Monitoring conformational change in the human erythrocyte glucose carrier: Use of a fluorescent probe attached to an exofacial carrier sulfhydryl

Biochemistry ◽  
1993 ◽  
Vol 32 (11) ◽  
pp. 2907-2915 ◽  
Author(s):  
James M. May ◽  
Joseph M. Beechem
Keyword(s):  
Fluorescent Probe ◽  
Conformational Change ◽  
Human Erythrocyte ◽  
Glucose Carrier

scholarly journals The interaction of 3-deoxy-3-fluoro-d-glucose with the hexose-transport system of the human erythrocyte

1973 ◽  
Vol 135 (4) ◽  
pp. 773-777 ◽  
Author(s):  
G. J. Riley ◽  
N. F. Taylor
Keyword(s):  
Kinetic Parameters ◽  
Erythrocyte Membrane ◽  
Transport System ◽  
Dissociation Energy ◽  
Human Erythrocyte ◽  
Optical Method ◽  
Hexose Transport ◽  
Human Erythrocyte Membrane ◽  
Significant Release ◽  
Glucose Carrier

1. By using an optical method the kinetic parameters of hexose transport across the human erythrocyte membrane were determined for several sugars. The series of half-saturated constants is as follows: 3-deoxy-3-fluoro-d-glucose = 3-O-methyl-d-glucose <d-glucose<d-mannose <3-deoxy-d-glucose<d-galactose<l-arabinose. 2. Estimations of the dissociation energy of the 3-deoxy-3-fluoro-d-glucose–carrier and d-glucose–carrier complexes suggest that the binding of the two sugars to the transport system is equivalent. 3. Incubation of the erythrocytes with 3-deoxy-3-fluoro-d-glucose results in a small but significant release of F−anion. Cells treated in this way lose their ability to transport glucose.

Human erythrocyte anion exchange site characterised using a fluorescent probe

Nature ◽  
1979 ◽  
Vol 282 (5738) ◽  
pp. 520-522 ◽  
Author(s):  
James A. Dix ◽  
A. S. Verkman ◽  
A. K. Solomon ◽  
L. C. Cantley
Keyword(s):  
Fluorescent Probe ◽  
Anion Exchange ◽  
Human Erythrocyte ◽  
Exchange Site

scholarly journals Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

1993 ◽  
Vol 291 (1) ◽  
pp. 103-107 ◽  
Author(s):  
H M Zhou ◽  
X H Zhang ◽  
Y Yin ◽  
C L Tsou
Keyword(s):  
Creatine Kinase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Emission Intensity ◽  
Enzyme Inactivation ◽  
Amino Groups ◽  
Guanidinium Chloride ◽  
Low Concentrations

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

Further Studies on Zn2+-Mediated Domain–Domain Communication in Human Erythrocyte Band 3

1998 ◽  
Vol 18 (5) ◽  
pp. 265-277
Author(s):  
Hong Xu ◽  
Xujia Zhang ◽  
Fu Yu Yang
Keyword(s):  
Conformational Change ◽  
Human Erythrocyte ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Anion Transport ◽  
Intrinsic Fluorescence ◽  
Transport Activity ◽  
Membrane Domain ◽  
Nmr Study ◽  
Anion Transport Activity

Human erythrocyte band 3 is purified and reconstituted into vesicles, forming right-side-out proteoliposomes. Zn2+ entrapped inside the proteoliposomes inhibits the anion transport activity of band 3, and removal of the cytoplasmic domain of band 3 is able to diminish Zn2+ inhibition. Thus, the inhibition of activity of band 3 results from the Zn2+ induced conformational change of the cytoplasmic domain, which in turn is transmitted to the membrane domain. The results of intrinsic fluorescence and its quenching by HB and the 35Cl NMR study indicate that the cytoplasmic domain is essential for the conformational change induced by Zn2+.SH-blocking reagents, CH3I and GSSG, are used to modify the cytoplasmic domain, where they specifically bind to Cys201 and Cys317. It is observed that the Zn2+ induced inhibition of anion transport activity is blocked. This demonstrates that Cys201 and Cys317 are required in Zn2+-mediated domain–domain communication.

scholarly journals Allosteric regulation: Illuminating allosteric conformational change with an environmentally sensitive fluorescent probe

2015 ◽  
Vol 37 (3) ◽  
pp. 54-57
Author(s):  
Robert B. Freedman ◽  
Alan D.B. Malcolm
Keyword(s):  
Glutamate Dehydrogenase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Allosteric Regulation ◽  
Structural Data ◽  
Biochemical Journal ◽  
Environmentally Sensitive ◽  
The 1960S ◽  
Classic Paper

Allosteric regulation was a hot topic in the 1960s, but there was very limited structural data on allosteric equilibria, and no solid information on the rates of allosteric conformational changes. In this Biochemical Journal Classic paper from 1969 George Radda and his first D.Phil. student, George Dodd determined the rate of allosteric transition in the regulatory enzyme glutamate dehydrogenase by a method new in the 1960s, the fluorescence of an environmentally sensitive extrinsic probe.

Evidence against the Involvement of Mutarotase in the D-Glucose Uptake Activity of Isolated Human Erythrocyte Membranes

1972 ◽  
Vol 50 (9) ◽  
pp. 1028-1030 ◽  
Author(s):  
Arthur Kahlenberg ◽  
Gary Miller
Keyword(s):  
Glucose Uptake ◽  
Membrane Transport ◽  
Glucose Transport ◽  
Human Erythrocyte ◽  
Transport Process ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes ◽  
Uptake Activity ◽  
Glucose Carrier

Mutarotase, the enzyme catalyzing the interconversion of the anomeric forms of D-glucose, has recently been suggested to be the membrane glucose carrier in human erythrocytes. However, hemoglobin-free human erythrocyte membranes possessing D-glucose uptake activity were found to be free of mutarotase activity. Mutarotase activity was detected in the membrane-free hemolysates of the cells. It is therefore concluded that the D-glucose uptake activity of isolated erythrocyte membranes is not due to the binding of the sugar to mutarotase, and that this enzyme is not involved in glucose transport in a manner compatible with most presently held concepts of the membrane transport process.

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