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Molecular Cell Biology

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Molecular Cell Biology

Postby Freckles » Wed Dec 07, 2011 11:24 pm

Can anyone help me with the answers to (b) and (c)?? I'm completely stuck :(

Problem 2 - Endocytosis of EGF and a fluid phase-marker (HRP) (70 marks)
Cells take up extracellular molecules by receptor-mediated endocytosis (RME) and by fluid
phase endocytosis (FPE). The efficiencies of these 2 pathways were compared experimentally by
incubation of the human A431 epithelial carcinoma cell line with epidermal growth factor (EGF)
to measure RME, and horseradish peroxidase (HRP) to measure FPE. A quantitative analysis
was carried out by incubating A431 cells with 40 nM ferritin-labelled EGF and 20 μM HRP
(which is at a 500-fold higher concentration than that for EGF) for various times. The cells were
then chemically fixed (cross-linked), stained for HRP-activity and the presence of the electrondense ferritin stain, and examined using the electron microscope. Both EGF and HRP were
present in small vesicles with an internal radius of 20 nm but EGF was very commonly detected
whereas the HRP was rarely detected.
The rates of EGF and HRP uptake were compared in Figure 1. HRP uptake was linear with
respect to both time and concentration: A431 cells took up HRP at a rate of 1 pmol/hr at 20 μM
HRP (Fig. 1A). EGF uptake showed an initial linear phase but reached a plateau at higher EGF
concentrations in the extracellular medium. Using appropriate formulas, answer the following:
(a) Explain why the shapes of the lines in the Fig. 1 are different for HRP and EGF. (10 marks)
(b) Calculate the number of EGF receptors on the surface of each A431 cell. (20 marks)
(c) Calculate how many HRP molecules get taken up into a single endocytic vesicle (radius, r =
20 nm) when the medium contains 1 mg/ml HRP (molecular weight of HRP = 40 000 daltons).
Note: The volume of a sphere = 4/3πr
3
(25 marks)
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Postby JackBean » Thu Dec 08, 2011 12:43 pm

b) for this you need the speed of uptake
c) if you know radius and formula for volume, you should be able to calculate volume, shouldn't you? If you know concentration and volume, you should be able to calculate the weight, shouldn't you? If you know the weight and molecule weight, you should be able to calculate the number of molecules, shouldn't you?
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Re: Molecular Cell Biology

Postby Freckles » Tue Dec 13, 2011 1:13 am

b) the speed of uptake is 4x106 pmol/hr

where do i go from here?

Thank you for your help :)
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Postby JackBean » Tue Dec 13, 2011 12:08 pm

if you knew speed of translocation by one receptor, then you could calculate the number of receptors
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Postby cbarlage » Tue Dec 20, 2011 5:53 pm

check out this cool animation on this topic!
http://www.youtube.com/watch?v=he_Qw4_v ... re=related
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Postby JackBean » Tue Dec 20, 2011 5:56 pm

really? How is it related?
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