Biochemical Pharmacology


where the molar extinction coefficient of TNB is 1.36 × 104 L/mol/cm. This is the absorbance of a 1 M solution in a 1 cm light path. The total volume in the cuvette is 3.17 mL, and there are (100 × 0.05) = 5 mg tissue in the cuvette. The factor of 106 is to convert mL to L.


The % inhibition is calculated as:


(8.2) numbered Display Equation


Questions



1. Express the drug-inhibited activities as a % of the control and construct semi-logarithmic plots of % control activity against –log [I] (Figure 8.1).

2. Measure the pI50 (−log IC50) for each drug tested.

3. Comment on (a) their relative potencies, (b) the mechanism of action of each of the drugs in this assay, (c) possible sources of interference with the method and (d) the suitability of these drugs for particular clinical applications.


Figure 8.1 Inhibition of AChE by tacrine, neostigmine and CCh. The rates of the enzyme reaction were measured from the slope.

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8.4 MONOAMINE OXIDASE INHIBITORS


MAO catalyses the deamination of a wide range of substrates including the neurotransmitters noradrenaline, 5-HT and dopamine. This mitochondrial enzyme has a wide tissue distribution, and occurs as two isoenzymes, MAOA and MAOB, that are different gene products. MAOA has a high affinity for the neurotransmitters noradrenaline and 5-HT, and is inhibited by clorgyline. MAOB is less specific and oxidizes dietary organic amines such as phenylethylamine and benzylamine. It is inhibited by deprenyl. Both enzymes deaminate tyramine and dopamine, and are inhibited by tranylcypromine. MAO can be assayed by several methods including oxygen uptake and radioisotopic measurements. In this practical, a fluorimetric method is used. The deamination of the substrate, kynuramine to the cyclic product 4-hydoxyquinoline (4-OHq), which fluoresces in alkaline solutions, is monitored (Morinan and Garratt, 1985).


Aim


This experiment is carried out in two parts:



1. To demonstrate the sub-cellular location of MAO. The relative specific activity (RSA) of MAO in sub-cellular fractions is compared with that of marker enzymes. It is demonstrated that MAO activity is highest in the mitochondrial fraction.

2. To demonstrate the specificity of MAO inhibitors for MAO isoenzymes. Three irreversible inhibitors (tranylcypromine, clorgyline and deprenyl (selegeline)), for MAOA and MAOB are tested in an enzyme preparation of whole rat brain.

8.4.1 Sub-cellular Distribution of MAO Activity


The aim of this experiment is to demonstrate that MAO (EC 1.4.3.4) is located in the mitochondrial fraction of a homogenate fractionated by differential and density gradient centrifugal fractionation. A 10% homogenate of rat brain in 0.32 M sucrose/1 mM EDTA, pH 7.2, is prepared using an Ultra-Turrax homogenizer (setting 6 for 20 s). This is centrifuged at 1000 g for 10 min at 4°C to give the nuclear pellet (P1). The supernatant (S1) is carefully removed and centrifuged at 10 000 g for 20 min to give the mitochondrial pellet (P2) and the supernatant (S2). Finally, the microsomal pellet (P3) and the soluble cytoplasmic components (S3) were prepared by centrifugation of S2 at 100 000 g for 60 min. P1 and P2, each resuspended in 15 mL of homogenizing medium together with P3 in 5 mL homogenizing medium and S3, are stored on ice until use in the MAO assays.


MAO Assay of Sub-cellular Fractions



1. Place 16 numbered 1.5 mL Eppendorff microcentrifuge tubes in a rack and pipette 870 μL of 10 mM potassium phosphate buffer, pH 7.2 into each of them. Dispense the following:

2. To tubes 1–4 add 100 μL P1

3. To tubes 5–8 add 100 μL P2

4. To tubes 9–12, add 100 μL P3

5. To tubes 13–16, 100 μL S3.

6. Place all the tubes in a rack and warm them to 37°C for 5 min. Then add 30 μL 3 mM kynuramine substrate (final concentration (92 μM)) to each tube.

7. After 15 min incubation, stop the reaction by adding 30 μL of 0.4 M perchloric acid. Cap and mix the tubes and then centrifuge them at 13 000 rpm. for 1 min in a benchtop microcentrifuge to sediment precipitated protein.

8. Carefully transfer 1 mL of the supernatant from each tube to plastic LP4 tubes and add 2 mL of 1 M NaOH. Mix and read the fluorescence of the product 4-hydroxyquinoline (4-OHq) at λex = 305 nm and λem = 380 nm.

4-hydroxyquinoline Standard (calibration) Curve


To calculate the amount of product formed by MAO in the sample tubes, a standard curve is prepared.



1. Dilute the stock 1 mM solution of 4-OHq by 100-fold (in a 25 mL plastic universal white capped tube) by adding 0.1 mL 1 mM 4-OHq to 10 mL 10 mM phosphate buffer. Cap and mix.

2. Pipette reagents (in triplicate) into Eppendorff microfuge tubes: as shown in Table 8.1.

3. Add 300 μL 0.4 M perchloric acid to each tube. Cap and mix.

4. Transfer 1 mL to test tubes containing 2 mL 1 M NaOH. Mix and read the fluorescence at the same wavelengths as used for the sample tubes.

5. Draw a calibration curve of nmol 4-OHq/tube against fluorescence (Figure 8.2).

Table 8.1 Preparation of standard concentrations of 4-OHq for the MAO assay.


Table08-1



Figure 8.2 Standard curve for 4-hydroxyquinoline

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Protein Assay


This is an adaption of the Lowry method which was first described by Markwell et al. (1978). Prepare a stock solution containing 100 μL/mL of bovine serum albumin, and prepare standard tubes containing 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg in 1 mL of distilled water. Make up the following dilutions of the sub-cellular fractions in distilled water: P1 and P2: 1/100 and 1/200; P3 and S3: 1/20 and 1/50. Add and mix 3 mL of Markwell C reagent (1% Reagent B in Reagent A) to duplicate 1 mL aliquots of each of these eight dilutions (a total of 16 tubes) and the standards. After 10 min at room temperature, add with immediate mixing 0.3 mL Reagent D (50% Folin-Ciocalteau Phenol reagent in distilled water). Leave the solutions at room temperature for a further 45 min before reading the absorbance at λ = 660 nm.


Calculations


Calculate the RSA of MAO in each sub-cellular fraction. A RSA value greater than 1 indicates an association of the marker with a particular fraction (see Table 8.2) and that the fraction is relatively pure from other organelles. The RSA is defined as:


Unnumbered Display Equation


Table 8.2 RSA data for enzymes and nucleic acid markers (Marchbanks, 1975). Fraction P1 is the nuclear fraction, P2, the mitochondrial fraction, P3, the microsomal fraction and S3 is the cytoplasmic fraction. The abbreviations used to denote enzymes are SDH, succinate dehydrogenase, NCcR, NADPH cytochrome c reductase (NADPH cytochrome P450 reductase) and LDH, lactate dehydrogenase.


Table08-1


8.4.2 Specificity of MAO Inhibitors for Isoenzymes


For this experiment, a crude homogenate of rat brain can be used. A 10% homogenate of rat brain in 10 mM phosphate buffer, pH 7.2, is prepared using an Ultra-Turrax homogenizer (setting 6 for 20 s). This is then centrifuged at 1000 g for 10 min to remove cell debris and nuclei, and the supernatant carefully removed and stored on ice until required. Acceptable results can be obtained if this homogenate stored at −80°C for several months.


Make seven 10-fold dilutions of the 1 mM stock concentration of each of the inhibitors. Label these as T4 (1 mM stock undiluted tranylcypromine, final incubation concentration 10−4 M), and T5–T11 for subsequent 10-fold dilutions (representing final incubation concentrations of 10−5–10−11 M) of tranylcypromine. Follow a similar procedure for clorgyline (C4–C11) and deprenyl (D4–D11). Note that a standard curve for the product of the reaction, 4-OHq, must also be prepared. This is done as detailed for the cell fractionation experiment (above). This should be done at this stage to prevent any delay at the end of the experiment. Standards should be read with the experimental samples.



1. Place 24 Eppendorff microcentrifuge (1.5 mL) tubes in a rack and label them as follows: three tubes for zero controls (Z1, Z2 and Z3) containing no inhibitor, and one each T4–T11 (for tranylcypromine dilutions), C4–C11 (for clorgyline dilutions) and D4–D11 (for deprenyl dilutions).

2. Add 100 μL of homogenate to each tube, followed by 870 μL of 10 mM phosphate buffer to the control tubes and 770 μL buffer to the tubes labelled T, C and D. Add 100 μL of each of dilutions of the inhibitors to the appropriate tubes.

3. Pre-heat for 5 min at 37°C.

4. Start reaction by adding 30 μL of 3.07 mM kynuramine (final concentration 92 μM). Incubate for 15 min at 37°C. Be accurate with the timing.

5. Stop the reaction by adding 300 μL of 0.4 M perchloric acid. Cap and mix and centrifuge at 13 000 rpm in a benchtop microcentrifuge for 1 min to remove precipitated protein.
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Jul 24, 2016 | Posted by in PHARMACY | Comments Off on Biochemical Pharmacology

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