Detection and characterization using circular dichroism and fluorescence spectroscopy of a stable intermediate conformation formed in the denaturation of bovine carbonic anhydrase with guanidinium chloride

Biochemistry ◽  
1982 ◽  
Vol 21 (23) ◽  
pp. 5918-5923 ◽  
Author(s):  
Robert W. Henkens ◽  
Barbara B. Kitchell ◽  
S. Chace Lottich ◽  
Paul J. Stein ◽  
Taffy J. Williams
Keyword(s):  
Circular Dichroism ◽  
Carbonic Anhydrase ◽  
Fluorescence Spectroscopy ◽  
Guanidinium Chloride ◽  
Bovine Carbonic Anhydrase ◽  
Stable Intermediate ◽  
Circular Dichroïsm

Spectroscopic and biophysical approaches for studying the structure and function of the P-glycoprotein multidrug transporter

1998 ◽  
Vol 76 (5) ◽  
pp. 695-708 ◽  
Author(s):  
Frances J Sharom ◽  
Ronghua Liu ◽  
Yolanda Romsicki
Keyword(s):  
Electron Microscopy ◽  
Circular Dichroism ◽  
Multidrug Resistance ◽  
Fluorescence Spectroscopy ◽  
Lipid Bilayers ◽  
Drug Transport ◽  
Circular Dichroism Spectroscopy ◽  
P Glycoprotein ◽  
Infra Red ◽  
Circular Dichroïsm

Multidrug resistance is a serious obstacle to the successful chemotherapeutic treatment of many human cancers. A major cause of multidrug resistance is the overexpression of a 170-kDa plasma membrane protein, known as P-glycoprotein, which appears to function as an ATP-driven efflux pump with a very broad specificity for hydrophobic drugs, peptides, and natural products. P-Glycoprotein is a member of the ABC superfamily and is proposed to consist of two homologous halves, each comprising six membrane-spanning segments and a cytosolic nucleotide binding domain. In recent years, P-glycoprotein has been purified and functionally reconstituted into lipid bilayers, where it retains both ATPase and drug transport activity. The availability of purified active protein has led to substantial advances in our understanding of the molecular structure and mechanism of action of this unique transporter. This review will focus on the recent application of fluorescence spectroscopy, infra-red spectroscopy, circular dichroism spectroscopy, electron microscopy, and other biophysical techniques to the study of P-glycoprotein structure and function.Key words: multidrug resistance, P-glycoprotein, fluorescence spectroscopy, infra-red spectroscopy, circular dichroism spectroscopy, differential scanning calorimetry, electron microscopy.

scholarly journals Optical properties and denaturation by guanidinium chloride and urea of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of four molecular forms of the enzyme

1977 ◽  
Vol 161 (2) ◽  
pp. 321-331 ◽  
Author(s):  
M Nieto ◽  
J A Ayala
Keyword(s):  
Circular Dichroism ◽  
Adenosine Triphosphatase ◽  
Quaternary Structure ◽  
Molecular Form ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Molecular Forms ◽  
Guanidinium Chloride ◽  
Micrococcus Lysodeikticus ◽  
Circular Dichroïsm

1. The fluorescence and circular dichroism of four homogeneous preparations of ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus differing in molecular structure and enzymic properties were examined at pH 7.5 and 25 degrees. Emission was maximum at 325 and 335 nm and the relative intensities at these wavelengths may be used to characterize the different ATPase preparations. The circular-dichroism spectra exhibited negative extrema at 208 and 220 nm, and the relative value of the molar ellipticity at these wavelengths was also different for each molecular form of the enzyme. 2. The four preparations undergo two consecutive major unfolding transitions in guanidinium chloride (midpoints at 0.94 and 1.5 M denaturant), with concomitant destruction of the quaternary structure of the protein. A comparatively minor alteration in the ATPase structure also occurred in 0.05-0.2M-guanidine and led to complete inactivation of the enzyme. The inactivation and the first unfolding transition were reversible by dilution of the denaturant; the transition with midpoint at 1.5M-guanidine was irreversible. 3. Similar results were obtained in urea, except that the successive transitions had midpoints at concentrations of denaturant of 0.4, 2.0 and 4.5M. Low concentrations of urea caused a noticeable activation of the enzyme activity and alterations of the electrophoretic mobility of the ATPase. 4. A model is proposed in which one of the major subunits, alpha, is first dissociated and unfolded reversibly by the denaturants, followed by the irreversible unfolding and dissociation of the other major subunit, beta, from subunit delta and/or the components of relative mobility 1.0 in dodecyl sulphate/polyacrylamide-gel electrophoresis (rho).

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