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1.
The kinetic investigation of hyaluronidases using physiologically relevant hyaluronic acid (HA or hyaluronan) substrate will provide useful and important clues to their catalytic behavior and function in vivo. We present here a simple and sensitive method for kinetic measurement of recombinant human hyaluronidase PH20 (rHuPH20) on HA substrates with sizes ranging from 90 to 752 kDa. The method is based on 2-aminobenzamide labeling of hydrolyzed HA products combined with separation by size exclusion–ultra performance liquid chromatography coupled with fluorescence detection. rHuPH20 was found to follow Michaelis–Menten kinetics during the initial reaction time. Optimal reaction rates were observed in the pH range of 4.5–5.5. The HA substrate size did not have significant effects on the initial rate of the reaction. By studying HA substrates of 215, 357, and 752 kDa, the kinetic parameters Km, Vmax, and kcat were determined to be 0.87–0.91 mg/ml, 1.66–1.74 nM s−1, and 40.5–42.4 s−1, respectively. This method allows for direct measurement of kinetics using physiologically relevant HA substrates and can be applied to other hyaluronidase kinetic measurements. 相似文献
2.
Enzyme kinetic parameters for rate equations are vital in metabolic network simulation, a major part of systems biology research efforts. Measurements of Michaelis–Menten kinetic parameters Km and Kcat have been performed for enzymes glucose-6-phosphate dehydrogenase (G6P DH) under crowded conditions using molecular crowding agents bovine serum albumin (BSA) and polyethylene glycol (PEG) of 8000 Da molecular weight. An increase in Kcat was observed at very low concentrations of crowding agent, and also at high crowder concentrations when the experiment was performed at 45 °C with PEG. The observed pattern in Kcat for G6P DH at high crowder concentrations has been explained via modelling using excluded volume theory. An increase in rate was observed at 45 °C for G6P DH versus 30 °C; this has been modelled via the Arrhenius equation. 相似文献
3.
A kinetic study of the effect of antimycin A on succinate oxidase from plant mitochondria produced sigmoidal curves for the reduction of cytochromes b560 and b585 and for the inhibition of succinate oxidase. In the stationary state the interaction of the various components of the respiratory chain (flavins, ubiquinone, cytochromes…) occurs in a sequential mode which allows the application of simple equations utilizing rate constants and cytochrome concentrations. In these equations, it is assumed that there exists an excess of ubiquinone relative to other components of the respiratory chain as suggested by Kröger & Klingenberg (1970) and that the reoxidation of b cytochromes is fast. The inhibition by antimycin A, characterized by non-linear inhibition curves for succin-oxidase and inhibitor fixation in complex III on a component other than cytochrome c1, is interpreted in terms of this model. This hypothesis presupposes the existence of an F factor between cytochrome b560 and cytochrome c1 as suggested by other authors. Utilizing these equations, theoretical curves for the inhibition of the reduction of cytochrome b560 have been constructed and the results agree with the experimental data. The kinetic behavior of cytochrome b566 during the induction of anaerobiosis suggests that it is not directly involved in the electron transfer chain but rather is either in thermodynamic equilibrium with cytochrome b560 or in a shunt between cytochrome b560 and factor F. From the experimental data, an equation is derived for the inhibition of the reduction of cytochrome b566 by antimycin A. The actual effects of ATP and mClCCP on succinoxidase agree well with those predicted by the model. 相似文献
4.
A steady state model for analyzing the cellular binding, internalization and degradation of polypeptide ligands 总被引:22,自引:0,他引:22
We demonstrate that the interaction of polypeptide ligands with cells under physiological conditions can be described by a set of steady state equations. These equations include four new rate constants: Vr, the rate of insertion of receptors into the cell membrane; Ke, the endocytotic rate constant of occupied receptors; Kt, the turnover rate constant of unoccupied receptors; and Kh, the rate constant of hydrolysis of internalized ligand. Several simple procedures are described for determining these constants. In experiments in which epidermal growth factor and human fibroblasts were used, the cell-ligand interactions followed the predictions of the steady state model. The utility of the steady state equations is demonstrated by establishing the kinetic basis of the commonly observed “down regulation” phenomenon and by quantitating the effect of methylamine on the endocytotic and degradation rates of epidermal growth factor. We also show that the slope of a “Scatchard plot” of steady state binding data is a complex constant including terms for the endocytotic rate of both occupied and unoccupied receptors. The X-intercept of such a plot is a function of the insertion rate of new receptors, the internalization rate of occupied receptors and the degradation rate of the internalized ligand. The steady state equations allow one to predict changes in cellular ligand binding resulting from alterations in the four rate constants. They also provide a foundation for computer simulations of ligand-cell interactions, which closely correspond to experimental data. These approaches should facilitate studies on the control of cellular activities by these polypeptide ligands. 相似文献
5.
The simple Langmuir isotherm is frequently employed to describe the equilibrium behavior of protein adsorption on a wide variety
of adsorbents. The two adjustable parameters of the Langmuir isotherm—the saturation capacity, orq
m, and the dissociation constant,K
d—are usually estimated by fitting the isotherm equation to the equilibrium data acquired from batch equilibration experiments.
In this study, we have evaluated the possibility of estimatingq
m andK
d for the adsorption of bovine serum albumin to a cation exchanger using batch kinetic data. A rate model predicated on the
kinetic form of the Langmuir isotherm, with three adjustable parameters (q
m,K
d, and a rate constant), was fitted to a single kinetic profile. The value ofq
m determined as the result of this approach was quantitatively consistent with theq
m value derived from the traditional batch equilibrium data. However, theK
d value could not be retrieved from the kinetic profile, as the model fit proved insensitive to this parameter. Sensitivity
analysis provided significant insight into the identifiability of the three model parameters. 相似文献
6.
7.
Chemical modifications of substrate peptides are often necessary to monitor the hydrolysis of small bioactive peptides. We developed an electrospray ionization mass spectrometry (ESI–MS) assay for studying substrate distributions in reaction mixtures and determined steady-state kinetic parameters, the Michaelis–Menten constant (Km), and catalytic turnover rate (Vmax/[E]t) for three metallodipeptidases: two carnosinases (CN1 and CN2) from human and Dug1p from yeast. The turnover rate (Vmax/[E]t) of CN1 and CN2 determined at pH 8.0 (112.3 and 19.5 s−1, respectively) suggested that CN1 is approximately 6-fold more efficient. The turnover rate of Dug1p for Cys-Gly dipeptide at pH 8.0 was found to be slightly lower (73.8 s−1). In addition, we determined kinetic parameters of CN2 at pH 9.2 and found that the turnover rate was increased by 4-fold with no significant change in the Km. Kinetic parameters obtained by the ESI–MS method are consistent with results of a reverse-phase high-performance liquid chromatography (RP–HPLC)-based assay. Furthermore, we used tandem MS (MS/MS) analyses to characterize carnosine and measured its levels in CHO cell lines in a time-dependent manner. The ESI–MS method developed here obviates the need for substrate modification and provides a less laborious, accurate, and rapid assay for studying kinetic properties of dipeptidases in vitro as well as in vivo. 相似文献
8.
Santiago-Castillo JA Covarrubias M Sánchez-Rodríguez JE Perez-Cornejo P Arreola J 《Channels (Austin, Tex.)》2010,4(5):422-428
In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously. 相似文献
9.
《Channels (Austin, Tex.)》2013,7(5):422-428
In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously. 相似文献
10.
Enzyme access, kinetic behavior, and protein–protein interactions are critical for explaining reaction of the metabolites
contained within the myriad compartments of biological systems. To explore these relationships, the reaction kinetics of oil
bodies versus oil emulsions as substrates for lipolytic reactions were measured. The initial rate of hydrolysis for the oil
body system was comparatively very low due to a brief latency period. However, the complete activation of the lipase at the
interface resulted in an enzyme–membrane complex that was catalytically enhanced 3–15-fold over the emulsion system for substrate
concentrations in the measured range of approximately 1–5.5 mM. This disparity is explained by the availability of substrate
to the enzyme active site (defined as the availability parameter “A”) which varies between the two substrates by 40-fold. A simple hyperbolic kinetic mechanism is proposed with K
m replaced by the parameter, A, to account for this phenomenon, leading to a maximum rate of approximately 1450 IU/mg protein. The interaction is verified
through separation of the enzyme–membrane complex which shows nearly double the activity towards an emulsified soybean oil
substrate (activity ratio of 5:3) when compared to the native enzyme. 相似文献
11.
Phytase (EC 3.1.3.–) hydrolyzes phytate (IP6) present in cereals and grains to release inorganic phosphate (Pi), thereby making it bioavailable. The most commonly used method to assay phytase, developed nearly a century ago, measures the Pi liberated from IP6. This traditional endpoint assay is time-consuming and well known for its cumbersomeness in addition to requiring extra caution for handling the toxic regents used. This article reports a simple, fast, and nontoxic kinetic method adaptable for high throughput for assaying phytase using IP6–lysozyme as a substrate. The assay is based on the principle that IP6 forms stable turbid complexes with positively charged lysozyme in a wide pH range, and hydrolysis of the IP6 in the complex is accompanied by a decrease in turbidity monitored at 600 nm. The turbidity decrease correlates well to the released Pi from IP6. This kinetic method was found to be useful in assaying histidine acid phytases, including 3- and 6-phytases, a class representing all commercial phytases, and alkaline β-propeller phytase from Bacillus sp. The influences of temperature, pH, phosphate, and other salts on the kinetic assay were examined. All salts, including NaCl, CaCl2, and phosphate, showed a concentration-dependent interference. 相似文献
12.
Emma Smith Rina Vekaria Katherine A. Brown Colin Longstaff 《Molecular and cellular biochemistry》2013,382(1-2):193-201
A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K D in the 10?8 M range and (ii) a kinetically rapid, low-affinity interaction with K D in the 10?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface. 相似文献
13.
Donald J. Winzor 《Analytical biochemistry》2013,441(2):214-217
This investigation completes the amendment of theoretical expressions for the characterization of antigen–antibody interactions by kinetic exclusion assay—an endeavor that has been marred by inadequate allowance for the consequences of antibody bivalence in its uptake by the affinity matrix (immobilized antigen) that is used to ascertain the fraction of free antibody sites in a solution with defined total concentrations of antigen and antibody. A simple illustration of reacted site probability considerations in action confirms that the square root of the fluorescence response ratio, RAg/Ro, needs to be taken in order to determine the fraction of unoccupied antibody sites, which is the parameter employed to describe the kinetics of antigen uptake in the mixture of antigen and antibody with defined initial composition. The approximately 2-fold underestimation of the association rate constant (ka) that emanates from the usual practice of omitting the square root factor gives rise to a corresponding overestimate of the equilibrium dissociation constant (Kd)—a situation that is also encountered in the thermodynamic characterization of antigen–antibody interactions by kinetic exclusion assay. 相似文献
14.
Estela Natacha Brandt Busanello Vannessa Gonçalves Araujo Lobato Ângela Zanatta Carolina Maso Viegas César Augusto João Ribeiro Moacir Wajner 《Life sciences》2014
Aims
Peroxisomal biogenesis disorders (PBD) are inherited disorders clinically manifested by neurological symptoms and brain abnormalities, in which the cerebellum is usually involved. Biochemically, patients affected by these neurodegenerative diseases accumulate branched-chain fatty acids, including pristanic acid (Prist) in the brain and other tissues.Main methods
In the present investigation we studied the in vitro influence of Prist, at doses found in PBD, on oxidative phosphorylation, by measuring the activities of the respiratory chain complexes I–IV and ATP production, as well as on creatine kinase and synaptic Na+, K+-ATPase activities in rat cerebellum.Key findings
Prist significantly decreased complexes I–III (65%), II (40%) and especially II–III (90%) activities, without altering the activities of complex IV of the respiratory chain and creatine kinase. Furthermore, ATP formation and synaptic Na+, K+-ATPase activity were markedly inhibited (80–90%) by Prist. We also observed that this fatty acid altered mitochondrial and synaptic membrane fluidity that may have contributed to its inhibitory effects on the activities of the respiratory chain complexes and Na+, K+-ATPase.Significance
Considering the importance of oxidative phosphorylation for mitochondrial homeostasis and of Na+, K+-ATPase for the maintenance of cell membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission in cerebellum. We postulate that these pathomechanisms may contribute to the cerebellar alterations observed in patients affected by PBD in which Prist is accumulated. 相似文献15.
《Bioscience, biotechnology, and biochemistry》2013,77(8):1446-1450
The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K m and V max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V max,app>V max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values. 相似文献
16.
B Chance 《The Journal of general physiology》1965,49(1):Suppl:163-Suppl:195
This paper considers the way in which the oxygen reaction described by Dr. Nicholls and the ADP control reactions described by Dr. Racker could cooperate to establish a purposeful metabolic control phenomenon in vivo. This has required an examination of the kinetic properties of the respiratory chain with particular reference to methods for determinations of oxygen affinity (Km). The constant parameter for tissue respiration is k
1, the velocity constant for the reaction of oxygen with cytochrome oxidase. Not only is this quantity a constant for a particular tissue or mitochondria; it appears to vary little over a wide range of biological material, and for practical purposes a value of 5 x 107 at 25° close to our original value (20) is found to apply with adequate accuracy for calculation of Km for mammalia. The quantity which will depend upon the tissue and its metabolic state is the value of Km itself, and Km may be as large as 0.5 µM and may fall to 0.05 µM or less in resting, controlled, or inhibited states. The control characteristic for ADP may depend upon the electron flux due to the cytochrome chain (40); less ADP is required to activate the slower electron transport at lower temperatures than at higher temperatures. The affinity constants for ADP control appear to be less dependent upon substrate supplied to the system. The balance of ADP and oxygen control in vivo is amply demonstrated experimentally and is dependent on the oxygen concentration as follows. In the presence of excess oxygen, control may be due to the ADP or phosphate (or substrate), and the kinetics of oxygen utilization will be independent of the oxygen concentration. As the oxygen concentration is diminished, hemoglobin becomes disoxygenated, deep gradients of oxygen concentration develop in the tissue, and eventually cytochrome oxidase becomes partially and then completely reduced. DPN at this point will become reduced and the electron flow diminished. The rate of ATP production falls and energy conservation previously under the control of the ADP concentration will now be controlled by the diffusion of oxygen to the respiratory enzymes in the mitochondria. Under these conditions the rate of reaction of cytochrome oxidase with oxygen and the reaction of cytochromes with one another become of key importance. The rise of ADP and the depletion of energy reserves evoke glycolytic activity, and failure of biological function may result. 相似文献
17.
This article describes a simple fluorescence method for the determination of tetradecyltrimethylammonium mono-oxygenase (TTAB mono-oxygenase) activity involving N-dealkylation of tetradecyltrimethylammonium bromide with concomitant production of trimethylamine (TMA). Activity was determined by measuring the formation of TMA using the morin reagent and aluminum (Al). Morin reacts with Al to form a fluorescent complex, Al-morin. In the presence of TMA, Al is tightly associated with TMA and cannot be sequestered by morin, thus providing evidence for formation of the Al-TMA complex. The concentration of TMA is estimated by calibration graphs constructed by plotting the fluorescence intensity of the Al-morin complex versus TMA concentration. The fluorescence intensities of the Al-morin complexes quenched by TMA are linearly dependent on both the time of the TTAB mono-oxygenase reaction and the amount of protein used in the reaction. The kinetic behavior is characterized by K0.5 = 4.26 × 10−4 M, and the apparent Hill coefficient (napp) = 2.24. These values are both comparable to those determined by GC-MS (K0.5 = 4.41 × 10−4 M and napp = 2.35). The advantages of this assay include rapid and efficient implementation and potential employment for routine accurate determinations of TTAB mono-oxygenase activity over a wide range of substrate concentrations. 相似文献
18.
Alexey A. Zeifman Fedor N. Novikov Victor S. Stroylov Oleg V. Stroganov Ghermes G. Chilov Alexander Y. Skoblov Anatoly I. Miroshnikov Yuri S. Skoblov 《FEBS letters》2014
2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki = 250 μM (95% CI: 106–405 μM) and dose-dependently increased the rate of the ATP hydrolysis with KM = 112 μM (95% CI: 28–195 μM). The kinetic scheme consistent with this experimental data is proposed. 相似文献
19.
Mohammad A. Kamal Abdulaziz A. Al‐Jafari 《Journal of biochemical and molecular toxicology》1999,13(1):41-46
We have designed this study to determine various kinetic parameters of camel retinal membrane‐bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N‐{{(methylamino)carbonyl}oxy} ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 (μM)−1, 1.14 (μM)−1, 0.216 μM, 0.016 min−1, 0.0741 (μM min)−1, 0.746 μM, and 4.42 μM for velocity constant (Kv), new inhibition constant (Knic), dissociation constant (Kd), carbamylation rate constant (k2c), overall carbamylation rate constant (k′2 ), 50% inhibition constant (KI50), and 99% inhibition constant (KI99), respectively. These unique methods may be used to estimate such kinetic parameters for time‐dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 41–46, 1999 相似文献
20.
Roland M. H. Gordon-Beresford Daniel Van Belle Jesús Giraldo Shoshana J. Wodak 《Proteins》1996,25(2):180-194
A thermodynamic cycle is used to describe barnase catalysis, which considers explicitly the presence of different ionic states of the catalytic residues Glu-73 and His-102 in barnase during the enzyme-substrate recognition process. Reinterpretation of published experimental data using rate equations derived from this cycle provides estimates of the ionization constants of these catalytic side chains, in the free enzyme and in the barnase-GpA complex. In addition, the electrostatic properties of the barnase-d(CGAC) crystal complex and of a barnase-5′3′(AAGAAp)-O-methyl ester modeled complex are investigated by means of a continuum approach to account for solvent polarization effects. Taking GpA as a reference substrate, it is shown that Increasing the length of the bound nucleotide induces pKa shifts in the catalytic side chains, which modulate the fraction of enzyme in the correct ionic form for achieving the transesterification reaction. The computed results are in good agreement with the experimental variation of the optimum pH of barnase activity. The present analysis underscores the influence of pH effects on the kcat and KM kinetic constants of barnase and provides the basic formalism for linking the effective kinetic parameters, which usually depend on the pH, to the theoretical estimates of the true kinetic constants. © 1996 Wiley-Liss, Inc. 相似文献