Question

Explain how you could use subcellular distribution data to identify the subcellular location of another enzyme for which its location is not yet known.

To address this issue, discuss the extension of the experiment you are doing to determine the subcellular location of another enzyme for which you have an assay, but its specific compartmentalization in the liver tissue is not known. Include attention to both % activity and specific activity histograms in your argument. How we validate the subcellular fraction procedure is not required here- we will assume that an ideal separation of organelles has been achieved.

Answer 1

In the experiment you can separate the organelles by ultracentrifugating. You can use an antibody for the enzyme you are looking for and then reveal the mark by flow cytometry or even western blot for each of the cell fractions separately to see where the enzyme is located.

As you have the subcellular location of an enzyme and you are looking for a different one, you can predict the subcellular location if the two enzymes have some type of relationship, for example if they interact at some point of their activity, you can expect to have a similar distribution. Also if you know the function of the enzyme you can predict if it is in the membrane or in any of the organelles.

To see if the two enzymes interact, you can use the technique of co-inmunoprecipitation giving different stimulus to the cells that activates the known enzyme and see if the not known enzyme interacts with it when activated or not activated. The data of the known enzyme should tell you where it is located when activated and if it interacts with the unkown enzyme you can predict it can be located in the same organelles.

Also you should take into consideration that you should use the data where you have the highest percentage of activity because the probability to find the unknown enzyme there is higher.

Depending on the function of the enzyme, you can also predict that the location on one cell type can be the same (or not) on another cell type, In this case the liver.