviations
MCA
Metabolic control analysis
-
NTA
Nitrilotriacetic acid
-
PCP
Pentachlorophenol
-
PID
Proportional integral derivative 相似文献
3.
A model of the C
3 photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers ( Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations
c
i
intercellular CO 2 concentration
-
C
infP
supJ
control coefficient for enzyme P with respect to flux J
- DHAP
dihydroxyacetonephosphate
- E4P
erythrose 4-phosphate
- F6P
fructose 6-phosphate
- FBP
fructose 1,6-bisphosphate
- FBPase
fructose 1,6-bisphosphatase
- G3P
glyceraldehyde 3-phosphate
- G1P
glucose 1-phosphate
- G6P
glucose 6-phosphate
- Pi
inorganic phosphate
- PCR
photosynthetic carbon reduction
- PGA
3-phosphoglyceric acid
- PPFD
photosynthetically active photon flux density
-
R
n
J
response coefficient for effector n with respect to flux J
- R5P
ribose 5-phosphate
- Rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- Ru5P
ribulose 5-phosphate
- RuBP
ribulose 1,5-bisphosphate
- S7P
sedoheptulose 7-phosphate
- SBP
sedoheptulose 1,7-bisphosphate
- SBPase
sedoheptulose 1,7-bisphosphatase
- SPS
sucrose-phosphate synthase
- Xu5P
xylulose 5-phosphate
-
n
P
elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P
This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated. 相似文献
4.
The present article briefly summarizes the theoretical studies made by the authors and co-workers on the effect of inborn enzyme deficiencies on oxidative phosphorylation in intact tissues and on the genesis of mitochondrial diseases. The dynamic computer model of oxidative phosphorylation developed previously allowed to extrapolate experimental data (especially: threshold curves describing the dependence of oxygen consumption and ATP turnover on activities/concentrations of particular oxidative phosphorylation enzymes) obtained for isolated muscle mitochondria in state 3 at saturating oxygen concentrations to more physiological conditions prevailing in intact tissues. In particular, theoretical studies demonstrated that the threshold value of the relative activity/concentration of a given mitochondrial complex, below which a significant decrease in the respiration rate takes place, increases with an increase in energy demand. This fact was proposed as a possible explanation of the tissue specificity of mitochondrial diseases. Additionally, a decreased oxygen concentration was shown to increase the threshold value (and flux control coefficient) for cytochrome oxidase. We subsequently developed a model called binary mitochondria heteroplasmy, in which there are only two subpopulations of mitochondria: one wild-type and one containing only defected molecules of a given enzyme. In this model we show that a defect has a pronounced effect on oxidative phosphorylation, significantly increasing the threshold value. It was also proposed that a parallel activation in the ATP supply-demand system during an increased energy demand significantly lessens the effect of enzyme deficiencies on oxidative phosphorylation (decreases the threshold value). Finally, the necessity of substrate activation may lead to an instability in the system and to appearance of a second threshold, below which respiration suddenly drops to zero, which is equivalent to the energetic death of a cell. 相似文献
5.
In chemostat cultures of Klebsiella pneumoniae (K. aerogenes) NCTC 418 we measured the concentrations of glucose and ammonium and we varied the ratio of the (limiting) concentrations of glucose and ammonium in the feed medium. By doing this at different dilution rates we found a range where growth rate varies with either concentration in the culture when the other concentration in the culture is held constant. This proves that within this range, dual-substrate controlled growth occurs. Dual substrate-controlled growth was accompanied by yield coefficients for glucose and for ammonium that were intermediate between the yield coefficients obtained for single glucose or single ammonium limitation. We quantified the control by either substrate in terms of the flux control coefficient with respect to that substrate, where flux refers to growth rate. Dualsubstrate controlled growth is reflected by the finding that both flux control coefficients exceed zero, simultaneously. In the transition of glucose to ammonium limitation, the control gradually shifts from glucose to ammonium.Abbreviations Symbol Units Meaning s
Steady-state concentration of substrate in the culture
- S r M
Concentration of substrate in reservoir medium
- Y gDWmol -1
Yield
- D h -1
Dilution rate
- h –1
Specific growth rate
- max h –1
Maximal growth rate
- C
2
Control coefficient, of s on
- J h -1
Rate or flux
- J ATP mmolgDW -1h -1
ATP synthesis rate
- a
Anabolism
- c
Catabolism
- l
Leak 相似文献
6.
Summary A method is described for the partial purification of pyruvate carboxylase from rainbow trout liver. The enzyme has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K + causes a decrease in the apparent K
m for HCO
3
–
. AcetylCoA activation shows positive cooperativity with K
a=0.072 mM and n
H=1.78 at pH 7.7, 2.5 mM free Mg 2+, 100 mM K +, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparent K
m values for MgATP and HCO
3
–
and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a U-type response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control the rate of pyruvate carboxylation in vivo.Abbreviations
DTT
dithiothreitol
-
PMSF
phenylmethylsulfonylfluoride 相似文献
7.
Summary The concentration dependence and the pH dependence of the phosphate transport across the red cell membrane were investigated. The unidirectional phosphate fluxes were determined by measuring the 32P-phosphate self-exchange in amphotericin B (5 mol/liter) treated erythrocytes at 25°C.The flux/concentration curves display an S-shaped increase at low phosphate concentrations, a concentration optimum in the range of 150 to 200 mm phosphate and a self-inhibition at high phosphate concentrations. The apparent half-saturation concentrations, P
(0.5), range from 50 to 70 mm and are little affected by pH. The self-inhibition constants, as far as they can be estimated, range from 400 to 600 mm. The observed maximal phosphate fluxes exhibit a strong pH dependence. At pH 7.2, the actual maximal flux is 2.1×10 –6 moles·min –1·g cells –1. The ascending branches of the flux/concentration curves were fitted to the Hill equation. The apparent Hill coefficients were always in the range of 1.5–2.0. The descending branches of the flux/concentration curves appear to follow the same pattern of concentration response.The flux/pH curves were bell-shaped and symmetric with regard to their pH dependence. The pH optimum is at approximately pH 6.5–6.7. The apparent pK of the activator site is in the range of 7.0 to 7.2, while the apparent pK for the inactivating site is in the range of 6.2 to 6.5. The pK-values were not appreciably affected by the phosphate concentration.According to our studies, the transport system possesses two transport sites and probably two modifier sites as indicated by the apparent Hill coefficients. In addition, the transport system has two proton binding sites, one with a higher pK that activates and one with a lower pK that inactivates the transport system. Since our experiments were executed under self-exchange conditions, they do not provide any information concerning the location of these sites at the membrane surfaces. 相似文献
8.
The interaction of the substrate-selective irreversible inhibitor J-508 [ N-methyl- N-propargyl-(1-indanyl)-ammonium hydrochloride] with the B form of human brain monoamine oxidase has been investigated, and the conditions necessary for this inhibitor to titrate the concentration of this enzyme form determined. It was found that the concentration of monoamine oxidase-B determined in this way was the same when either benzylamine or -phenethylamine was used to assay for activity, which would indicate that this enzyme form is not heterogeneous. Furthermore, the variation in activity from sample to sample was found to be due to a variation in the concentration of available monoamine oxidase-B active centers, rather than due to a variation in the molecular turnover numbers of this enzyme form towards its amine substrates. 相似文献
9.
Summary Hood and Dare cultivars of soybean, Glycine max (L.) Merr., vary in their foliar response to ozone. The physiological basis of this variation was investigated as a function of leaf age through an analysis of ozone flux data, leaf developmental morphology, and analogue modelling techniques. At all concentrations (0.25–0.58 l l -1) and exposure times (1–4 h), resistance to O 3 flux in the gas phase of the diffusive pathway (i.e., boundary layer and stomate) did not account fully for variation in pollutant uptake rates into the leaf interior. Ozone molecules experienced a residual resistance to diffusion that is not shared by effluxing water vapor molecules. Residual resistance to O 3 flux increased with pollutant concentration and exposure time and was associated with age-dependent differences in foliar O 3 response. Leaf morphology data, including stomatal frequency and the ratio of internal to external surface area, did not help explain cultivar or age-dependent differences in O 3 flux. The extent of foliar injury was not consistently related to the magnitude of O 3 flux into the leaf interior. An analysis of the residual resistance to O 3 flux suggests that the gas and liquid phase pathways for O 3, water vapor, and carbon dioxide are not identical. 相似文献
10.
Summary
Atriplex gmelini and Chenopodium album were grown in mixed stands with various combinations of plant density and mixing ratio, and irrigated with seawater of different concentrations ( f) to formulate the effect of changing concentration on the competitive relationship between the species.In single-species stands, the mean plant weight ( w)plant density () relation for each level of seawater concentration could well be described by Shinozaki-Kira's reciprocal equation of crowding effect. On the other hand, the response of w to f followed Hozumi-Shinozaki's formulation for an optimum growth factor at respective levels of .By introducing the density conversion factor ( q) that enabled the conversion of the density of one species to that of the other species on the basis of their effects on growth of respective species, the results of mixed culture experiments could be successfully formulated by similar reciprocal equations. The dependence of q and coefficient values of the equations on seawater concentration was also formulated in a way similar to the case of pure stands.Based on all these quantitative relations, a comprehensive formulation was developed to describe the effects of plant density and seawater concentration on the growth of two species in mixed stands. The behavior of species biomass in mixed stands was then examined by means of the formulation.It was thereby demonstrated that the relative dominance of two species in a mixed stand was strongly affected not only by total plant density and density ratio between the two species but also by concentration of irrigated seawater. Even the optimum seawater concentration that resulted in the maximum species biomass differed between pure and mixed cultures. 相似文献
11.
An extracellular chitin deacetylase activity has been purified to homogeneity from autolyzed cultures of Aspergillus nidulans. This enzyme is an acidic glycoprotein with a pI of 2.75 and a 28% (wt/wt) carbohydrate content. The apparent M
r
of the enzyme estimated by SDS/PAGE and Superose 12 (f.p.l.c.) was around 27,000. The enzyme had an optimum pH at 7.0 and was stable in the pH range 4.0–7.5. Its optimum temperature of reaction was 50°C, and it was stable from 30° to 100°C after 1 h of preincubation. The enzyme hydrolyzed glycol chitin and oligomers of N-acetylgucosamine and to a lesser extent chitin, colloidal chitin, carboxymethylchitin, and an -1 3,1 6- N-acetylgalactosamine-galactan among other substances with amido groups, but the enzyme did not hydrolyze peptide bonds. The role of this enzyme could be deacetylation of chitin oligosaccharides during autolysis, after action of endochitinase on cell walls. 相似文献
12.
In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors affecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP 3 concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg 2+ ion but was inhibited at Mg 2+ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I 50 = 39 mM) was much lower than that of the neutral salts (I 50 240 mM). The enzyme exhibited a broad pH optimum between pH 6.5–8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Q 10 value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.Abbreviations CAMP
adenosine 3:5-cyclic monophosphate
- cGMP-guanosine
3:5-cyclic monophosphate
- H-89N
[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCI
- IP 3
d-myo-inositol 1,4,5-triphosphate
- I 50
the concentration of inhibitor required to reduce the velocity to one half its original value
- PKA
cAMP dependent protein kinase
- PKAc
PKA catalytic subunit
- PKA-I
PKA inhibitor protein
- PKC
calcium and phospholipid-dependent protein kinase
- PKC-I
PKC inhibitor protein
- PKG
cGMP dependent protein kinase
- mU
nmol of phosphate transferred per minute 相似文献
13.
We have previously developed a dynamic flux balance analysis of Saccharomyces cerevisiae for elucidation of genome-wide flux response to furfural perturbation (Unrean and Franzen, Biotechnol J 10(8):1248–1258, 2015). Herein, the dynamic flux distributions were analyzed by flux control analysis to identify target overexpressed genes for improved yeast robustness against furfural. The flux control coefficient (FCC) identified overexpressing isocitrate dehydrogenase (IDH1), a rate-controlling flux for ethanol fermentation, and dicarboxylate carrier (DIC1), a limiting flux for cell growth, as keys of furfural-resistance phenotype. Consistent with the model prediction, strain characterization showed 1.2- and 2.0-fold improvement in ethanol synthesis and furfural detoxification rates, respectively, by IDH1 overexpressed mutant compared to the control. DIC1 overexpressed mutant grew at 1.3-fold faster and reduced furfural at 1.4-fold faster than the control under the furfural challenge. This study hence demonstrated the FCC-based approach as an effective tool for guiding the design of robust yeast strains. 相似文献
14.
In this research the characteristics of free (partially purified) and immobilized (mould pellets of Absidia griseola) -galactosidase have been investigated. Inhibition studies of the enzyme showed that p-nitrophenol and sucrose do not have any inhibitory effect on the enzyme, but that galactose is a competitive inhibitor. In the immobilized form, inhibition was lower than in the free enzyme and the level of inhibition decreased as the temperature increased. The activity and stability of free and immobilized enzyme were investigated with respect to temperature, and the results showed that the optimal temperature range of the free enzyme was 45–50 °C, while the immobilized enzyme had an optimum at 55–60 °C. The optimum pH for the free enzyme was 6.0 and the value was decreased to 5.0 by immobilizing. The experimental effectiveness factors were found to be represented as a single function of the modified Thiele modulus, including parameters such as pellet size, enzyme concentration in the pellets and substrate concentration. Both experimental and theoretical data concerning effectiveness factors are nearly the same. 相似文献
15.
-Ketothiolase from Zoogloea ramigera I-16-M was purified 140-fold to electrophoretic homogeneity. The bacterium appeared to contain a single isoenzyme of -ketothiolase with a molecular weight of 190000, as determined by Sephadex G-200 gel filtration. The monomer molecular weight was 44000, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme thus appeared to be a tetramer with identical subunits.The enzyme showed a pH optimum of 7.5 in the condensation reaction, and 8.5 in the thiolysis reaction. The enzyme employed a Bi Bi ping pong mechanism for the forward thiolysis reaction. The apparent K
m
value for acetoacetyl coenzyme A in the thiolysis reaction was 10 M, and that for coenzyme A was 8.5 M. The apparent K
m
value for acetyl coenzyme A in the condensation reaction was 0.33 mM. The condensation reaction was inhibited by coenzyme A concentrations lower than 0.1 mM.The enzyme was stable in the presence of dithiothreitol and other SH-compounds, but was strongly inhibited by 0.4 mM p-chloromercuribenzoate.Non-Standard Abbreviation PHB
poly--hydroxybutyrate 相似文献
16.
The total Na+/K + ATP-ase activity of the thick ascending limb of the loop of Henlé may be stimulated by arginine-vasopressin (AVP). Lysine-vasopressin (LVP), oxytocin (OT), and arginine-vasotocin (AVT) produce less than 5% of the enzyme activity induced by the same concentration of AVP. Physiological concentrations of a mixture of other hormones with known activity on the kidney (T 3, T 4 aldosterone, angiotensin II, and OT) did not significantly increase total Na+/K + ATP-ase activity. Specific AVP antiserum consistently removed greater than 90% of the stimulatory effect of plasma. The concentration of AVP in plasmas from dehydrated subjects was greater than l0 times that of the same subjects hydrated. Intra-assay coefficient of variation was 35% and 52% from 200 l and 20 l of plasma respectively. The interassay coefficient of variation was 53% and 55% from plasma pools with high and low AVP content. 相似文献
17.
Bifidobacteria assimilated raffinose about 4-fold more effectively than other intestinal bacteria, and -galactosidase was active in all strains of Bifidobacteria tested. The enzyme activity of Bifidobacterium breve grown on raffinose was highly and specifically increased. Its activity was 30-fold higher than that of B. breve grown on glucose. Melibiose was also effective for production of the enzyme. The enzyme was purified to homogeneity from B. breve. It is a homodimer with Mr of about 160 kDa, and its optimum pH for activity of 5.5–6.5. The enzyme showed strict substrate specificity for -galactoside although it had slight activity for -glucoside. It hydrolysed stachyose, melibiose ( Km = 2 mM) and raffinose ( Km = 0.7 mM). 相似文献
18.
Biliverdin reductase was characterized and purified from the liver of Atlantic salmon ( Salmo salar) using a novel enzymatic staining method. The properties of the enzyme are quite different from those of mammals. The purified enzyme is a monomeric protein with a molecular weight of approximately 68 kD and an isoelectric point of around 3.8. The enzyme can utilize both NADH and NADPH as coenzyme, but the kinetic properties of the NADH-dependent and the NADPH-dependent enzyme activities are different: K
m value for biliverdin IX is 0.6 M in the NADPH system, while it is 6.8 M in the NADH system. Both enzyme activities are inhibited by excess biliverdin IX, but the NADPH-dependent enzyme activity is far more susceptible. The optimum pH for activity is 5.5 with NADPH and 6.0 with NADH. The optimum reaction temperature is 35°C. 相似文献
19.
Summary Pairs of cultured amniotic cells and maternal fibroblasts (feto-maternal pairs) were studied for hexosaminidase A (HXA) and arylsulfatase A (ASA) activity. These lysosomal enzyme activities are genetically deficient in Tay-Sachs disease and metachromatic leukodystrophy, respectively. After HXA was standardized by relating it to hexosaminidase B (HXB) activity, a feto-maternal correlation coefficient of r=0.51 ( n=32; 95% confidence limits 0.197–0.73) was found for the HXA/HXB activity quotients. This coefficient was near the 0.5 value theoretically valid for mother-child pairs, suggesting that the studied activities reflect essentially the genetic variability. The studies of ASA revealed a high variability of individual activities, which was reduced in two steps: (1) The ASA activity was related to the mean of two lysosomal reference enzyme activities, total hexosaminidase and acid -galactosidase. (2) Since the square root of ASA activity was found to follow more closely the variation of the reference activities, the square root of ASA activity over the mean reference activity was taken as a more standardized measure of ASA activity, and the quotient was treated statistically. Positive feto-maternal correlation of standardized ASA activity was obtained after the elimination of three pairs with extreme values. A correlation coefficient of r=0.42 ( n=26; 95% confidence limits 0.039–0.695) resulted.The implications of these correlation studies for the problem of heterozygote identification by quantitative enzyme assays in families deficient in HXA and ASA activity were considered. 相似文献
20.
Summary A -cyclodextrin glucosyltransferase was purified from alkalophilic Bacillus sp. No. 562 over 64-fold with a yield of 32%. Its molecular size was estimated to be 170 kDa by gel filtration and 82 kDa by SDS-PAGE, with a pI of 7.2. The enzyme showed optimum activity at 65 °C and pH 7.0. It was stable from 0 to70 °C and from pH 7.0 to 11.0. The enzyme was specifically inhibited by Fe2+ and Fe3+. 相似文献
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