If one has to look at the variations in Affinity (Km) and uptake rate (V or Vmax) of a receptor-ligand uptake phenomena, and compare it across different doses of the ligand, how will it be done?
I want to look at a ligand (neurotransmitter) and want to assess the uptake rate as well as the binding affinity over varying concentration of the ligand in cell line. So goal is to get the data on these across a WT receptor and mutant receptors. Whether varying ligand concentration over a constant time and temperature vary the affinity or rate of the reaction? The assay is radioactivity based so incorporation of radio labeled ligand in a specific ratio with unlabeled is added to the cells expressing either Wt or mutant receptors. So how to go about Km calculations? Will it fit to MM equations?
Hi Kalpita,
If you perform a radiolabelled substrate transport assay at a range of different substrate concentrations you can plot the initial uptake rate (V) as a function of the concentration of substrate that you added. Whether this data will fit to the Michaelis-Menten equation or not, depends on the mechanism of you neurotransmitter transporter. From the fit you can get values for Km and Vmax, those you can compare amongst the different mutants to look for trends. Keep in mind that a Km value is not necessarily the same as a Kd for binding. If you need Kd values specifically, you will need another assay where you eliminate substrate transport.
Hi,
Actually I have been doing the approach mentioned by you and have compared. Yes it doesnot really fit to MM equation in untransfected which is expected but it does fit to it for WT except one point in the graph. I am curious whethr the approach is right or the data has problem. I am interested in affinity so I hope looking at Km is fine. Should I be looking at Kd too? does looking at Kd complement the data in any case?
Page and I analysed an interesting case where MM kinetics held over part of the concentration range but at very low concentrations the rate of diffusion up to the transporter began to dominate and the kinetics became linear. West IC & Page MGP (1984) J. Theoret. Biol. 110, 11 - 19
If your data does not fit the MM equation, it could be that you're looking at a cooperative process. I cannot judge from here whether that is the case, or that your data is not accurate enough. Regarding the Kd/Km issue: in general it is a good thing to have as much data as possible, so measuring the Km might be worth spending some time on. But it depends on what you exactly want to explain with your data if you really need additional information.
Hi Guss.
What I want to explain is that, the expression the protein on the membrane responsible for the ligand binding and uptake is not the only factor for the variablity of the uptake but affinity plays a role in some mutants too.
Say in comparison to my WT, my mutants might not show a differential expression but their affinities to the ligand might vary or might not at a particular or varying conc. I need to assess do they vary in Km, Vmax or not and correlate the uptake data over different conc. and different constructs.
I would tentatively suggest that V m + Km is enough at this stage. What matters to an organism is rate of operation (at various concentrations); i.e. there is turnover happening. Kd seems to me much harder and I don't know how you would do it except possibly with a non-transported substrate and competition studies. If MM kinetics hold over a limited range of concentration (but fail to hold over a wider range), I suggest that you derive the parameters from the limited range where MM holds.
Your mutant may express a mixture of receptors, especially with different Km values. The combined effects of more than one receptor will probably turn out to be deviating from a fit according to MM equation for a single receptor
Hi,
Eggehard. That is a possibity. In my WT, I do sit a decnt fit but in mutants I don't see. What approach needs to be followed here then. Any suggestions?
Fit the experimental data for two overlapping MM kinetics with different Kms and Vmax. Adjust these parameters to have a best fit. This approach assumes that you have only two different kinds of receptors. I would chose the wild type receptor as one of them and use the corresponding Km and Vmax in the first approach. Then vary the parameters for the putative mutant receptor until best fit.
Good luck,
Eggehard Holer
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