scholarly journals The determination of binding parameters when the total and free substrate concentrations are not approximately equal

1979 ◽  
Vol 179 (3) ◽  
pp. 697-700 ◽  
Author(s):  
N Gains
Keyword(s):  
Substrate Concentration ◽  
Graphical Method ◽  
Enzyme Concentration ◽  
Binding Parameters ◽  
Free Concentration ◽  
Equilibrium Binding ◽  
Substrate Concentrations

By using a standard graphical method values of Km and V may be found that are independent of the conditions and assumptions that the total substrate concentration approximates to its free concentration and that Km is much larger than the enzyme concentration. The procedure is also applicable to the determination of equilibrium binding parameters of a ligand to a macromolecule.

Study on the Process of the Reaction Catalyzed by Polyphenol Oxidase from Purple Sweet Potato

2014 ◽  
Vol 898 ◽  
pp. 153-156 ◽  
Author(s):  
Lu Gao ◽  
Li Chun Zhao ◽  
Ji Dong Duan ◽  
Yan Li Tao
Keyword(s):  
Sweet Potato ◽  
Polyphenol Oxidase ◽  
Substrate Concentration ◽  
Phosphate Buffer ◽  
Phosphate Buffer Solution ◽  
Enzyme Concentration ◽  
Buffer Solution ◽  
Purple Sweet Potato ◽  
The Relationship ◽  
Substrate Concentrations

The polyphenol oxidase (PPO) was extracted from fresh purple sweet potato (PSP) by phosphate buffer solution, and spectrophotometry method was applied in the experiment. The process of the reaction catalyzed by PPO with different substrate concentrations and the relationship between enzyme concentrations and PPO activity were mainly studied here. The result showed that the effect of enzyme concentration on PPO activity was stronger than that of substrate concentration on PPO activity.

Effects of substrate concentration on results of determination of prostatic acid phosphatase with thymolphthalein monophosphate.

1983 ◽  
Vol 29 (1) ◽  
pp. 148-151 ◽  
Author(s):  
A Kessner ◽  
E J Woodard ◽  
G N Bowers
Keyword(s):  
Catalytic Activity ◽  
Acid Phosphatase ◽  
Substrate Concentration ◽  
Prostatic Acid Phosphatase ◽  
Combined Effects ◽  
Mixed Substrate ◽  
Order Of Magnitude ◽  
Substrate Concentrations

Abstract The relation between concentration of thymolphthalein monophosphate substrate and catalytic activity was investigated for the determination of prostatic acid phosphatase. This study, an extension of previously reported work (Clin. Chem. 27: 1372, 1981), shows that lot-to-lot variation in purity of thymolphthalein monophosphate preparations is reflected in substrate-velocity curves. Plateau regions in these curves at 1.5-2.5 g/L result from the combined effects of (a) substrate concentrations that are an order of magnitude below Km and (b) a further decrease in available substrate caused by formation of substrate aggregates in the presence of serum. To simplify the identification of superior lots of thymolphthalein monophosphate, we give a mixed-substrate protocol for testing different lots.

scholarly journals A method for determining kinetic parameters at high enzyme concentrations

1982 ◽  
Vol 203 (1) ◽  
pp. 339-342 ◽  
Author(s):  
C J Halfman ◽  
F Marcus
Keyword(s):  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Graphical Method ◽  
Free Ligand ◽  
Enzyme Concentration ◽  
Fructose 1,6 Bisphosphatase ◽  
High Enzyme ◽  
Substrate Inhibitor ◽  
The Relationship

A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.

MEASUREMENT OF STEROID BINDING PROTEINS

1970 ◽  
Vol 65 (1_Suppl) ◽  
pp. S104-S121 ◽  
Author(s):  
E. E. Baulieu ◽  
J. P. Raynaud ◽  
E. Milgrom
Keyword(s):  
Kinetic Parameters ◽  
Binding Proteins ◽  
Binding Specificity ◽  
Binding Parameters ◽  
Target Organs ◽  
Biological Mixtures ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT A brief review of the characteristics of steroid binding proteins found in the plasma and in some target organs is presented, followed by some general remarks on binding »specificity« and binding parameters. Useful techniques for measuring binding parameters at equilibrium are reported, both those which keep the equilibrium intact and those which implicate its disruption. A concept is developed according to which the determination of a specific steroid binding protein is based on the »differential dissociation« of the several steroid binding complexes present in most biological mixtures. Methods which allow determination of the kinetic parameters of the binding systems are also presented. Various representations of the binding and therefore different modes of graphic representation and calculation are discussed, including the recent »proportion graph« method.

A Graphical Method for Storage Coefficient Determination from Quasi-Steady State Flow Data

1996 ◽  
Vol 27 (4) ◽  
pp. 247-254 ◽  
Author(s):  
Zekâi Şen
Keyword(s):  
Steady State ◽  
Graphical Method ◽  
Pumping Test ◽  
Quasi Steady State ◽  
Flow Data ◽  
Steady State Flow ◽  
Storage Coefficient ◽  
State Flow ◽  
Aquifer Test

A simple, approximate but practical graphical method is proposed for estimating the storage coefficient independently from the transmissivity value, provided that quasi-steady state flow data are available from a pumping test. In the past, quasi-steady state flow distance-drawdown data have been used for the determination of transmissivity only. The method is applicable to confined and leaky aquifers. The application of the method has been performed for various aquifer test data available in the groundwater literature. The results are within the practical limits of approximation compared with the unsteady state flow solutions.

Multiplication of a Klebsiella pneumoniae Strain in Water at Low Concentrations of Substrates

1988 ◽  
Vol 20 (11-12) ◽  
pp. 117-123 ◽  
Author(s):  
D. van der Kooij ◽  
W. A. M. Hijnen
Keyword(s):  
Amino Acids ◽  
Water Treatment ◽  
Klebsiella Pneumoniae ◽  
Substrate Concentration ◽  
Treatment System ◽  
Water Treatment System ◽  
Low Concentrations ◽  
Ks Values ◽  
Substrate Concentrations

A K.pneumoniae strain, isolated from a water treatment system, was tested in growth measurements for its ability to multiply at substrate concentrations of a few micrograms per liter. The organism multiplied on mixtures of carbohydrates and amino acids at a substrate concentration of 1 µg of C of each compound per liter. Tests with individual compounds revealed that especially carbohydrates were utilized at low concentrations. The Ks values obtained for maltose and maltopentaose were 53 µg of C/l and 114 µg of C per liter, respectively. The significance of the growth of K.pneumoniae at low substrate concentrations is discussed.

Respirometry for determination of the influents SS-concentration

1996 ◽  
Vol 33 (1) ◽  
pp. 311-323 ◽  
Author(s):  
A. Witteborg ◽  
A. van der Last ◽  
R. Hamming ◽  
I. Hemmers
Keyword(s):  
Experimental Design ◽  
Activated Sludge ◽  
Substrate Concentration ◽  
Full Scale ◽  
Activated Sludge Process ◽  
Respiration Rates ◽  
On Line ◽  
Large Measurement ◽  
Set Up

A method is presented for determining influent readily biodegradable substrate concentration (SS). The method is based on three different respiration rates, which can be measured with a continuous respiration meter which is operated in a cyclic way. Within the respiration meter nitrification is inhibited through the addition of ATU. Simulations were used to develop the respirometry set-up and decide upon the experimental design. The method was tested as part of a large measurement programme executed at a full-scale plant. The proposed respirometry set-up has been shown to be suitable for a semi-on-line determination of an influent SS which is fully based on the IAWQ #1 vision of the activated sludge process. The YH and the KS play a major role in the principle, and should be measured directly from the process.

scholarly journals Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Gianluca Trinco ◽  
Valentina Arkhipova ◽  
Alisa A. Garaeva ◽  
Cedric A. J. Hutter ◽  
Markus A. Seeger ◽  
...  
Keyword(s):  
Kinetic Mechanism ◽  
Protein Quantification ◽  
Detergent Solution ◽  
Sodium Ions ◽  
Binding Studies ◽  
Equilibrium Binding ◽  
Uptake Rates ◽  
Bona Fide ◽  
Inside Out

AbstractIt is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.

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