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Proteins of the Red Blood Cell Membrane.

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Proteins of the Red Blood Cell Membrane Clare Oliver October 24th 2003 The aim of this experiment was to find the major proteins that make up the plasma membranes of cells, their whereabouts within the membranes, and what effect various pre-treatments would have on these proteins. It was the erythrocyte membrane that was studied in this experiment because red blood cells can easily be extracted from whole blood and the membranes themselves are not contaminated, as the inside of the red blood cell does not contain any organelles, and therefore no internal membranes (Lodish et al. 2000). The plasma membranes of cells are composed of a lipid bilayer and protein molecules, which form a barrier around the inner components of the cell and ensure its cell processes are separate from those in the outside environment. This is essential to the survival of the cell. The proteins within the membrane carry out many different roles, all of which are vital to the life of the cell, and there are different types for each role. There are so many membrane proteins that it is thought they may make up as much as 30% of all proteins that are produced in the body (Alberts et al. 2002). Proteins are present in the cell membrane to act as signals or sensors, to create ion gradients and act as pores. The red blood cell membrane, like all plasma membranes, contains a wide range of proteins. However, many of the proteins found in red blood cells are distinct because of their function in the body. Many of these proteins have been identified, and their role in the cell membrane discovered. The most widely found are spectrin, glycoporin and Band 3. These proteins make up 60% of the total amount of protein found in the membrane. Spectrin is a cytoskeletal protein that is long and fibrous, and seems to only be found in erythrocyte membranes (Kruckeberg et al. ...read more.


0.027 0.044 0.117 0.039 0.121 0.039 0.040 0.041 0.041 1.0 0.028 0.046 0.121 0.039 0.121 0.040 0.042 0.043 0.045 1.5 0.029 0.046 0.122 0.039 0.119 0.038 0.045 0.047 0.048 2.0 0.029 0.048 0.126 0.039 0.119 0.038 0.048 0.049 0.049 2.5 0.031 0.049 0.127 0.039 0.119 0.040 0.052 0.052 0.053 3.0 0.032 0.050 0.129 0.039 0.119 0.039 0.053 0.054 0.056 Time Centrifuged papain/acetate-treated Centrifuged PBS-treated 0 0.042 0.024 0.032 0.049 0.030 0.036 0.5 0.042 0.026 0.034 0.048 0.029 0.037 1.0 0.043 0.027 0.034 0.047 0.030 0.038 1.5 0.043 0.027 0.034 0.049 0.031 0.037 2.0 0.042 0.026 0.034 0.049 0.031 0.037 2.5 0.042 0.027 0.034 0.049 0.031 0.036 3.0 0.042 0.027 0.034 0.049 0.031 0.037 Table 1 - Acetlycholinesterase Assay- Raw data giving values of absorbance collected from the 5 different samples (each in triplicate) over 3 min. Time Neostigmine-treated Papain/acetate-treated PBS-treated Centrifuged papain/acetate-treated Centrifuged PBS-treated 0 0.059 0.063 0.036 0.033 0.038 0.5 0.063 0.066 0.041 0.034 0.038 1.0 0.065 0.067 0.043 0.035 0.038 1.5 0.066 0.065 0.046 0.035 0.039 2.0 0.068 0.066 0.049 0.034 0.039 2.5 0.069 0.066 0.052 0.034 0.039 3.0 0.070 0.066 0.055 0.034 0.039 Table 2 - Acetlycholinesterase Assay- Averages of the change in absorbance over time for each pre-treated sample. Figure 2: Comparing the acetylcholinesterase activity in pre-treated red blood cell ghosts. Fig 2: The absorbance of pre-treated red blood cell ghosts showing how the amount of the activity of acetylcholinesterase was affected by each specific pre-treatment. The activity of any enzyme is written as ?mol/l/min, but the results that were obtained were absorbance over 3min. The gradients were taken for each sample and calculated using the formula: Y2 - Y1 / X2 - X1 PBS sample calculation of acetylcholinesterase activity The gradient for the PBS sample was: 0.019/3 = 6.3 x 10-3 (absorbance per min) The Beer-Lambert Formula - A = ?.c.l This was used to calculate the concentration change over time: c = A/?.l c = 6.3 x 10-3/ 13,600x1 c = 4.63 x 10-7 mol/800?l/min This must be converted to mol/l/min: (4.63 x 10-7) ...read more.


As papain is a proteinase (which hydrolyses proteins), when the samples were extracted with PBS/Saponin and all the proteins released, they would be hydrolysed by the papain, and only very small proteins would be in the sample and therefore run through that lane in the gel. The PBS precipitate showed faint bands (small amounts of the proteins), as this had been treated with the PBS/Saponin extraction, which appears to destroy most of the proteins in the sample. All of the untreated samples showed protein bands. These samples, unlike all of the others, were not treated with PBS/Saponin. This reagent is a surfactant and breaks down all of the lipid bilayer of the membrane, and appears to also cause a disruption in the running of the proteins in the gel. On the samples that could be seen, only three distinct proteins could be identified that were 250,000, 230,000 and 100,000 molecular weight. These correspond to ?-spectrin, ?-spectrin and Band 3 (Alberts et al. 2002). The smaller protein, glycophorin (30,000 molecular weight), which makes up such a large proportion of the proteins in the membrane, was not shown on the gel as it had entered the solvent front. A conclusion as to where the proteins were on the red blood cell membrane could not be reached. As only two distinct proteins could be seen on any lanes in the gel, even though there had been different pre-treatments, no comparisons could be made. These two proteins are found in very different areas of the membrane, as spectrin can be found in the cytoskeleton, and Band 3 in the outer layer of plasma membrane (Alberts et al. 2002). A further investigation would have to be carried out to achieve results from which the location of the proteins within the plasma membrane could be determined. A higher percentage gel could be used in further work so that smaller proteins (like glycophorin) could be shown. A 2-dimensional gel could also be used to get a better resolution of the proteins, first using NEPHGE (non-equilibrium pH gradient electrophoretic technique, then SDS-PAGE electrophoresis (Kruckeberg et al. 1980). ...read more.

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