Characterization of a vanadate-based transition-state-analog complex of phosphoglucomutase by kinetic and equilibrium binding studies. Mechanistic implications

Biochemistry ◽  
1990 ◽  
Vol 29 (11) ◽  
pp. 2790-2801 ◽  
Author(s):  
William J. Ray ◽  
Joseph M. Puvathingal
Keyword(s):  
Transition State ◽  
Binding Studies ◽  
Transition State Analog ◽  
Equilibrium Binding

scholarly journals Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases

1997 ◽  
Vol 6 (2) ◽  
pp. 444-449 ◽  
Author(s):  
Genfa Zhou ◽  
Gopalakrishnan Parthasarathy ◽  
Thayumanasamy Somasundaram ◽  
Andrea Ables ◽  
Lance Roy ◽  
...  
Keyword(s):  
Transition State ◽  
Inclusion Bodies ◽  
Arginine Kinase ◽  
Transition State Analog ◽  
Phosphagen Kinases ◽  
Crystal Characterization ◽  
Kinase A

scholarly journals Efficient screening for catalytic antibodies using a short transition-state analog and detailed characterization of selected antibodies

1999 ◽  
Vol 261 (1) ◽  
pp. 108-114 ◽  
Author(s):  
Naoko Takahashi ◽  
Hiroyuki Kakinuma ◽  
Katsumi Hamada ◽  
Kazuko Shimazaki ◽  
Kyoko Takahashi ◽  
...  
Keyword(s):  
Transition State ◽  
Catalytic Antibodies ◽  
Detailed Characterization ◽  
Transition State Analog ◽  
Efficient Screening ◽  
Short Transition

Characterization of vanadate-based transition-state-analog complexes of phosphoglucomutase by spectral and NMR techniques

Biochemistry ◽  
1990 ◽  
Vol 29 (11) ◽  
pp. 2770-2778 ◽  
Author(s):  
William J. Ray ◽  
John W. Burgner ◽  
Carol Beth Post
Keyword(s):  
Transition State ◽  
Transition State Analog ◽  
Nmr Techniques

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

scholarly journals Ultrafiltration Method for Plasma Protein Binding Studies and Its Limitations

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 382
Author(s):  
Camelia-Maria Toma ◽  
Silvia Imre ◽  
Camil-Eugen Vari ◽  
Daniela-Lucia Muntean ◽  
Amelia Tero-Vescan
Keyword(s):  
Protein Binding ◽  
Plasma Protein Binding ◽  
Plasma Protein ◽  
Specific Binding ◽  
Critical Role ◽  
Volume Ratio ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Key Aspects

Plasma protein binding plays a critical role in drug therapy, being a key part in the characterization of any compound. Among other methods, this process is largely studied by ultrafiltration based on its advantages. However, the method also has some limitations that could negatively influence the experimental results. The aim of this study was to underline key aspects regarding the limitations of the ultrafiltration method, and the potential ways to overcome them. The main limitations are given by the non-specific binding of the substances, the effect of the volume ratio obtained, and the need of a rigorous control of the experimental conditions, especially pH and temperature. This review presents a variety of methods that can hypothetically reduce the limitations, and concludes that ultrafiltration remains a reliable method for the study of protein binding. However, the methodology of the study should be carefully chosen.

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.

Sign in / Sign up

Export Citation Format

Share Document