Flashcards in Lab 6 Deck (10)
yeast centerfuge steps (whatspellet whats super
Sediment 1 nuclei & intact cells
Sediment 2 mitochondria
Sediment 2a after washing:mitochondria
Sediment 3: microsomes
Supernatant 3:soluble material of homogenate
what indicates enzyme activity is present
An increase in absorbance at 410 nm
The enzyme assay is based on the principle that
alkaline phosphatase will convert the
substrate in the assay mix, p-nitro phenyl phosphate [pnpp], to p-nitro phenol [pnp] by cleaving off the terminal phosphate group. This assay will continue to work as long as there is an excess of pnpp, and the pH is kept around pH 8-9. The linear increase in absorbance that you observe is due to the fact that pnp absorbs light much more strongly, at 410 nm than does pnpp; so, as the pnp accumulates in the cuvette, the absorbance increases.
One unit of enzyme activity is
the amount of enzyme which causes an increase in absorbance of 0.01 absorbance
units in 1 minute.
change in absorbance/change in time
change to ml, times b total volume then *100
Based on your results, make a conclusion regarding the location of alkaline phosphatase in yeast cells.
Based on the results of this experiment, it can be determined that the alkaline phosphatase is in the nuclear supernatant, but more specifically in the post mitochondrial supernatant of the nuclear supernatant, as this had the highest absorbance value. Alkaline phosphate converts pnpp (p-nitro phenyl phosphate) to pnp (p-nitro phenol) which absorbs light much more strongly at 410nm then pnpp. Therefore higher absorbance means more pnp, which means more alkaline phosphatase activity. The post mitochondrial supernatant is made of soluble material of homogenate after the other material has been deleted out. This is mostly soluble proteins. (can only say where its not: nucleus/mito. Could be cytoplasm or assorted with other organelle present in post mito supernatant.
Phosphatases are widespread in cells. Suppose you were trying to locate another of the phosphatase family – acid phosphatase – in cells. You already suspect that this enzyme is located primarily in lysosomes, but you would like to have direct evidence of this. Briefly describe how you might conduct an experiment to determine if your assumption is correct.
To find the the activity of acid phosphatase, you would follow steps very similar to this lab. First homogenize the cell by vortexing with glass beads for 6 minutes in 30 second intervals, keeping it on ice for 30seconds between each vortex. Then do differential centrifugation to separate out materials. First an 800 x g for 15min at 4degrees celsius. This will separate into a nuclear pellet and a nuclear supernatant. Resuspend the nuclear pellet which currently contains mitochondria, chloroplasts, lysosomes, and peroxisomes. The lysosomal fraction can be pelleted by centrifugation at 12,000 x g for 5 minutes. The lysosomes will now be in the pellet. Take 1ml of sample from the various fractions collected (the yeast homogenate, the nuclear supernatant, nuclear pellet, lysosome pellet and post lysosome supernatant) and assay for acid phosphatase activity with a spectrometer. If the lysosome pellet has the highest absorbance reading then you can confirm that the enzyme is located primary in lysosomes.
This experiment required you to isolate organelles in order to investigate them further. The specific technique that was used to do this is called what? (