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1.
Diffusion rates of cell surface antigens of mouse-human heterokaryons. I. Analysis of the population 总被引:7,自引:3,他引:4
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《The Journal of cell biology》1977,75(2):475-482
The rate of appearance, in a newly formed heterokaryon population, of cells bearing completely intermixed mouse and human surface antigens may be used to estimate diffusion constants for antigens on individual cells. From this estimate, it appears that the surface antigens in most cells do not diffuse at the rate expected, but rather move more slowly, by a factor of ten or more, than expected from either measured or calculated diffusion constants for proteins freely mobile in the plane of a lipid membrane. Differences in diffusion rates between cells are not due to effects of Sendai virus, or of trypsin. Restrictions on diffusion are apparently not due to cytochalasin B- or Colcemid- sensitive elements. 相似文献
2.
Dithionite causes the depletion of dioxygen from suspensions of erythrocytes by reduction of the external dioxygen and not by diffusion into the cell. The molar enthalpy for the reduction shows a small difference with respect to the values found for free hemoglobin; and the normal stoichiometry of 2 moles dithionite/mole dioxygen found there is not observed with erythrocytes. At low hematocrit, the stoichiometry is 2.6:1 and decreases to 1.5:1 at high hematocrit. The change is not due to differences in the hemoglobin saturation or to an inability of dithionite to reduce all dioxygen present at the higher hematocrit. Neither catalase nor peroxidase added to the extracellular volume significantly alters the stoichiometry or the enthalpy of dioxygen reduction by dithionite. Addition of superoxide dismutase, however, restores the normal stoichiometry at high hematocrit and further increases the stoichiometry at low hematocrit. The calorimetrical signal of hydrogen peroxide, clearly seen with free dioxygen, is not present with erythrocytes. In all these cases the total heat evolved is the same. 相似文献
3.
In heme degradation catalyzed by the reconstituted heme oxygenase system, 8 to 9 mol of dioxygen and 11 to 12 mol of NADPH were consumed per mol of hemin lost, and about half the amount of dioxygen consumed could be accounted for by the production of hydrogen peroxide, which accumulated in the reaction mixture. Production of hydrogen peroxide in the heme oxygenase reaction did not appear to be due to the bimolecular dismutation of superoxide anions but rather seemed to be due to dissociation of a "peroxo" species formed on heme or intermediates of heme degradation. The hydrogen peroxide produced appeared to cause a considerable degree of non-specific degradation of heme (not leading to the formation of biliverdin) and also caused an inactivation of heme oxygenase. By taking into account the amount of dioxygen incorporated into hydrogen peroxide and some other factors, it could be deduced that 3 mol of dioxygen is consumed for the formation of 1 mol of biliverdin in the heme oxygenase reaction. 相似文献
4.
Rates of energy transfer between tryptophans and hemes in hemoglobin, assuming that the heme is a planar oscillator.
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Using the Förster equations we have estimated the rate of energy transfer from tryptophans to hemes in hemoglobin. Assuming an isotropic distribution of the transition moments of the heme in the plane of the porphyrin, we computed the orientation factors and the consequent transfer rates from the crystallographic coordinates of human oxy- and deoxy-hemoglobin. It appears that the orientation factors do not play a limiting role in regulating the energy transfer and that the rates are controlled almost exclusively by the intrasubunit separations between tryptophans and hemes. In intact hemoglobin tetramers the intrasubunit separations are such as to reduce lifetimes to 5 and 15 ps/ns of tryptophan lifetime. Lifetimes of several hundred picoseconds would be allowed by the intersubunit separations, but intersubunits transfer becomes important only when one heme per tetramer is absent or does not accept transfer. If more than one heme per tetramer is absent lifetimes of more than 1 ns would appear. 相似文献
5.
The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5 degrees C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25 degrees C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15 degrees C was observed for S1, S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25 degrees C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15 degrees C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented. 相似文献
6.
Tryptophan photolysis is responsible for gramicidin-channel inactivation by ultraviolet light 总被引:3,自引:0,他引:3
The decay of gramicidin fluorescence resulting from ultraviolet exposure was compared to the decay of conductance from gramicidin-containing planar bilayer membranes under the same conditions of illumination. The decay rate was the same for both processes. The fluorescence decay was identical whether gramicidin was dissolved in methanol or incorporated into lipid vesicles, indicating that the peptide conformation does not affect the sensitivity of gramicidin to photolysis. The correlation of fluorescence decay and conductance decay imply that conductance loss from gramicidin-doped membranes illuminated with ultraviolet light is due to photochemical modifications of the channel tryptophans rather than simply to disturbance of the conformation of gramicidin channels. 相似文献
7.
The kinetics of O2 binding to a vacant coordination site on the cobalt(II) ion have been determined, revealing a radical-like character for the reaction. Reversible oxygenation of Co(II) cyclidenes (C4, C5, C6, C8, C12-bridged and unbridged) was studied by a cryogenic stopped-flow method. In the presence of axial base, kinetic parameters are insensitive to the nature of the solvent, and negative entropies of activation suggest that dissociation of a solvent molecule is not the rate-determining step for the dioxygen binding process. This is in contrast to the behavior of previously studied Co(II) complexes. A very low activation energy (1–4 kcal mol−1), typical of diffusion controlled processes, was found for dioxygen binding. The binding rate constants for the highest affinity complexes (108 M−1 s−1) are comparable to the values for natural dioxygen carriers. The size of the lacuna primarily affects the dioxygen binding rates, while the axial bases influence the dioxygen dissociation rates. 相似文献
8.
Sivaramakrishnan Ramadurai Andrea Holt Lars V. Schäfer Dirk T.S. Rijkers J. Antoinette Killian Bert Poolman 《Biophysical journal》2010,99(5):1447-1454
We investigated the effect of amino acid composition and hydrophobic length of α-helical transmembrane peptides and the role of electrostatic interactions on the lateral diffusion of the peptides in lipid membranes. Model peptides of varying length and composition, and either tryptophans or lysines as flanking residues, were synthesized. The peptides were labeled with the fluorescent label Alexa Fluor 488 and incorporated into phospholipid bilayers of different hydrophobic thickness and composition. Giant unilamellar vesicles were formed by electroformation, and the lateral diffusion of the transmembrane peptides (and lipids) was determined by fluorescence correlation spectroscopy. In addition, we performed coarse-grained molecular-dynamics simulations of single peptides of different hydrophobic lengths embedded in planar membranes of different thicknesses. Both the experimental and simulation results indicate that lateral diffusion is sensitive to membrane thickness between the peptides and surrounding lipids. We did not observe a difference in the lateral diffusion of the peptides with respect to the presence of tryptophans or lysines as flanking residues. The specific lipid headgroup composition of the membrane has a much less pronounced impact on the diffusion of the peptides than does the hydrophobic thickness. 相似文献
9.
Glucose oxidase catalyzes the oxidation of glucose by molecular dioxygen, forming gluconolactone and hydrogen peroxide. A series of probes have been applied to investigate the activation of dioxygen in the oxidative half-reaction, including pH dependence, viscosity effects, 18O isotope effects, and solvent isotope effects on the kinetic parameter Vmax/Km(O2). The pH profile of Vmax/Km(O2) exhibits a pKa of 7.9 +/- 0.1, with the protonated enzyme form more reactive by 2 orders of magnitude. The effect of viscosogen on Vmax/Km(O2) reveals the surprising fact that the faster reaction at low pH (1.6 x 10(6) M-1 s-1) is actually less diffusion-controlled than the slow reaction at high pH (1.4 x 10(4) M-1 s-1); dioxygen reduction is almost fully diffusion-controlled at pH 9.8, while the extent of diffusion control decreases to 88% at pH 9.0 and 32% at pH 5.0, suggesting a transition of the first irreversible step from dioxygen binding at high pH to a later step at low pH. The puzzle is resolved by 18O isotope effects. 18(Vmax/Km) has been determined to be 1.028 +/- 0.002 at pH 5.0 and 1.027 +/- 0.001 at pH 9.0, indicating that a significant O-O bond order decrease accompanies the steps from dioxygen binding up to the first irreversible step at either pH. The results at high pH lead to an unequivocal mechanism; the rate-limiting step in Vmax/Km(O2) for the deprotonated enzyme is the first electron transfer from the reduced flavin to dioxygen, and this step accompanies binding of molecular dioxygen to the active site. In combination with the published structural data, a model is presented in which a protonated active site histidine at low pH accelerates the second-order rate constant for one electron transfer to dioxygen through electrostatic stabilization of the superoxide anion intermediate. Consistent with the proposed mechanisms for both high and low pH, solvent isotope effects indicate that proton transfer steps occur after the rate-limiting step(s). Kinetic simulations show that the model that is presented, although apparently in conflict with previous models for glucose oxidase, is in good agreement with previously published kinetic data for glucose oxidase. A role for electrostatic stabilization of the superoxide anion intermediate, as a general catalytic strategy in dioxygen-utilizing enzymes, is discussed. 相似文献
10.
Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification. 总被引:2,自引:0,他引:2
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M. R. Bray P. E. Johnson N. R. Gilkes L. P. McIntosh D. G. Kilburn R. A. Warren 《Protein science : a publication of the Protein Society》1996,5(11):2311-2318
The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose. 相似文献
11.
The quenching of tryptophan fluorescence by N-bromosuccinamide, studied by the fluorescence stopped-flow technique, was used to compare the reactivities of tryptophan residues in protein molecules. The reaction of N-bromosuccinamide with the indole group of N-acetyltryptophanamide, a model compound for bound tryptophan, followed second-order kinetics with a rate constant of (7.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1 at 23 degrees C. The rate does not depend on ionic strength or on the pH near neutrality. The non-fluorescent intermediate formed from N-acetyltryptophanamide on the reaction with N-bromosuccinamide appears to be a bromohydrin compound. The second-order rate constant for fluorescence quenching of tryptophan in Gly-Trp-Gly by N-bromosuccinamide was very similar, (8.8 +/- 0.8) . 10(5) dm3 . mol-1 . s-1. Apocytochrome c has the conformation of a random coil with the single tryptophan largely exposed to the solvent. The rate constant for the fluorescence quenching of the tryptophan in apocytochrome c by N-bromosuccinamide was (3.7 +/- 0.3) . 10(5) dm3 . mol-1 . s-1. The fluorescence quenching by N-bromosuccinamide of the tryptophan residues incorporated in alpha-chymotrypsin at pH 7.0 showed three exponential terms from which the following rate constants were derived: 1.74 . 10(5), 0.56 . 10(5) and 0.11 . 10(5) dm3 . mol-1 . s-1. This protein is known to have eight tryptophan residues in the native state, six residues at the surface, and two buried. Three of the surface tryptophans have the indole rings protruding out of the molecule and may account for the fastest kinetic phase of the quenching process. The intermediate phase may be due to three surface tryptophans whose indole rings point inwards, and the slowest to the two interior tryptophan residues. 相似文献
12.
Christian Trampitsch Anita Slavica Waander Riethorst Bernd Nidetzky 《Journal of Molecular Catalysis .B, Enzymatic》2005,32(5-6):271-278
2,6-Dichloroindophenol (DCIP) is shown to be utilised efficiently as electron acceptor replacing dioxygen in the reaction of Trigonopsis variabilis d-amino acid oxidase (TvDAO) with d-methionine as the substrate. The specificity constant for DCIP reduction at 30 °C is one-twelfth that of oxygen conversion into hydrogen peroxide. Time course analysis of simultaneous consumption of DCIP and dioxygen, recorded on-line by absorption and non-invasive fluorescence quenching, respectively, pinpoints the preferential utilisation of dioxygen; and reveals a maximum DCIP conversion rate that is independent of the initial concentration of dioxygen. A robust direct assay of TvDAO activity has been developed that does not require anaerobic reaction conditions. It was down-scaled to microtitre plate format and overcomes practical limitations of other assays due to the low affinity of TvDAO for dioxygen (Km ≈ 0.7 mmol L−1). 相似文献
13.
Zargarian L Le Tilly V Jamin N Chaffotte A Gabrielsen OS Toma F Alpert B 《Biochemistry》1999,38(6):1921-1929
The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface. 相似文献
14.
A laccase from the fungus Trametes villosa (TviL) was investigated in order to elucidate the reaction mechanism of the reduction of dioxygen to water performed by this blue multi-copper oxidase. The ability of TviL to activate dioxygen was studied by stopped-flow experiments and under steady-state conditions. In the stopped-flow experiments TviL was reduced with a small excess of 4-hydroxyphenylacetic acid and afterwards the re-oxidation process was monitored by stopped-flow techniques by mixing with excess dioxygen. The reaction between reduced TviL and dioxygen was studied in the temperature range 10-35 degrees Celsius and with the concentration of dioxygen between 30 and 240microM. The observed rate constant k(obs) is found to be linear dependent on the dioxygen concentration and the observed second-order rate constant for the re-oxidation of reduced TviL is, at 25 degrees Celsius, determined to be 1.14x10(6)M(-1)s(-1). The activation energy, E(a), is from the same data determined to be 22kJmol(-1). Oxidation of different phenols (4-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, guaiacolsulfonic acid and hydroquinone) under steady state conditions was investigated at concentrations of dioxygen ranging from 60 to 250microM. This line of experiments showed that TviL follows a ping-pong mechanism, and an observed second-order rate constant for the reaction with dioxygen of 7.1x10(5)M(-1)s(-1) at 25 degrees Celsius was found with 4-hydroxyphenylacetic acid as reducing substrate. The two kinetic methods resulted in observed rate constants of equal magnitudes for the reaction with dioxygen, which suggests that the rate limiting step(s) is (are) included in both the reactions studied by the two different techniques. 相似文献
15.
Dioxygen activation by putidamonooxin. The oxygen species formed and released under uncoupling conditions 总被引:1,自引:0,他引:1
In the presence of substrates not favourable for hydroxylation, more than 80% of the dioxygen consumed by purified, reconstituted 4-methoxybenzoate monooxygenase appears in the reaction mixture as hydrogen peroxide. We have investigated whether under these conditions (a) reduced putidamonooxin, the oxygenase of this enzyme system, either autoxidizes in the presence of dioxygen, with liberation of superoxide anion radicals which then disproportionate to H2O2 and O2, or (b) dioxygen is reduced by two sequential single-electron steps leading to the active oxygen species that forms hydrogen peroxide directly when inactivated by protonation. Quantitative estimation of O-2 radicals, with either succinylated ferricytochrome c or epinephrine used as O-2 scavengers, revealed that only about 6% of the total electron flux channelled via putidamonooxin to dioxygen led to the monovalent reduction on dioxygen. This means that not more than 3% of the hydrogen peroxide found under uncoupling conditions arises from the rapid bimolecular disproportionation of initially formed O-2 radicals. Inconsistent results were obtained when lactoperoxidase was used as an O-2 trap. Our measurements indicate that the conversion of lactoperoxidase into compound III is an inappropriate method of detecting any O-2 radicals that may be found by the uncoupled 4-methoxybenzoate monooxygenase. The stoichiometry of about 1:1 for O2 uptake: H2O2 formation indicates that under uncoupling conditions H2O is virtually not formed. The role of [FeO2]+ as the active oxygenating species of putidamonooxin is discussed. 相似文献
16.
The model for carbon monoxide or dioxygen recombination with heme proteins developed by the group at the University of Illinois is reexamined. We propose that the carbon monoxide or dioxygen molecule enters the protein at essentially a diffusion-limited rate determined by the solvent viscosity and that the protein offers no important barriers to this entry. The viscosity dependence of the entry rate k(ED), its magnitude (1 x 10(10) M(-1)s(-1), and the rate of quenching of triplet states of protoprophyrin IX in apomyoglobin by dioxygen are used as supporting evidence. Comparison is made to the model of a fluctuating protein developed by G. Weber. 相似文献
17.
The dioxygen stretch bands in infrared spectra for solutions of oxy species of human hemoglobin A and its separated subunits, human mutant hemoglobin Zurich (beta 63His to Arg), rabbit hemoglobin, lamprey hemoglobin, sperm whale myoglobin, bovine myoglobin, and a sea worm chlorocruorin are examined. Each protein exhibits multiple isotope-sensitive bands between 1160 and 1060 cm-1 for liganded 16O2, 17O2, and 18O2. The O-O stretch bands for each of the mammalian myoglobins and hemoglobins are similar, with frequencies that differ between proteins by only 3-5 cm-1. The spectra for the lamprey and sea worm hemoglobins exhibit greater diversity. For all proteins an O-O stretch band expected to occur near 1125 cm-1 for 16O2 and 17O2, but not 18O2, appears split by approximately 25 cm-1 due to an unidentified perturbation. The spectrum for each dioxygen isotope, if unperturbed, would contain two strong bands for the mammalian myoglobins (1150 and 1120 cm-1) and hemoglobins (1155 and 1125 cm-1). Two strong bands separated by approximately 30 cm-1 for each oxy heme protein subunit indicate that two major protein conformations (structures) that differ substantially in O2 bonding are present. The two dioxygen structures can result from a combination of dynamic distal and proximal effects upon the O2 ligand bound in a bent-end-on stereochemistry. 相似文献
18.
The giant Antarctic pycnogonid Decolopoda australis Eights takes up oxygen by diffusion across the integument, particularly of the legs. The circulatory system is feeble and haemolymph pressure changes are induced by leg movements during locomotion rather than by cardiac action. Heart rate is about30–40 beats min-1 between 1 and 5 °C; it becomes irregular above 5 °C and ceases (reversibly) at6–7 °C.
The integumentary structure appears to facilitate gaseous exchange. Although the cuticle (c. 200 μm thick) is chitinous, it is perforated (over 35% of the internal surface) by circular/ ellipsoidal tissue-filled pits which are separated from the external environment by thin layers of chitinous cuticle no more than4–6 μm thick. The pits occur in all parts of the body and appendages (except arthrodial membranes). It is suggested that the primary function of the tissue within the pits is to form a route for gaseous diffusion, which bypasses the relatively thick impermeable chitinous layers of the rest of the cuticle. Calculations suggest that the pits reduce resistance to gaseous diffusion by about 90%.
To function as a respiratory surface, the general body surface has to be kept clean. The cleaning action of the ovigers appears to be effective, except in the regions between the contiguous lateral processes of the body where detrital material accumulates. 相似文献
The integumentary structure appears to facilitate gaseous exchange. Although the cuticle (c. 200 μm thick) is chitinous, it is perforated (over 35% of the internal surface) by circular/ ellipsoidal tissue-filled pits which are separated from the external environment by thin layers of chitinous cuticle no more than4–6 μm thick. The pits occur in all parts of the body and appendages (except arthrodial membranes). It is suggested that the primary function of the tissue within the pits is to form a route for gaseous diffusion, which bypasses the relatively thick impermeable chitinous layers of the rest of the cuticle. Calculations suggest that the pits reduce resistance to gaseous diffusion by about 90%.
To function as a respiratory surface, the general body surface has to be kept clean. The cleaning action of the ovigers appears to be effective, except in the regions between the contiguous lateral processes of the body where detrital material accumulates. 相似文献
19.
Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1), as the terminal enzyme of the mammalian mitochondrial electron transport chain, has long been known to catalyze the reduction of dioxygen to water. We have found that when reductively activated in the presence of dioxygen, the enzyme will also catalyze the oxidation of carbon monoxide to its dioxide. Two moles of carbon dioxide is produced per mole of dioxygen, and similar rates of production are observed for 1- and 2-electron-reduced enzyme. If 13CO and O2 are used to initiate the reaction, then only 13CO2 is detected as a product. With 18O2 and 12CO, only unlabeled and singly labeled carbon dioxide are found. No direct evidence was obtained for a water-gas reaction (CO + H2O----CO2 + H2) of the oxidase with CO. The CO oxygenase activity is inhibited by cyanide, azide, and formate and is not due to the presence of bacteria. Studies with scavengers of partially reduced dioxygen show that catalase decreases the rate of CO oxygenation. 相似文献
20.
Francine Malaisse-Lagae Véronique Liemans Willy J. Malaisse 《Molecular and cellular biochemistry》1989,89(1):57-67
Summary The rate of conversion of D-glucose 6-phosphate to D-fructose 6-phosphate as catalyzed by yeast phosphoglucoisomerase is about fourfold lower when 3H, rather than 1H, is present on the C2 of D-glucose 6-phosphate. This difference appears to be due mainly to a change in maximal velocity, rather than affinity. Phosphoglucoisomerase also distinguishes between 1H and 3H in terms of either their intramolecular transfer from C2 to C1 or their incorporation from water on the C1 of D-fructose 6-phosphate. 相似文献