Lab 8: Intracellular Localization of Alkaline Phosphatase in Yeast

Question 1

Differential Centrifugation

Answer 1

process where organelles and subcellular components can be separated based on MASS and DENSITY for further investigation

Question 2

in differential centrifugation, what property of the organelles allow them to be separated?

Answer 2

organelles are separated based on MASS and DENSITY. larger organelles can be removed first through lower centrifugal force

Question 3

Microsomes

Answer 3

membranous fraction of cells which includes plasma membrane, ER, and golgi.

Question 4

4 Methods of homogenization

Answer 4

1) homogenizer
2) mortar and pestle
3) chemical homogenization (ex/ lyse chloroplasts with NaCl to create leaky membranes)
4) grind with glass beads

Question 5

when you centrifuge a homogenate for the first time at a relatively low speed, what is contained in the pellet? in the supernatant?

Answer 5

nuclear supernatant contains other organelles besides the nucleus. Contains mitochondria and vacuoles and lysosomes etc.

nuclear pellet contains nuclei and whole cells that were not lysed by the homogenizer

Question 6

once you centrifuge the nuclear supernatant, what is in the supernatant? the pellet?

Answer 6

the new supernatant is the post-mitochondrial supernatant containing microsomes and organelles that are smaller than the mitochondria

the pellet is the mitochondrial pellet, which contains majority of the mitochondria, maybe SOME CHLOROPLASTS AND PEROXISOMES.

Question 7

If you centrifuge the post-mitochondrial supernatant, what is the pellet?

Answer 7

microsomes. contains small membranous material from ER and golgi, maybe some ribosomes.

Question 8

the ____ you spin, and the ____ the spin is, the smaller the particles you are able to obtain in the pellet during differential centrifugation

Answer 8

the FASTER you spin, and the LONGER the spin is, the smaller the particles you are able to obtain in the pellet during differential centrifugation

Question 9

What substrate did we add to the alkaline phosphatase in order to measure it’s activity colorimetrically?

Answer 9

we added pnitrophenyl phosphate (pnpp) which gets converted to p-nitrophenol (pnp) when alkaline phosphatase cleaves of the phosphate, resulting in a color change that absorbs light stronger than pnpp.

Question 10

conditions in order for alkaline phosphatase to react with pnpp

Answer 10

1) excess of pnpp

2) pH of 8-9 (basic)

Question 11

if you reacted alkaline phosphatase with pnpp, and a large increase in absorbance was seen, what can you infer?

Answer 11

the large increase in absorbance is pnpp being dephosphorylated to pnp, which absorbs strongly, indicating that there is a lot of PNP present. A lot of PNP present means the alkaline phosphatase is very active, or that there is most likely a lot of enzyme in the sample.

Question 12

After measuring pnp formation (alkaline phosphate activity) for three minutes via spectrophotometry, it was determined that the change in absorbance was 0.0588 Abs/min. If one enzyme unit was the amount of enzyme required to change absorbance by 0.01 in 1 minute, and 100 micro liters of alkaline phosphatase solution was added to the cuvette to start with, how many enzyme units are present in the entire sample of homogenate (30.2mL) that the 100 microliters of alkaline phosphatase came from?

Answer 12

1776.24 Enzyme units. check notes for calculations.

Question 13

100 microliters of nuclear supernatant was placed in a cuvette with pnpp and the change in absorbance was determined to be 0.053Abs/min. The totaly amount of nuclear supernatant isolated in the experiment was 25.5 mls. an Enzyme unit is the amount of enzyme required to change 0.01Abs in 1 minute. What is the total enzyme units in the entire sample of nuclear supernatant extracted? If the total enzyme units for the homogenate was 1776.24, what percentage of enzyme does this fraction contain?

Answer 13

1351.5 U, that’s 76.5% of the enzymes in the homogenate.

Question 14

the total enzyme units present in 30.2 ml of homogenate is 1776.24. We used 100 microliters in the cuvette to determine absorbance. How many enzyme units are in the cuvette? What would the absorbance per min be? Assume 1 enzyme unit is the amount of enzyme needed to change 0.01 abs in one minute.

Answer 14

5.8816 enzyme units in the cuvette, 0.0588 abs/min.

Question 15

from this experiment, how could you tell which fraction from differential centrifugation had the most alkaline phosphatase?

Answer 15

the one with the most total enzyme units (highest percentage of homogenate units)

Question 16

You realized that the nuclear supernatant (76%) had high activity, but when you centrifuged it, the post mitochondrial supernatant had an activity of 56% while the mitochondrial pellet had an activity of 3%. Where are possible organelles that the enzyme may be located in?

Answer 16

Since the nuclear supernatant has a high activity, it is not in the nuclear pellet. Therefore, the enzyme is not in the nucleus. The mitochondrial pellet also had low activity, indiciating that the enzyme is NOT in the mitochondria. The nuclear supernatant had high activity, but because the enzyme is not in the mitopellet, it is known to still be in the post-mitochondrial supernatant. Therefore, the enzyme COULD BE in which ever organelles are still remaining in the post-mitochondrial supernatant, such as chloroplasts, lysosomes, vesicles or microsomes.

Question 17

What organelles are in the post-mitochondrial SUPERNATANT?

Answer 17

lyosomes, vesicles, microsomes, chloroplasts

Question 18

How could you achieve more separation in differential centrifugation?

Answer 18

-undergo more centrifugation at intermediate steps to isolate the target organelles

Question 19

if you were to centrifuge the post-mitochondrial supernatant, what would be the pellet ? the new supernatant? What would you isolate if you centrifuged the new supernatant obtained from the post-mitochondrial supernatant?

Answer 19

pellet: microsomes, membranous portions of ER and Golgi
supernatant; small homogenated material that is soluble. ribosomes and cytosol could be isolated if you were to centrifuge the new supernatant.

Question 20

Why didn’t subsequent fractions’ enzyme activity add up? ex/ why didnt the post mito supernatant and mito pelle activity add up to the nuclear supernatant activity that the two components came from?

Answer 20

1) protein loss due to transfer
2) denaturation due to temp or pH change
3) loss of co factors.

Question 21

alkaline phosphatase is considered to be a _____ because it relies on magnesium or zinc cofactors to function

Answer 21

considered to be a metalloenzyme