Cellular Locations of Metabolic Enzymes

CONTENTS

Practical No. Page

1. Introduction to microscopy and measurement of cellular structures using an eye piece graticule/Haematoxylin and Eosin Staining of cheek

cells [to be peer-assessed during session itself] 2

2. Observation of chromosomes undergoing mitosis in onion root tips [to be peer-assessed during session itself] 7

3. Measurement of enzyme activity in different subcellular fractions obtained from liver cells (over 2 weeks).

9

Assessment portfolio (submit electronically via Turnitin – hand-in deadline: 29/1/18)

Write-up practical No. 3 in the form of a scientific paper i.e. Title, Abstract (300 words), Introduction (300 words) Methods (300 words), Results

(300 words [plus graphs where appropriate]), Discussion (300 words), Conclusion (bullet points) and References (Preferably Harvard style – you

should aim to have at least 5-6 references). (NB word counts shown in brackets are the maximum amounts you are allowed per section.)

1. INTRODUCTION TO MICROSCOPY
Bright-Field Light Microscopy (Bright Field)
Light rays from an illumination source are focused on the specimen to be examined by a condenser lens. The light rays leaving the specimen (mounted

onto a slide, and placed onto the stage) are focused into a magnified image by two lenses placed at either end of a tube. The lens near to the

specimen is called the objective lends and the one near the eye is called the ocular (or eyepiece) lens.

The extent to which a microscope can distinguish fine details in the specimen as separate, distinct image points is termed resolution.

In the light microscope:
Resolution (symbol: d) = 0.61 
n sin 
 is the wavelength of the light used to illuminate the microscope
n is the refractive index of the transmitting medium surrounding the specimen/filling the space between specimen and objective lens.
 is the half angle of the cone of light entering the objective lens from the specimen
0.61 is a constant describing the degree to which image points can overlap and still be recognised as separate points by the observer.

Since resolution is a measure of the ability of a microscope to image fine details, the quantity ‘d’ becomes smaller as resolution improves.

Therefore for best resolution ‘0.61 ’ should take on the smallest possible value, and ‘n sin ’ the largest possible value (eg. the value of n can

be pushed to its maximum by placing a drop of immersion oil [refractive index 1.5] in the space between the objective lens and the specimen).

The best resolution possible with a light microscope is 0.2m or 200nm because for visible light,  = 450nm. However, in a fluorescence microscope,

resolution is enhanced to 0.1m or 100nm, because ultraviolet light has a  of 250 nm.

A) MEASUREMENT AND CALCULATION OF DIMENSIONS USING AN EYE PIECE GRATICULE (approx. 1 hour). (NB. Work in pairs; always clean eye pieces and

lens with microscope lens tissue (never use any other type of tissue)).

Using a light microscope it is possible to accurately measure the sizes of objects using an eye piece graticule and a stage micrometer. The

graticule fits inside the eye piece of the lens, and has a fine scale etched upon it; the eye piece graticule’s scale has to be calibrated using a

stage micrometer slide (basically a microscope slide with a fine scale of 1mm, divided into 100 graticule units, imprinted on it).

1. Using the 10x objective, focus on the stage micrometer slide scale. You will find that approximately 100 eyepiece units (e.p.u.) is

equivalent to 100 graticule units (1mm). Therefore 1 e.p.u. is equal to 1/100 x 1mm = 0.01mm or 10m (microns).

2. Similarly, using the 40x objective, you can focus on the stage micrometer’s 1mm scale; you should find that approximately 100 e.p.u. is

equivalent to 25 graticule units (0.25mm). Therefore, 1 e.p.u. is equal to 1/100 x 0.25mm = 0.0025mm or 2.5m (microns).

3. You have already completed a ‘Microscopy’ competency test exercise using stage micrometers. However, if you wish to refresh your memories (&

if time allows), you can take the opportunity in the space below to determine whether the conversion factors given in steps 1 and 2 are correct for

your microscope.. (Show your calculations in both cases).

• Work out the conversion factor for the x10 objective on your microscope:

• Now work out the conversion factor for the x40 objective on your microscope:

• If an object has a diameter of 17 graticule units with the x40 objective, what is its actual diameter in microns?

• If an object is 28microns in length, how many eyepiece graticule units will its length be when viewed with the x10 objective?
B. HAEMATOXYLIN AND EOSIN STAINING OF CHEEK CELLS (approx. 2h 30min)

In this practical session, we will use the calculations/conversions performed last week using an eye piece graticule and a stage micrometer in order

to produce scale drawings of buccal epithelial cells obtained via a ‘cheek scrape’ protocol.

Consenting to Participate in this Practical
• For consent to be valid:
• The participant should understand what the activity involves, & what the risks are.
• The participant should give consent voluntarily, and should have the capacity to agree to the activity.
• Written consent serves as valid evidence that consent has been given
• You will cover ‘consent’ as a general topic in the ‘Analytical Research Methods’ module, but in this case, the introduction to this

practical, & the informed consent form currently being circulated, provide a means by which you can give informed consent to participate.
• (NB. You do not have to participate in this practical, and can withdraw at any time, if you so wish.)

‘Cheek Scrape’ Protocol (wear gloves at all times):

1. Collect a cheek scrape from your mouth using a wooden spatula; prepare a smear and allow to air dry, then fix in 95% ethanol for 1-2 mins.

2. Transfer the specimen to 50% alcohol for a few seconds and then wash in distilled water

3. Stain in Harris’s haematoxylin 10 mins.

1. Wash in water.

2. Destain the cytoplasm by very briefly washing the slide (1-2 secs) in acid alcohol (1% HCl + 70% ethanol).

3. Wash in water.

4. Counterstain the cytoplasm in 1% eosin for 3 mins

5. Wash in water for 1 min.

6. Mount specimen in DPX resin (in the fume cupboard)

Make scale-drawings of the samples you have prepared. (NB. If you have not succeeded in preparing samples, please note that an inventory of

microscope slides is available; you can sign out slides, and use them as the basis for your scale-drawings.)

Draw your observations at x100 and x400 magnification. Indicate on your diagram cell sizes, and of the length of 2-3 nuclei – this should be used in

order to calculate the mean size of a nucleus (NB. all measurements MUST be in microns (uM)).

Notes/Scale Drawings for Practical 1.

NB. Questions based on this session have been included in an online quiz, which will become available to students on the APS4003 Moodle site from

the end of the session on the Friday morning until midnight the following Wednesday. Students will need to submit their answers before Wednesday

night, & then will be able to view their individual online feedback via Moodle on Thursday. An approx. 20-30 min slot at the beginning of the next

practical session (ie. on the following Friday) will be used to field any queries, & to use the big screens in the teaching lab to take the entire

group through the quiz.

2) OBSERVATION OF CHROMOSOMES UNDERGOING MITOSIS IN ONION ROOT TIPS

To put into practice what was covered before the break, we will now use the calculations/conversions performed previously (see above) using an eye

piece graticule and a stage micrometer in order to produce scale drawings of chromosomes undergoing mitosis obtained via an ‘onion root tip’

protocol.

‘Onion Root Tip’ Protocol (wear gloves)

1. Cut off about 2 mm of root tip from onion.

2. Fix root tips in Carnoy’s fluid for 10 mins in a Bijou tube.

3. Remove root tip from Carnoy’s fluid and place in a Petri dish containing distilled water. Wash in water for 2 minutes.

4. Place a root tip in 1ml of 1M HCl in an Eppendorf tube and incubate at 60C for 5 mins

5. Pour contents of tube into a petri dish and carefully pick out the root tips with forceps. Place the root tips in an Eppendorf tube

containing aceto-orcein and leave in the dark for 10 mins.

6. Place the root tip on a slide in a drop of 45% acetic acid and cover with a cover slip.

7. Squash the softened, stained root tip by lightly tapping on the cover slip with a pencil. The root tip should spread out as a pink mass.

8. View using x 400 light microscopy. You should be able to see chromosomes in various stages of mitosis.

9. Make scale-drawings in pencil of the samples you have prepared, using x 400 magnification (make at least 2 drawings showing different stages

of mitosis; include indications of the dimensions of the cells undergoing mitosis, and calculate the mean length of the cells you have drawn). (NB.

all measurements MUST be in microns (uM)).

(NB. If you have not succeeded in preparing samples, please note that an inventory of microscope slides is available; you can sign out slides, and

use them as the basis for your scale-drawings.)

Notes/Scale Drawings for Practical 1.

NB. Questions based on this session have been included in an online quiz, which will become available to students on the APS4003 Moodle site from

the end of the session on the Friday morning until midnight the following Wednesday. Students will need to submit their answers before Wednesday

night, & then will be able to view their individual online feedback via Moodle on Thursday. An approx. 20-30 min slot at the beginning of the next

practical session (ie. on the following Friday) will be used to field any queries, & to use the big screens in the teaching lab to take the entire

group through the quiz.

3. Cellular Locations of Metabolic Enzymes

Introduction

Within the cell, enzymes/multienzyme systems have characteristic intracellular locations. For example, in eukaryotic cells glycolysis occurs in the

cytoplasm, the citric acid cycle occurs in the matrix of the mitochondrial membrane, and electron transport and oxidative phosphorylation occur on

the inner mitochondrial membrane. However, this metabolic compartmentalisation does not necessarily mean that these systems act in complete

independence, as transport mechanisms allow transport of metabolic intermediates between different cellular compartments.

The objective of this practical is to fractionate liver cells, and to determine the cellular locations of some important metabolic enzymes.

Practical 3, Session 1

Prac 1a. Preparation of Liver Homogenate and Cell Fractions

Theory – When liver is homogenised in isotonic media by mild procedures, the cell nuclei and mitochondria remain relatively intact. These

subcellular structures can then be centrifuged out of the homogenate, leaving behind in the supernatant all the soluble components of the liver –

including the cytoplasmic enzymes. (NB. However, mitochondria prepared in this way do not exhibit many of the properties demonstrable in more

carefully isolated preparations. The membranes are ‘leaky’, with the result that many small molecules can permeate the mitochondrial membranes).

Mitochondria may then be completely lysed by addition of the detergent Triton, which lyses the mitochondrial membranes and liberates all of the

proteins present in the matrix of the mitochondria.

Method

1. Homogenise 30g of fresh liver in isotonic KCl (9ml per gram of tissue).

2. Strain homogenate through several layers of surgical gauze.

3. Centrifuge homogenate at 3500 rpm for 10 mins remove and retain supernatant (A), wash pellet, resuspend in 10 ml isotonic saline and label

liver particulate (B).

4. Pipette 2 ml of liver particulate (mix tube before pipetting as particles will sediment) into a clean test tube and add 0.1 ml of 2% Triton

and mix. Label this fraction as lysed liver particulate (C).

5. You should have three fractions: A Liver Supernatant, B Liver Particulate and C Liver Lysed.

Store all fractions on ice.

Prac 1b. Determination of Lactate and Malate Dehydrogenase

Theory-
i) Malate dehydrogenase (MDH) catalyses the reaction:-

Malate + NAD+  NADH + H+

MDH activity can be assayed by measurement of the production of NADH. In this experiment, the NADH formed is used to reduce a dye which when reduced

is red-coloured; hence NADH formation (and thus MDH activity) can be determined using a spectrophotometer. In fact two electron acceptors are

actually employed – phenazine methosulphate (PMS) accepts electrons from NADH and transfers them to the electron acceptor iodophenyl nitrophebyl

tetrazolium chloride (INT), which turns red on accepting these electrons.

NADH PMS Formazan (red colour)

NAD+ PMSH2 INT

ii) Similarly, Lactate dehydrogenase (LDH) catalyses the reaction:-

Lactate + NAD+  pyruvate + NADH + H+

The NADH formed in this reaction (and thus LDH activity) can also be determined spectrophotometrically via monitoring of generation of a red colour,

using a PMS, INT cocktail.

Method

1. Label 9 test tubes:
(1: Lactate/Fraction A; 2: Lactate/Fraction B; 3: Lactate/Fraction C; 4: Malate/Fraction A; 5: Malate/Fraction B; 6: Malate/Fraction C; 7:

Water/Fraction A; 8: Water/Fraction B; 9: Water/Fraction C).

2. Pipette 2 ml of the INT/PMS cocktail into each test tube.

3. Add 0.1 ml of lactate (into tubes 1-3, to assay for LDH), 0.1 ml of malate (into tubes 4-6, to assay MDH), or 0.1ml of ddH2O (to act as

controls). Mix well in all cases.

Start the enzyme reaction by adding 250l* of Fraction A to tubes 1, 4 and 7; 25l* of Fraction B to tubes 2, 5 and 8; or 25l* of Fraction C to

tubes 3, 6 and 8. Mix well in all cases. [*Note: Depending on the enzyme activity in each of your fractions, you may need to alter the volume of

fraction used, and re-run your assay (eg. if the absorbance at the end of the assay is very high, the INT has probably been used up before the end

of the assay, and therefore the assay should be re-run using a smaller volume of fraction)].

4. After 5 mins, stop the reaction by adding 3 ml of n-propanol/1 M HCl to each of the tubes. Mix well in all cases.

5. Measure the absorbance of each tube at 540 nm for each incubation.

6. Subtract the absorbance seen in the respective ‘water-control’ sample from that seen in each fraction, in order to calculate the change in

absorbance/min/ml in each case.

If time allows, you should perform these enzyme assays in triplicate, and calculate mean values in each case.

Practical 3, Session 2

Estimation of Protein Concentration

Theory Polypeptide concentration may be estimated from the colour of a chelate formed at room temperature between copper in alkaline solution and

the nitrogen atoms of peptide bonds. This is termed the Biuret reaction. Bovine serum albumin (BSA) is used to standardize the colour reaction. Each

protein has a unique amino acid composition and a slightly varying colour yield will be given per unit mass of polypeptide. An assay of unknown

protein by this method gives results which are really expressed in terms of the equivalent concentration of BSA.

Method

1. You are provided with a protein solution of concentration 10 mg/ml. Prepare 5 standards with concentrations varying from 0 to 10 mg/ml.

2. Mix 2ml of each standard or Fraction (A. B or C) with 3 ml of Biuret reagent (i.e. you should have 8 tubes!)

3. Incubate for 30 min and read the absorbance at 600 nm. [*HINT:- all the test solutions must be within the absorbance range of your standard

curve. Depending on the protein concentration in each of your fractions, you may need to alter the volume of fraction used, and re-run your assay].

4. Calculate the concentration of protein in mg/ml of each fraction.

5. Combine the LDH or MDH activity data (obtained in Session 1) with the protein concentration data (obtained in Session 2) in order to obtain

specific activity data (i.e. enzyme activity per unit mass of protein).

Notes for Practical 3.

WE ACCEPT