Question

You are a directed studies student (undergraduate student conducting research in a lab for course credit) and your supervisor asks you to use a cryopreserved cell culture stock to determine where an enzyme is located in the cell and to measure its activity.  

The cryopreserved yeast or E. coli cells have been genetically modified to express a synthetic enzyme. During the synthesis of this enzyme it is thought that the enzyme could be incorporated into different membranes within the cell.

The enzyme produces a by product that reacts with 2, 6 diphenolindophenol that can be measured when the enzyme concentration is at least 20 ug/mL.

Explain to your supervisor all the steps necessary, starting with thawing the cells, to determine where in the cell the enzyme might be located and how you would measure its activity.

Answer 1

Thawing protocol:
Remove the cryovial containing the frozen bacterial cells from liquid nitrogen storage and immediately placed in waterbath at 37^oC. Quickly thaw the cells by gently swirling the vial in 37^oC until there is no bit of ice on the cells. The thawed cells are diluted slowly, using pre-warmed growth medium. Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes. After the centrifugation, decant the supernatant without disturbing the cell pellet. The cells are resuspended in complete growth medium into the appropriate culture vessel and into the recommended culture environment.

The enzymes are found on the rigid cell wall of the yeast cells. The action of these enzymes are associated with the changes in the cell wall nature of the yeast. The nature of the compounds released during autolysis pointed out the activities of protease, mannase, lipases, glucanases and phospholipases. The freeze-pressing of the cells yield cell wall membranes preperations that are relatively intact and forms a good method in isolation of membrane-associated enzymes.

Enzyme assays are used to study the rates of enzyme catalysed reactions, and the enzyme activity can be measured by different assays such as,
Stopped assay: It is used to measure the activity of an enzyme that one must measure how much product is being formed over a given period of time or, in certain cases, how much substrates have been used up, which should be the same thing. The ways of enzyme assay can be done by Stopped assay- Measure reducing sugars (F + G) with the Somogyi–Nelson reagent (chemical)
Continuous assays: The alternative to a stopped assay is a continuous assay, which is more convenient. The action of the enzyme itself can be followed by changes in absorbance at 340 nm. This assay converts the glucose to glucose 6-phosphate, which is then oxidized using NAD+ gives glucose using heterokinase gives glucose 6-p reacts with glucose 1,6-biphosphate and gives to 6-P gluconate which is measured at 340 nm.