Kanes, Katherine J. (1989) Band 3 structure and function : [superscript]35 C1 NMR and topographical investigations. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-05302007-081241
Band 3 is the anion exchange protein in red blood cells. It is the most abundant protein in the erythrocyte membrane and is the most heavily used ion transport protein in vertebrates. Physiologically, it transports Cl- into or out of the red blood cell and then transports HCO3- in the opposite direction so that electroneutrality is maintained on both sides of the membrane.
The anion exchange mechanism of band 3 is unique among the ion transport proteins. It transports anions by a 'ping-pong' mechanism, meaning it is a gated protein which effects the one-for-one exchange of anions across the membrane. It is also unusual because it transports a wide variety of anions in a very efficient manner (up to 200 sec-1). An arginine has been implicated in the binding and transport of chloride across the red blood cell membrane. The primary goal of this work was to determine the location of that arginine. A second goal was to investigate divalent anion binding to the active site.
35Cl NMR was used to investigate the competition of chloride with divalent anions at the chloride binding site of band 3. These studies were performed to determine if divalent anions compete with chloride for binding at the active site. These investigations indicate that molybdate, sulfate, and sulfate's analogue selenate interfere with chloride's binding to the transport site. Hydrogen phosphate and its analogue hydrogen arsenate also appear to compete with chloride for binding at the transport site. However, it appears that chloride binding is only fractionally inhibited by these two dianions. This is demonstrated by the inability of hydrogen phosphate and hydrogen arsenate to saturate the transport site and completely inhibit transport site linebroadening. pH profiles of chloride competition with divalent anions were also obtained. It appears that these large, inorganic, hydrated, approximately spherical molecules can reach the transport site in the band 3 channel but not as effectively as chloride.
The goal of the biochemical studies was to determine the location of the band 3 arginine anion binding site. The complete sequence of human band 3 is was not known until very recently, but it has been available for chickens and mice since 1986. On the basis of these sequences, Vogelaar and Chan have modeled transmembrane helices to determine the number of times the protein traverses the membrane(1). In order to insure that an arginine preferentially labeled by 14C-phenylglyoxal was indeed at a transmembrane peptide and to verify the model, many of the transmembrane sequences have been isolated. This was accomplished by modification of a technique developed to separate hydrophobic synthetic peptides(2). The band 3 transmembrane helices are tightly associated and very similar in hydrophobicity. Of a probable total of 14 transmembrane helices, the N-termini and/or C-termini have been determined for 6 of them. Because x-ray crystallography has been difficult to achieve for membrane systems (rhodopsin and the reaction center are two that that have been crystallized), this method provides a simple and relatively inexpensive method of studying membrane protein topography.
Finally, 14C-phenylglyoxal has shown the location of at least one arginine in band 3 when labeled by the method of Zaki(3). (By this method, two to three arginines are labeled per band 3 monomer.) The location of the second arginine has been restricted to two possible other transmembrane peptides (modeled helices 10 and 14). The active site arginine is probably at position R748 in the mouse erythrocyte band 3 sequence.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||10 May 1989|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||31 May 2007|
|Last Modified:||26 Dec 2012 02:49|
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