scholarly journals Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins

2012 ◽  
Vol 426 (2) ◽  
pp. 103-105 ◽  
Author(s):  
Boris Repen ◽  
Erwin Schneider ◽  
Ulrike Alexiev
Keyword(s):  
Membrane Proteins ◽  
Malachite Green ◽  
Atp Hydrolysis ◽  
Malachite Green Assay

An improved malachite green assay of phosphate: Mechanism and application

2011 ◽  
Vol 409 (1) ◽  
pp. 144-149 ◽  
Author(s):  
Juan Feng ◽  
Yuan Chen ◽  
Jun Pu ◽  
Xiaolan Yang ◽  
Chun Zhang ◽  
...  
Keyword(s):  
Malachite Green ◽  
Malachite Green Assay

The In Vitro Anti-Cancer Activities of 17βH-Neriifolin Isolated from Cerbera odollam and its Binding Activity on Na+, K+-ATPase

2020 ◽  
Vol 21 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Nurhanan M. Yunos ◽  
Asiah Osman ◽  
Muhammad H. Jauri ◽  
Nor J. Sallehudin ◽  
Siti Syarifah Mohd Mutalip
Keyword(s):  
Cell Death ◽  
Binding Energy ◽  
Cell Lines ◽  
Malachite Green ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Α Subunit ◽  
Anti Cancer ◽  
Malachite Green Assay

Background: 17βH-neriifolin, a cardiac glycoside compound had been successfully isolated from Cerbera odollam leaves based on the bioassay guided-isolation procedure. The aim of these studies were to determine the in vitro anti-cancer and binding effects of 17βH-neriifolin on Na+, K+-ATPase. Methods: The in vitro anti-cancer effects were evaluated using Sulphorhodamine B and Hoescht 33342 assays. The Na+, K+-ATPase assay was carried out using Malachite Green assay. In silico molecular docking studies and in vitro malachite green assay were used to predict the binding activities of 17βH-neriifolin on Na+, K+-ATPase and ouabain was also included as for comparison studies. Results: The compound was tested against breast (MCF-7, T47D), colorectal (HT-29), ovarian (A2780, SKOV-3) and skin (A375) cancer cell lines that gave IC50 values ranged from 0.022 ± 0.0015 to 0.030 ± 0.0018 μM. The mechanism of cell death of 17βH-neriifolin was further evaluated using Hoescht 33342 assay and it was found that the compound killed the cancer cells via apoptosis. 17βHneriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were - 8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. Conclusion: The results had confirmed the anti-proliferative effects exerted by 17βH-neriifolin in the breast, colorectal, ovarian and skin cancer cell lines. 17βH-neriifolin had shown to cause apoptotic cell death in the respective cancer cell lines.17βH-neriifolin and ouabain both bound at α-subunit in Na+, K+-ATPase and their binding energy were -8.16 ± 0.74 kcal/mol and -8.18 ± 0.48 kcal/mol respectively. This is the first report to reveal that 17βH-neriifolin managed to bind to the pocket of α-subunit of Na+.K+-ATPase.

Rational Identification of Hsp90 Inhibitors as Anticancer Lead Molecules by Structure Based Drug Designing Approach

2020 ◽  
Vol 20 (3) ◽  
pp. 369-385 ◽  
Author(s):  
Sayan D. Gupta ◽  
Pappu S. Swapanthi ◽  
Deshetti Bhagya ◽  
Fernando Federicci ◽  
Gisela I. Mazaira ◽  
...  
Keyword(s):  
Schiff Bases ◽  
Cell Lines ◽  
Malachite Green ◽  
Cancer Cell ◽  
Mtt Assay ◽  
Research Work ◽  
Cancer Cell Lines ◽  
Docking Studies ◽  
Hsp90 Inhibitors ◽  
Malachite Green Assay

Background: Heat shock protein 90 (Hsp90) is an encouraging anticancer target for the development of clinically significant molecules. Schiff bases play a crucial role in anticancer research because of their ease of synthesis and excellent antiproliferative effect against multiple cancer cell lines. Therefore, we started our research work with the discovery of resorcinol/4-chloro resorcinol derived Schiff bases as Hsp90 inhibitors, which resulted in the discovery of a viable anticancer lead molecule. Objective: The objective of the study is to discover more promising lead molecules using our previously established drug discovery program, wherein the rational drug design is achieved by molecular docking studies. Methods: The docking studies were carried out by using Surflex Geom X programme of Sybyl X-1.2 version software. The molecules with good docking scores were synthesized and their structures were confirmed by IR, 1H NMR and mass spectral analysis. Subsequently, the molecules were evaluated for their potential to attenuate Hsp90 ATPase activity by Malachite green assay. The anticancer effect of the molecules was examined on PC3 prostate cancer cell lines by utilizing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay methodology. Results: Schiff bases 11, 12, 20, 23 and 27 exhibiting IC50 value below 1μM and 15μM, in malachite green assay and MTT assay, respectively, emerged as viable lead molecules for future optimization. Conclusion: The research work will pave the way for the rational development of cost-effective Schiff bases as Hsp90 inhibitors as the method employed for the synthesis of the molecules is simple, economic and facile.

scholarly journals Domains of alpha-SNAP required for the stimulation of exocytosis and for N-ethylmalemide-sensitive fusion protein (NSF) binding and activation.

1996 ◽  
Vol 7 (5) ◽  
pp. 693-701 ◽  
Author(s):  
R J Barnard ◽  
A Morgan ◽  
R D Burgoyne
Keyword(s):  
Membrane Proteins ◽  
Fusion Protein ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Atp Hydrolysis ◽  
Coiled Coil ◽  
Protein Protein Interactions ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Adrenal Chromaffin

The binding of alpha-SNAP to the membrane proteins syntaxin, SNAP-25, and synaptobrevin leads to the recruitment of the N-ethylmaleimide-sensitive fusion protein (NSF). ATP hydrolysis by NSF has been suggested to drive conformational changes in one or more of these membrane proteins that are essential for regulated exocytosis. Functional evidence for a role of alpha-SNAP in exocytosis in adrenal chromaffin cells comes from the ability of this protein to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized cells. Here we examine the effect of a series of deletion mutants of alpha-SNAP on exocytosis, and on the ability of alpha-SNAP to interact with NSF, to define essential domains involved in protein-protein interactions in exocytosis. Deletion of extreme N- or C-terminal regions of alpha-SNAP produced proteins unable to bind to syntaxin or to stimulate exocytosis, suggesting that these domains participate in essential interactions. Deletion of C-terminal residues abolished the ability of alpha-SNAP to bind NSF. In contrast, deletion of up to 120 N-terminal residues did not prevent the binding of NSF to immobilized alpha-SNAP and such mutants were also able to stimulate the ATPase activity of NSF. These results suggest that the C-terminus, but not the N-terminus, of alpha-SNAP is crucial for interactions with NSF. The involvement of the C-terminus of alpha-SNAP, which contains a predicted coiled-coil domain, in the binding of both syntaxin and NSF would place the latter two proteins in proximity in a ternary complex whereupon the energy derived from ATP hydrolysis by NSF could induce a conformational change in syntaxin required for exocytosis to proceed.

scholarly journals Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H(+)-ATPase complex.

1994 ◽  
Vol 5 (9) ◽  
pp. 1039-1050 ◽  
Author(s):  
K J Hill ◽  
T H Stevens
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Atp Hydrolysis ◽  
Integral Membrane Protein ◽  
Vacuolar Membrane ◽  
Proton Pumping ◽  
Carboxy Terminus ◽  
Lysine Residues ◽  
A Subunit

The yeast vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral membrane subunits involved in ATP hydrolysis and integral membrane subunits involved in proton pumping. The yeast vma21 mutant was isolated from a screen to identify mutants defective in V-ATPase function. vma21 mutants fail to assemble the V-ATPase complex onto the vacuolar membrane: peripheral subunits accumulate in the cytosol and the 100-kDa integral membrane subunit is rapidly degraded. The product of the VMA21 gene (Vma21p) is an 8.5-kDa integral membrane protein that is not a subunit of the purified V-ATPase complex but instead resides in the endoplasmic reticulum. Vma21p contains a dilysine motif at the carboxy terminus, and mutation of these lysine residues abolishes retention in the endoplasmic reticulum and results in delivery of Vma21p to the vacuole, the default compartment for yeast membrane proteins. Our findings suggest that Vma21p is required for assembly of the integral membrane sector of the V-ATPase in the endoplasmic reticulum and that the unassembled 100-kDa integral membrane subunit present in delta vma21 cells is rapidly degraded by nonvacuolar proteases.

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