Activity of lactate dehydrogenase in mammalian tissues

please help me with qs 3,4,5 and 6

 

thankyouu

D. Naughton
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LS20

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0 Practical 1

Activity of Lactate Dehydrogenase in Mammalian Tissues

Lactate dehydrogenase (LDH) catalyses the reversible reduction of pyruvate to lactate using NADH as a coenzyme.

CH3.CO.COOH + NADH + H+

CH3.CHOH.COOH + NAD+

The activity of LDH can be measured by continuously following the decrease in absorbance at 3

4

0 nm using pyruvate as the substrate. If the pyruvate concentration is varied, in the presence of a saturating concentration of NADH, the kinetic parameters of the enzyme with respect to pyruvate can be determined.

MATERIALS

(a)

0.1

mol/l phosphate buffer pH

7

.4

(b)
2.7 mmol/l sodium pyruvate, freshly made in phosphate buffer, pH 7.4

(c)
3.5 mmol/l NADH, freshly made in phosphate buffer, pH 7.4

(d)
Cytosols (50 ml) from rat liver, heart and fast muscle* [These cytosols have been diluted 800 fold in the phosphate buffer]

(e) Ice, for storage of NADH solution and the cytosols

(f) Recording spectrophotometers (recording at 1 cm/min and a full scale = 1.0 OD), linked to a 37ºC water bath

* Tissue was weighed, then homogenised in Tris buffer containing 0.25 mol/l sucrose. A 20% (w/v) homogenate was prepared for liver and heart and a 10% (w/v) for muscle. Cytosols were prepared by centrifuging at 100,000 x g for 1 hour at 4ºC.

METHOD

Prepare SEVEN tubes as shown below, initially omitting the pyruvate solution.

0.1

0.3

0.1

0.3

0.1

0.3

0.1

0.3

0.1

0.3

0.1

0.3

Tube No.	Phosphate buffer (ml)	NADH solution (ml)	Cytosol‡ (ml)	Pyruvate solution (ml)
1	2. 6							0.1							0.3	0.0
2	2.5	0.1
3	2.4	0.2
4	2.3	0.3
5	2.1	0.5
6	1.9	0.7
7	1.6	1.0

‡ Working in pairs select two of the three types of cytosol provided. OBTAIN RESULTS FOR THE THIRD CYTOSOL FROM ANOTHER GROUP

Equilibrate the number of tubes you can monitor in a single ‘run’ on the spectrophotometer at 37ºC for 10 minutes.
This number will vary according to the spectrophotometer used.

Rapidly add the appropriate volume of pyruvate (kept at 37ºC), transfer to a cuvette in a thermostatically heated cell holder of the UV spectrophotometer and observe the change in absorbance at 340 nm with time.

References

Stryer Biochemistry (3rd Ed. ONLY) and Manchester (1994) Biochemical Education
22(2) [to be found on LS2050 StudySpace in the Practical 1 documents folder]

NAME:

I.D. No.

LS2050 Practical 1:

Activity of Lactate Dehydrogenase in Mammalian Tissues

This assessment consists of six questions. You must answer all questions to obtain maximum marks. There are a total of 60 marks to be awarded.

You should also attach: (i) A table containing the raw data for all three cytosols (acknowledging the group(s) that provided any data that you yourself did not collect), (ii) a table containing the calculated data used to create the Michaelis Menten plot and the linearised plot, (iii) the hand drawn Michaelis Meneten plot and (iv) the hand drawn linearised plot.

1. Write the equation for the reaction being studied, SHOWING STRUCTURES, and explain how the rate of the reaction is monitored in this experiment [do not just describe the protocol]. (5 marks)

2. FOR EACH OF THE THREE SETS OF DATA plot the rates of reaction in (A340/min against pyruvate concentration (mmol/l) ([S]) in the final assay volume. Explain your calculations by presenting a sample calculation for [S] below. (10 marks)

3. For each set of data convert the rate data from (A340/min to nmol NADH oxidised/min/mg tissue. The molar extinction coefficient of NADH at 340nm ((340) is 6300 litres/mol/cm, the reaction volume is 3 x 10-3 l, there are 109 nmoles/mole and the diluted cytosols contain 0.25 mg tissue/ml [liver & heart] or 0.125 mg tissue/ml [muscle]. Therefore 1 (A340/min equals 6349 nmol NADH oxidised/min/mg [heart & liver] or 12699 nmol NADH oxidised/min/mg [muscle]. Present an example calculation below, but tabulate each set of data. (10 marks)

4. Transform each set of the above data to obtain an
appropriate
linear plot [tabulate the manipulated data with correct units] and hence calculate the Km and Vmax values. Present the Km and Vmax for each cytosol below, including units. (15 marks)

5. Compare the Vmax and Km values in the three tissues and explain any similarities or differences in the three values. (10 marks)

6. Explain how you could characterise/determine the specific isozymes present in the three cytosolic preparations. (10 marks)
[for this answer you may extend onto the next page].