In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis

2005 ◽  
Vol 383 (1) ◽  
pp. 92-97 ◽  
Author(s):  
Sandro Hillebrand ◽  
Wanius Garcia ◽  
Marcelo Delmar Cantú ◽  
Ana Paula Ulian de Araújo ◽  
Manami Tanaka ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Enzyme Kinetics ◽  
Gtpase Activity ◽  
Kinetics Studies

Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells

1994 ◽  
Vol 107 (8) ◽  
pp. 2249-2257 ◽  
Author(s):  
T. Asada ◽  
H. Shibaoka
Keyword(s):  
Motor Activity ◽  
Molecular Basis ◽  
Plant Cells ◽  
Cell Plate ◽  
Gtpase Activity ◽  
High Concentration ◽  
Higher Plant ◽  
Main Components ◽  
Brain Tubulin

As part of our efforts to understand the molecular basis of the microtubule-associated motility that is involved in cytokinesis in higher plant cells, an attempt was made to identify proteins with the ability to translocate microtubules in an extract from isolated phragmoplasts. Homogenization of isolated phragmoplasts in a solution that contained MgATP, MgGTP and a high concentration of NaCl resulted in the release from phragmoplasts of factors with ATPase and GTPase activity that were stimulated by microtubules. A protein fraction with microtubule-dependent ATPase and GTPase activity caused minus-end-headed gliding of microtubules in the presence of ATP or GTP. Polypeptides with microtubule-translocating activity cosedimented with microtubules that had been assembled in vitro from brain tubulin and were dissociated from sedimented microtubules by addition of ATP or GTP. After cosedimentation and dissociation procedures, a 125 kDa polypeptide and a 120 kDa polypeptide were recovered in a fraction that supported minus-end-headed gliding of microtubules. The rate of microtubule gliding that was caused by the fraction that contained the 125 kDa and 120 kDa polypeptides as main components was 1.28 microns/minute in the presence of ATP and 0.50 microns/minute in the presence of GTP. This fraction contained some microtubule-associated polypeptides in addition to the 125 kDa and 120 kDa polypeptides, but a fraction that contained only these additional polypeptides did not cause any translocation of microtubules. Thus, it appeared that the 125 kDa and 120 kDa polypeptides were responsible for translocation of microtubules. These polypeptides with plus-end-directed motor activity may play an important role in formation of the cell plate and in the organization of the phragmoplast.

Application of Capillary Electrophoresis To the In Vitro Assessment of Drug Metabolism

1995 ◽  
Vol 23 (3) ◽  
pp. 432S-432S
Author(s):  
CM Hill ◽  
KE Herbert ◽  
MD Evans ◽  
J Lunec
Keyword(s):  
Capillary Electrophoresis ◽  
Drug Metabolism ◽  
In Vitro Assessment

scholarly journals Quinazolinones as Competitive Inhibitors of Carbonic Anhydrase-II (Human and Bovine): Synthesis, in-vitro, in-silico, Selectivity, and Kinetics Studies

2020 ◽  
Vol 8 ◽  
Author(s):  
Ajmal Khan ◽  
Majid Khan ◽  
Sobia Ahsan Halim ◽  
Zulfiqar Ali Khan ◽  
Zahid Shafiq ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Active Sites ◽  
Binding Mode ◽  
Competitive Inhibitor ◽  
Significant Inhibition ◽  
Carbonic Anhydrase Ii ◽  
Complete Agreement ◽  
Active Inhibitor ◽  
Kinetics Studies

Carbonic anhydrase-II (CA-II) is associated with glaucoma, malignant brain tumors, and renal, gastric, and pancreatic carcinomas and is mainly involved in the regulation of the bicarbonate concentration in the eyes. CA-II inhibitors can be used to reduce the intraocular pressure usually associated with glaucoma. In search of potent CA-II inhibitors, a series of quinazolinones derivatives (4a-p) were synthesized and characterized by IR and NMR spectroscopy. The inhibitory potential of all the compounds was evaluated against bovine carbonic anhydrase-II (bCA-II) and human carbonic anhydrase-II (hCA-II), and compounds displayed moderate to significant inhibition with IC50 values of 8.9–67.3 and 14.0–59.6 μM, respectively. A preliminary structure-activity relationship suggested that the presence of a nitro group on the phenyl ring at R position contributes significantly to the overall activity. Kinetics studies of the most active inhibitor, 4d, against both bCA-II and hCA-II were performed to investigate the mode of inhibition and to determine the inhibition constants (Ki). According to the kinetics results, 4d is a competitive inhibitor of bCA-II and hCA-II with Ki values of 13.0 ± 0.013 and 14.25 ± 0.017 μM, respectively. However, the selectivity index reflects that the compounds 4g and 4o are more selective for hCA-II. The binding mode of these compounds within the active sites of bCA-II and hCA-II was investigated by structure-based molecular docking. The docking results are in complete agreement with the experimental findings.

An online immobilized α-glucosidase microreactor for enzyme kinetics and inhibition assays

RSC Advances ◽  
2015 ◽  
Vol 5 (70) ◽  
pp. 56841-56847 ◽  
Author(s):  
Dong-Mei Liu ◽  
Juan Chen ◽  
Yan-Ping Shi
Keyword(s):  
Capillary Electrophoresis ◽  
Enzyme Kinetics ◽  
Inhibition Kinetics

A novel online α-glucosidase-immobilized microreactor was developed by immobilizing α-glucosidase on capillary inner wall. The microreactor combination with capillary electrophoresis was applied in studying enzyme kinetics and inhibition kinetics.

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