73. Investigation of subcellular cell fractions

Question 1

What is the aim of Investigation of subcellular cell fractions?

Answer 1

• Eukaryotic cells contain several subcellular compartments (nucleus, mitochondria, ER, Golgi, lysosomes, peroxisomes and cytosol) and each of them contain highly specific macromolecules which can only be found in that particular compartment.
• Therefore, these macromolecules can be regarded as organelle-specific markers.
• The aim of this lab is to verify that these markers indeed are specific for the particular fraction (compartments).

Question 2

II. Markers in Investigation of subcellular cell fractions
1. What can markers be?

Answer 2

Markers can, for instance, be nuclear laminas and cytosolic actin filaments, enzymes and nucleic acids

Question 3

II. Markers in Investigation of subcellular cell fractions
2A. Which techniques can we use to identify these markers?

Answer 3

1. Immunostaining (immunofluorescence)
2. Immunoblot (Western Blot)
3. Biochemical assays

Question 4

II. Markers in Investigation of subcellular cell fractions
2B. What is the role of Immunostaining (immunofluorescence)?

Answer 4

Immunostaining (immunofluorescence): can detect markers in intact cells and tissues.
- Proteins detected by antibodies, DNA by DAPI (fluorescent dye)

Question 5

II. Markers in Investigation of subcellular cell fractions
2C. What is the role of Immunoblot (Western Blot)?

Answer 5

• Proteins will be isolated from subcellular extracts, resolved on SDS page (divides samples into their weights) and then transferred to a Western blot

Question 6

II. Markers in Investigation of subcellular cell fractions
2D. What is the role of Biochemical assays?

Answer 6

Enzymes from subcellular fractions are incubated in presence of specific substrates and product formation is monitored by spectrophotometer/fluorimetry (􏲋isolation of proteins)

Question 7

II. Markers in Investigation of subcellular cell fractions
3. How should we perform detection of markers?

Answer 7

• Detection of markers will be performed in isolated subcellular fractions, by a fractionation method called differential centrifugation.
• It will exploit the different densities of intracellular organelles􏲋cellular compartments will be separated from crude cell extracts by centrifuging them at increasing rpms (revolutions per minute).
• Sedimentation rate is specific for each compartment:

Question 8

II. Markers in Investigation of subcellular cell fractions
3. How should we perform detection of markers?

Answer 8

• Detection of markers will be performed in isolated subcellular fractions, by a fractionation method called differential centrifugation.
• It will exploit the different densities of intracellular organelles
  => cellular compartments will be separated from crude cell extracts by centrifuging them at increasing rpms (revolutions per minute).
• Sedimentation rate is specific for each compartment:

Question 9

III. Investigation of subcellular cell fractions
1. How do we investigate nucleus fraction?

Answer 9

• Genomic DNA is the nuclear marker
• Acid hydrolysis of DNA will give deoxyribose, which is quantified with diphenylamine that yields a colored condensation product (Schiff base = blue)

Question 10

III. Investigation of subcellular cell fractions
2. How do we investigate mitochondria fraction?

Answer 10

• The succinate dehydrogenase is an enzyme of the inner mitochondrial membrane
• The enzyme will from its substrate, succinate, remove 2 hydrogen atoms to give the product, fumarate
• These hydrogen atoms can reduce a FAD prosthetic group tightly bound to the enzyme
• To measure the enzyme activity, a reagent able to reoxidize the FADH2 will be added. During this reaction, the color of the reagent will change
• The reagent used is INT chloride, its formazan is red (low solubility)
• To make INT formazan soluble, it will be solubilized by the FTF mixture
  􏲄 Formic acid (decreases the pH)
  􏲄 Triton X (detergent)
  􏲄 Formaldehyde (reacts with AA residue)
  => By adding the FTF mixture, we will be able to see the red solution (INT formazan becomes soluble)

Question 11

III. Investigation of subcellular cell fractions
3A. How to we investigate “microsomes” fraction with ER marker?

Answer 11

• The microsomal fraction contains vesicles which are mostly cell extraction artefacts
  of large intracellular membrane systems such as the ER and Golgi membrane
• It may also contain peroxisomes and cell membrane fragments

ER marker:
- Marker is UDP glucuronyl transferase
- An artificial yellow-colored substrate (p-nitrophenol) is used together with uridine-diphospho-glucuronosyltransferase (UDP-GT) to catalyze the transfer of the glucuronic acid component of UDP-glucuronic acid to hydrophobic molecules
=> to produce more hydrophilic compounds that are easier to excrete from the body
=> The product of the reaction is bilirubin mono- or diglucuronide, which is water- soluble and non-toxic

Question 12

III. Investigation of subcellular cell fractions
3B. How to we investigate “microsomes” fraction with Cell membrane marker?

Answer 12

• Marker is 5’ nucleotidase
• Determination of 5’ nucleotides levels by Fiske-Subbarow colorimetric phosphate assay
• 5’nucleotidase will be assayed from sub cellular fractions in the presence of Mg2+ - ions using AMP in (pH 8,5) glycine-NaOH buffer

Question 13

III. Investigation of subcellular cell fractions
4. How to we investigate “cytosol” fraction?

Answer 13

• The marker is arginase
• In the mammalian liver, arginase is localized in the cytoplasm where it catalyzes the last enzymatic reaction of the urea cycle