Determination of kinetic constants in the general case of cooperative type or Michaelis enzymes inhibited by their substrate: theoretic analysis

For an enzyme (E) susceptible to substrate (S) inhibition, (S) can bind on one hand to (E) and on the other hand to (ES), leading to the dead-end complexes (SE) and (SES). In the general case where the (E)/(S) interaction obeys the Hill equation, the theoretical maximum velocity VM can be estimated when n not equal to 1, from the determination of velocities v beta at substrate concentrations S beta = Sm beta where Sm is the value corresponding to the actual maximum velocity vm. The Hill coefficient (n) as well as the constants KS, KSE and KSES corresponding to the respective dissociations of the complexes (ES), (SE) and (SES) are then determined from the equation: Ln (v/(VM-v] = nLnS-LnKS(1 + Sn/KSE + S2n/KS KSES) and its two asymptotes.     

Studies of Karyotypes in Three Wild and Four Cultivated Species of Gossypium

Karyotypes in seven species of Gossypium, G. thurberi, G. advidsonii, G. raimondii of D group; G. herbaceum, G. arboreum of A group; G. hirsutum and G. ba- rbadense of AD group were studied in 1983. It can be simplified as follows: G. thurberi 2n = 2x = 26 = 24m + 2Sm (2SAT); G. davidsonii 2n= 2x = 26 20m+6Sm(4SAT); G. raimondii 2n=2x= 26= 20m+6Sm(2SAT); G. herbaceum 2n = 2x = 26 = 18m + 4Sm+4St(4SAT); G. arboreum 2n = 2x = 26 = 18m + 6Sm (2SAT) + 2St(2SAT); G. hirsutum 2n = 4x =52 = 32m + 18Sm(4SAT) +2St (2 SAT); G. barbadense 2n = 4x = 52 = 38m + 12Sm (2SAT) + 2St(2SAT). This paper also deals with the supplier in A group and D group of tetraploids.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

三种野生和四个栽培棉种的核型研究

作者对棉属 D 组的瑟伯氏棉(Gossypium thurberi)、戴维逊氏棉(C.davidsonii)、雷蒙德氏棉(G.raimondii)、A 组的草棉(G.herbaceum)、中棉(G.arboreum)、AD 组的陆地棉(G.htrsutum)和海岛棉(C.barbadense)等7个种的核型进行了研究。各个种的核型可简式为:瑟伯氏棉2n=2x=26=24m 2Sm(2SAT);戴维逊氏棉20=2x=26=20m 6Sm(4SAT);雷蒙德氏棉2n=2x=26=20m 6Sm(2SAT);草棉2n=2x=26=18m 4Sm 4St(4SAT);中棉2n=2x=26=18m 6Sm(2SAT) 2St(2SAT);陆地棉2n=4x=52=32m 18Sm(4SAT) 2St(2SAT);海岛棉20=4x=52=38m 12Sm(2SAT) 2St(2SAT)。此外,作者对四倍体种的 A 组和 D 组的供体问题进行了讨论。     

Eat in or take away? How phosphatidylinositol 4-kinases feed the phospholipase C pathway with substrate

Phosphatidylinositol 4-kinases (PI4Ks) catalyze the first step in the synthesis of phosphoinositide pools hydrolysed by phosphoinositide-dependent phospholipase C (PI-PLC) and thus constitute a potential key regulation point of this pathway. Twelve putative PI4K isoforms, divided as type-II (AtPI4KIIγ1-8) and type-III PI4Ks (AtPI4KIIIα1-2 and AtPI4KIIIβ1-2), have been identified in Arabidopsis genome. By a combination of pharmalogical and genetic approaches we recently evidenced that AtPI4KIIIβ1 and AtPI4KIIIβ2 contribute to supply PI-PLC with substrate and that AtPI4KIIIα1 is probably also involved in this process. Given the current knowledge on PI-PLC and type-III PI4Ks localization in plant cells it raises the question whether type-III PI4Ks produce phosphatidylinositol 4-phosphate at the site of its consumption by the PI-PLC pathway. We therefore discuss the spatial organization of substrate supply to PI-PLC in plant cells with reference to recent data evidenced in mammalian cells.     

Bromopyruvate as an affinity label for baker's yeast flavocytochrome b2. Kinetic study of the inactivation reaction.

Bromopyruvate was shown to completely inactivate cytochrome b2 in a reaction that obeyed the kinetic criteria required for affinity labels: it inactivated flavocytochrome b2 according to saturation kinetics, and the inactivation reaction was competitively inhibited by the substrate or competitive inhibitors. Inactivation was irreversible. The behaviour of both forms of flavocytochrome b2 (lintact and proteolytically cleaved) was examined. It was found that the reduced cleaved enzyme was not inactivated by bromopyruvate; this phenomenon can probably be ascribed to a structural change undergone upon reduction. The value of the lactate dissociation constant of intact cytochrome b2 cytochrome b2 was determined in competition experiments with bromopyruvate. By comparison with the divergent published values for the Ks of the cleaved from, it appears that only those that differ from the Km by a factor of two or three are reasonable. This study opens the way for the identification of an active site residue and localization in the peptide chain of the bifunctional enzyme.     

Serologic reactivities of the 23-kDa integral membrane proteins of schistosomes.

The 23-kDa integral membrane proteins of Schistosoma mansoni and Schistosoma japonicum (Sm23 and Sj23) are Ag of some interest in terms of both antiparasite vaccination and immunodiagnosis. We have raised an antiserum against a recombinant fusion protein expressing the extracellular hydrophyllic domain of Sm23 (Sm23HD-pGEX) and used this serum, as well as other antibody reagents reacting with Sm/Sj23, in immunochemical analyses. The immunogenicity and antigenicity of Sm23HD-pGEX, and the surprising lack of cross-reactivity between Sm23 and Sj23 support the hypothesis that Sm/Sj23 are host-like molecules with a very limited number of B cell epitopes that are likely to reside in the extracellular hydrophilic domain. We also present evidence that, unlike the highly immunogenic Sj23, Sm23 is not immunogenic in chronically infected mice. Moreover, we confirm a surface location for Sj23 in adult worms, in S. japonicum.     

Ultrastructures and evolutionary modalities of flagellar and ciliary systems in protists

Summary— As both the ultrastructure and function of flagella and cilia have been for the main part remarkably conserved during Eukaryote, evolution, the question arises as to whether the variations observed at the organite ultrastural level, or at the level of the development of a flagellar or ciliary cellular system could be considered as systematic or phylogenetic criteria. With regard to the fundamental structure, the known variations concern: 1) the kinetosome (length, position and number of cartwheels, number of triplets, and respective lengths of the microtubules); 2) the transition zone (various structural types); 3) the axoneme (number of doublets, central tubules, arms); 4) the paraxonemal formations (presence, position, structure); 5) the membrane (intramembranous particles; intramembranous particles; addition of components, mastigonemes, scales); 6) the fibres associated with the kinetosomes. Some of these variations are characteristic of taxa, and are considered as phylogenetic markers. Regarding the variations in the number of ciliary or flagellar units per cell, the following can be distinguished: cells with only one kinetosome (Ks), carrying one flagellum; cells with 2 neighbouring Ks (primary Ks = 2A), only one Ks, or the 2 Ks, bearing a flagellum; cells with numerous flagella or cilia. We consider that this configuration can result from: a) addition of new Ks around the primary Ks, forming a primary group (PG): either by replication of the primary couple (2 × 2A in Karotomorpha; 2 × 2A in Phaeopolykrikos; 4(2 × 2A) in Polkrikos), or by addition of new Ks (N) without copy or replication of the primary Ks (2A + 2N in Polytomella; 2A +3N in Tetratrichomonas; 2A + 3N in Tetratrichomonas; 2A + 4N in Hexamastix; 2A + 6N in Pyramimonas octopus). b) amplification of each constituent of the primordial couple in opalinids. c) amplification of the primary group [yx(AA + 2N) with y = 2 in diplomonadida and y > 2 in calonymphidae. d) appearance in morphogenetic fields situated outside the primary group (but possibly related to it) of new Ks which then form secondary groups (SG) where they are arranged in polarized rows composed of juxtaposed cortical units (monokinetids). In this case, PG can be of type 2A+2N (1PG+1SG in lophomonadida; 1PG+2SG in some trichonymphina), or can lose some Ks, resulting in reduced PG (4 reduced PG+4GS in Staurojoenina, n reduced PG + nGS in spirotrichonymphina). The PG may even disappear; either partially leaving remnants in the form of MTOCs in Stephanopogon, or completely, as in cilliates where the general ciliature would represent on the SG. From the above, it is seen that: a) the pluriflagellar state in protists depends on only a small number of factors; b) the same factors are used in different groups; c) different states have been obtained within different groups. An analysis of the respective evolution of kinetosomes and centrioles is proposed.     

Kinetic mechanism of guinea pig neutrophil 5-lipoxygenase

The kinetic mechanism of guinea pig neutrophil 5-lipoxygenase was investigated using a continuous spectrophotometric assay that monitors product diene formation at 236 nm due to substrate oxygenation. Progress curves for reactions with both arachidonic acid and eicosapentaenoic acid are characterized by 1-3-min lag phases in the attainment of steady-state velocities and product inhibition, as indicated by the total cessation of the reaction prior to complete depletion of substrate. The dependence of the steady-state velocity on arachidonic acid concentration appears to follow Michaelis-Menten kinetics, with Vmax = 4.2 +/- 0.4 nmol of 5-hydroxy-6,8,11,14-eicosatetraenoic acid/min/mg of protein and Ks = 25 +/- 4 microM. The addition of Ca2+ results in an overall activation: lag phases are shortened to 10-20 s, Vmax increases to 24 +/- 2 nmol/min/mg of protein, and Ks decreases to 7.7 +/- 1.7 microM; and a change in a mechanism to one involving substrate inhibition (Kss = 13 +/- 1 microM). The observed activation by Ca2+ has a half-maximal response at around 30 microM. In the presence of Ca2+, ATP causes an increase in Vmax to 30 +/- 4 nmol/min/mg of protein without changing Ks or Kss and a reduction of the lag to less than 5 s. The half-maximal response for ATP is 31 +/- 7 microM. Oxygenation of eicosapentaenoic acid in the presence of Ca2+ and ATP occurs with similar kinetics, except for significantly less substrate inhibition: Vmax = 31 +/- 6 nmol/min/mg of protein, Ks = 7 +/- 1 microM, and Kss = 33 +/- 2 microM. This is the first report suggesting a kinetic mechanism for 5-lipoxygenase, which accounts for substrate inhibition, regulation by Ca2+, and ATP and substrate specificity.     

Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme.

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.     

Further theoretical refinement on the internal thermodynamics of perfect enzymes.

By solving simultaneously the equation for ''uniform binding'' [Albery & Knowles (1976) Biochemistry 15, 5631-5640] and the equation for ''differential binding'' [Chin (1983) J. Am. Chem. Soc. 105, 6502-6503], I derived the following simple equation for perfect enzymes (with single substrate and single product) under irreversible conditions: K2 = beta(1 + Rs)/1-beta(1 + Rs) where K2 is the internal equilibrium constant and beta is the Brönsted coefficient of the elementary catalytic step, and Rs is defined as [S]0/Ks, with [S]0 being the physiological substrate concentration and Ks being the substrate dissociation constant. The equation suggests that the perfect enzyme can have different internal thermodynamic properties depending on physiological conditions.     

重组巴氏毕赤酵母恒化培养动力学及代谢迁移特性研究

通过对甲醇营养型毕赤酵母基因工程菌以碳源甘油为限制性基质进行恒化培养动力学试验 ,结果认为 :(1 )细胞光密度与其干、湿重呈线性关系 ,当细胞光密度 (OD60 0 )为 1 0 0时细胞湿重 (WCW)为 1 2 8 3g L ,细胞干重 (WDW)则为 2 2 9g L ;(2 )基因工程菌P .pastoris的生长与限制性基质甘油残留浓度的关系符合Monod关系式 ,通过 1 μ对 1 S进行线性回归得 μmax=0 .366h- 1,Ks=0 .1 82 3g L ,经参数推导甘油最大菌体得率系数YG =0 .54g g ,菌体维持生长消耗底物系数m =0 .0 0 69g (g·h) ;氧最大菌体系数YX O2 =30 .96g moL ,菌体维持生长时消耗氧系数mO2 =0 .0 0 0 8mol (g·h) ,最适理论稀释速率Dm =0 .341h- 1;(3)从氨水的消耗速率和呼吸商 (RQ)的变化认为随着比生长速率 (μ)的增大 ,甘油代谢流从糖原异生和磷酸戊糖途径线性地向糖酵解和三羧酸循环途径进行代谢迁移 ,即糖酵解和三羧酸循环途径的代谢流量在线性地增大     

Romanowsky dyes and the Romanowsky-Giemsa effect. 4. Binding of azure B to DNA

We investigated the binding of azure B to DNA (calf thymus) over a wide range of concentrations of the dye (CF) and the nucleic acid (CN) using absorption spectroscopy [CF and CN represent the total concentrations of the ye (F) and the mononucleotide units (N) of the DNA, respectively]. The binding isotherms of the dye to DNA in aqueous solutions were determined. In addition, we analysed the composition of insoluble DNA/azure B precipitates that are formed in presence of an excess of azure B. These precipitates are of particular interest, because Giemsa staining is usually performed using high dye concentrations. Azure B easily forms dimers in aqueous solutions. When determining the binding isotherms, the equilibrium between free monomers and dimers must be taken into account. Therefore, we determined the dimerisation constant (Kd) of azure B from the concentration dependency of its absorption spectra in water at the standard temperature T = 298 K (25 degrees C), Kd = 6.5 X 10(3) M-1 (experimental conditions: tris buffer, pH 7.2; concentration of Na ions, CNa = 0.002 M). As the CNa value increases, the dimerisation constant rises rapidly. When the azure B concentration is very low and there is an excess of DNA, ordinary Scatchard and Langmuir isotherms are observed. Monomer dye cations are bound to DNA, these cations being in equilibrium with free monomers in the solution. In order to obtain the Scatchard binding constant (Ks) and the binding parameter (n) spectroscopically, it is necessary to determine the extinction coefficient (epsilon Fb) of the monomer bound (b) dye molecules (F) at one analytical wave number (upsilon a). The three constants can be determined simultaneously using an iterative technique that combines Scatchard isotherms and the Benesi-Hildebrand extrapolation, CN----infinity. We obtained Ks = 1.8 X 10(5) M-1 and n = 0.18 (25 degrees C; tris buffer, pH 7.2; CNa = 0.002 M). At very low dye (CF) and competitor (CNa) concentrations, only 18% of the anionic binding sites of the DNA are capable of binding the dye cations. With increasing CNa values the concentration of bound azure B cations decreases rapidly. The Na cations displace the bound dye cations and act as a competitor. The Ks value also greatly depends on the competitor concentration (CNa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.     

Kinetic analysis of simultaneous 2,4-dinitrotoluene (DNT) and 2, 6-DNT biodegradation in an aerobic fluidized-bed biofilm reactor.

We previously reported on the mineralization of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in an aerobic fluidized-bed bioreactor (FBBR) (Lendenmann et al. 1998 Environ Sci Technol 32:82-87). The current study examines the kinetics of 2, 4-DNT and 2,6-DNT mineralization at increasing loading rates in the FBBR with the goal of obtaining system-independent kinetic parameters. At each steady state, the FBBR was subjected to a set of transient load experiments in which substrate flux in the biofilm and bulk substrate concentrations were measured. The pseudo-steady-state data were used to estimate the biokinetic parameters for 2,4-DNT and 2,6-DNT removal using a mechanistic mathematical biofilm model and a routine that minimized the sum of the squared residuals (RSS). Estimated kinetic parameters varied slightly for each steady-state; retrieved parameters for qm were 0. 83 to 0.98 g DNT/g XCOD d for 2,4-DNT removal and 0.14 to 0.33 g DNT/g XCOD d for 2,6-DNT removal. Ks values for 2,4-DNT removal (0. 029 to 0.36 g DNT/m3) were consistently lower than Ks values for 2, 6-DNT removal (0.21 to 0.84 g DNT/m3). A new approach was introduced to estimate the fundamental biofilm kinetic parameter S*b,min from steady-state performance information. Values of S*b,min indicated that the FBBR performance was limited by growth potential. Adequate performance of the examined FBBR technology at higher loading rates will depend on an improvement in the growth potential. The obtained kinetic parameters, qm, Ks, and S*b,min, can be used to aid in the design of aerobic FBBRs treating waters containing DNT mixtures.     

Identification of immunodominant Th1-type T cell epitopes from Schistosoma japonicum 28 kDa glutathione-S-transferase, a vaccine candidate

Th1-type cytokines produced by the stimulation of Th 1-type epitopes derived from defined schistosome-associated antigens are correlated with the development of resistance to the parasite infection. Schistosoma mansoni 28 kDa glutathione-S-transferase （Sm28GST）, a major detoxification enzyme, has been recognized as a vaccine candidate and a phase II clinical trial has been carried out. Sheep immunized with recombinant Schistosoma japonicum 28GST （Sj28GST） have shown immune protection against the parasite infection. In the present study, six candidate peptides （P1, P2, P3, P4, P7 and P8） from Sj28GST were predicted, using software, to be T cell epitopes, and peptides P5 and P6 were designed by extending five amino acids at the N-terminal and C-terminal of P1, respectively. The peptide 190-211 aa in Sj28GST corresponding to the Th1-type epitope （190-211 aa） identified from Sm28GST was selected and named P9. The nine candidate peptides were synthesized or produced as the fusion protein with thioredoxin in the pET32c（＋）/BL21（DE3） system. Their capacity to induce a Th1-type response in vitro was measured using lymphocyte proliferation, cytokine detection experiments and flow cytometry. The results showed that P6 （73-86 aa） generated the strongest stimulation effect on T cells among the nine candidate peptides, and drove the highest level of IFN-γ, and IL-2. Therefore, P6 is a functional Thl-type T cell epitope that is different from that in Sm28GST, and will be useful for the development of effective vaccines which can trigger acquired immunity against S. japonicum. Moreover, our strategy of identifying the Thl-type epitope by a combination of software prediction and experimental confirmation provides a convenient and cost-saving alternative approach to previous methods.     

The pre-eminence of k(cat) in the manifestation of optimal enzymic activity delineated by using the Briggs-Haldane two-step irreversible kinetic model.

The suggestion by Fersht [(1974) Proc. R. Soc. London Ser. B 187, 397-407] that enzymes that provide maximal rates of catalysis should be characterized by values of Ks, the dissociation constant of the enzyme-substrate complex, greater than 10 times the value of the ambient substrate concentration has been examined. 2. For such enzymes, Ks is not relevant, and attention is best focused on the relative numerical values of k(cat). (in units of s(-1) and the substrate molarity. It is necessary only that the former be about 10(10)-10(11) times the latter to ensure that the rate of product formation be diffusion-limited and thus maximal.     

Kinetic comparison of seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria.

Seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria, including Pseudomonas, Alcaligenes, and Bordetella spp., were compared on the basis of growth kinetics. Estimates of maximum growth rate (mu max, k1) and half-saturation growth constant (Ks, k3) were obtained by fitting substrate depletion curves to a four-parameter version of the integrated Monod equation. Estimates of Ks ranged from 2.2 micrograms/ml (10 microM) to 33.8 micrograms/ml (154 microM), and estimates of mu max ranged from 0.20 h-1 (Td = 3.5 h) to 0.32 h-1 (Td = 2.2 h). Estimates of mu max, but not Ks, were affected by changes in initial inoculum density. Maximum growth rates (mu max) were also estimated from turbidity measurements. They ranged from 0.10 h-1 (Td = 6.9 h) to 1.0 h-1 (Td = 0.7 h). There was no correlation between estimates of mu max derived from substrate depletion curves and those derived from turbidity measurements (P = 0.20).