Chapter

Material And

Method

2.1 Experimental Animal

Mice have been selected for the present

sudy as they have physiological systems and

responses similar to those of man. Their small

size, ease of handling, housing,maintainance,

early puberty, and relatively high position in

the evolutionary scale also entice us to select

them as a test animal to study. They have also

a remarkable genetic similarities to human.

Identification and systematic position of

mouse in the animal kigdom is given below.

Classification

Phylum Chordata

Sub-phylum Vertebrata

Class Mammalia

Sub-class — Theria

Infra- class Eutheria

Order Rodentia

Species Mus musculus

Mice were collected from Dr.

Panjabrao Deshmukh Medical College,

Amravati and maintained and cared in the labo­

ratory, in cages, made up of plastic galvanized

iron, with raw dust bed at bottom. Food and

water supplied ad libitum to the animals.

Only the healthy pairs (a male and fe­

male) were housed in the separate cages. The

temperature of the house was maintained in

the range of 20 to 25 c. The twelve hours

of darkness and twelve hours of lightning was

provided in the room for the optimal growth

and reproduction. The animals were fed on

commercially available pellete diet as given in

table 2.1.1

2.1.1 Composition of semisynthetic diet

for Mice.

S.No. Ingredient gm/kg.

1.

2.

3.

4.

5.

6.

7.

8.

Wheat Flour

Roasted Bengalgram Flour

Ground nut Flour

Skimmed milk powder

Casein (min. 80% protein)

Refined Ground nut oil

*Salt mixture with starch

**Vitamine Mixture

150.00

570.00

100.00

50.00

40.00

40.00

48.00

48.00

10000

: Material And Methods 43

^Composition of salt Mixture

S.No. Mineral gm/lOOkg

of diet

1. Potassium Phosphate

2. Calcium Carbonate

3. Sodium Choloride

4. Magnesium sulphate

5. Ferrous sulphate

6. Manganese sulphate

7. Potassium iodide

8. Zinc sulphate

9. Copper suphate

10. Cobah Chloride

1556.000

1525.600

557.200

229.200

108.000

16.040

3.160

2.192

1.908

0.092

3999.392

**Composition of Vitamin Mixture

S.No. Vitamin

1. DL-Tocoferol

2. Menaphthone

3. Thiamine (Bl)

4. Riboflavin(B2)

5. Pyridoxin (B6)

gm/lOOkg

of diet

6.000

0.150

0.400

0.500

0.600

6. Niacin 1.000

7. Pentothenic acid 1.200

8. Cyanocobalamine(B12) 0.00005

9. Folic acid 0.100

10. p.Amino benzoic acid 10.000

11. Biotin 0.040

12. Inositol 10.000

13. Choline chloride 100.000

14. Starch 70.009

200.00

{i.e. 200 gm of vitamine mixture suspended in

starch is used for every 100 kg. of the diet}.

Excess food and fecal material were

removed from cages once a day or twice.

Water was changed twice a day. Saw dust was

changed twice a week. Cages were cleaned

with disinfectant every time. Mice were trans­

ferred to fresh, cleaned, disinfected cages.

2.2 Morphological and Physiological

Information about Mice.

Mice are very delicate creatures. Length

of tail is about 7.5 cm. Weight of adult male is

about 35 to 40 gm and weight of adult female

is about 25-30 gm. Adult mice require 4 to 5

gm. of food per day and 6 ml water per day.

Daily protein, fat, sugar requirement of adult

• Material And Methods 44

mice is 16 to 24%, 4 to 6 % and 45-55%

respectively. Gestation period of mice is 19

to 21 days. (24-30 days in post-partum mat­

ing)-

6 to 12 pups are delivered at each de­

livery. Weight of one day pup is 1 gm. It is hair­

less, hair develops from 3rd day age. On the

10th day, pup is fully covered by hair. On 12th

day, eyes and ear opens. From 13th to 14th

day, pups start eating solid food.

From 21th day, pup is kept in separate

cage, when it attain the weight of 10 to 14

gm.

Pup attain puberty at 4 to 6 days age.

Mating occur when the mice attain the age of

45 to 60 days.

Adult female has 5 pairs of nipples,

3 are thoracic and 2 are abdominal. Duration

of estrous cycle is of 4 days.

Life span of each mice is about 1V to

2'/j years. Mice can live at the temperature

between 20 to 25°c. The optimum tempera­

ture for them is 22°c.They require humidity in

the range of 45 to 55 %.

2.2.1 Physiological Information :

Daily urine output of Mice is about

4-7ml/day. Rectal temperature is 36.5°c.

Heart rate is about 328-728/min. Rate

of respiration about 84 to 230/ min. Systolic

blood pressure is 113mm/Hg and diastolic is

81 mm/Hg and diastolic is 81 m m/Hg.

R.B.C.number is 6 x lO*- to 11 X lOVmm^

and total W.B.C. count is 5 x 1 O to 10x10V

mm^ Heamoglobin content is 4 to 8 gm.

Colour of urine is clear yellow and Ph is about

6.

Milk contain 75% water, 10 to 12% fat,

3% sugar and 9% protein.

Besides being genetically similar to

humans, mice are small and inexpensive to

maintain. Their short life span and rapid

reproductive rate make it possible to study

disease processes in many individuals

throughout their life cycle.

Aside from their physical traits, mice are

commonly viewed as pests and vehicles of

disease. Despite favoring Mickey Mouse,

most people do not regard real mice as

appealing as they would a dog. This typical

perception of mice decreases the likelihood

of sympathy for any pain and suffering of mice

are used in experimental research, testing and

education.

Mice are used to evaluate the

•Material And Methods 45

safety of new chemicals or products such as

household cleaners and pesticides that may be

potentially toxic to humans. Mice are also used

to assess the safety of drugs and vaccines

made for medical use.

Toxicity tests are performed to meas­

ure the effects of limited or repeatd, long-term

exposure of an animal to a particular sub­

stance. Other tests measure the extent to

which the substance damages cells and causes

cancer, mutations in DNA, and birth defects.

The LDjQ test, developed in 1927, made

it possible to derive a numerical index of tox­

icity reflecting the lethal dose of a test sub­

stance. The test substance was administered

to the animal by feeding, injecting, inhalation,

or application to the skin.

Mice are used in biomedical research

as models of human beings in order to under­

stand to human body, determine the effects of

diseases, and develop treatments for diseases.

The nude mouse is used to study cancer. Its

immunodeficient status allows human tumors

to be grafted onto the mouse without rejec­

tion. This procedure allows for the study of

specific human cancers and the testing of new

treatments.

Mice and other mammals are used in

biological, medical and veterinary education.

High school and college students commonly

perform dissections on cats, fetal pigs, or other

animals to learn about anatomy. Students in

medical and veterinary schools use animals to

learn and practice surgical and other medical

procedure. Far fewer mice and other animals

are used in teaching than in testing and re­

search.

Mice are very delicate creatures and,

like all other small pets, should be handled

carefully.Mice are lifted not by the tip of their

tails as this is very painful. Hold near the base

of the tail close to their bodies and are lifted

into the hand.

The life expectancy of the average

mouse in the wild is generally short and can

be calculated in weeks or months. Pet mice,

however, can live up to 3 or 5 years. They

have in-built homing instincts but are almost

certainly colour-blind, viewing their world only

in black and white.

Like most rodents, mice need to take in

little water. They produce almost all they re­

quire under normal circumstances by "burn­

ing" the carbohydrates in the food they eat and

• Material And Methods 46

using the water that is released in their bodies

as a by-product of the "burning".This meta-

boHc water is produced by larger animals, in­

cluding man. But to these larger animals, it is

a minor source of water as their relatively

massive bodies demand for more water than

such an internal spring can provide. Desert

species, such as the spiny mouse, can survive

happily without any external source of water.

As an extra water conservation measure, such

species produce only limited amounts of drop­

pings and a highly concentrated urine. Some

spiny mice can live purely on tiny quantities of

sea-water and even your ordinary house mouse

can get by almost indefinitely without liquids.

This does not mean that you should ever

leave your same mice without a source of

freash, clean water. They are domesticated

varieties which may not be as hardy as their

wild cousins.

S- (1,2-dicarboxylethyl)

2.3 Pesticide used

2.3.1 Malathion:

Commercial Name : Malathion.

Chemical Name :0,0-dimethyl.

Chemical Fonnula :C,„H,.0,PS^ fO 19 6 2

Structural Formula

CH30

CH30

O

-S-CH COC2H5

CH2COC2H5

O

Physical Properties : Clean, brown coloured

viscous liquid with

mercepton like odour.

Solubility : Sparingly soluble in

water and most of the

organic solvent. 5%

DP, 50% EC, 20%

WP.

Manufacturar : Cynamid India Limited

P.O , Atul, Valsad-

396020.(Gujrat State).

Biological Properties: Malathion is an

organophosphosphorous based insecticide

which has good activity against pest tike stem

borer, thrips, vassids, leaf roUel, leaf miner.

'•- Material And Methods 47

arniy worm, reet cotton bug. It has prooved

to be very useful for the control of pest of

cotton, sugar cane, tobacco, jawar, bhindi,

vegetables etc.

2.3.2 Dieldrin :

1. Commercial Name: Dieldrin (Contains 85%

HEOD).

2.ChemicalName: 1,2,3,4,10,10,-Hexa

chloro-6,7-epoxy-l ,4,4a,5,6,7,8,-8a-

octahydroendo-l,4-exo-5,8-dimethano

naphthalene (HEOD)

3. Chemical Formula : C,,H„C,^ O

4. Structural Formula :

5. Physical properties : Dieldrin is one of the

more stabal chlorinated

hydrocarbons.

6. Solubility: Soluble in water.

0.195 mg/L at temp.25°C shake

flask method Bigger etal 1974.

7 Manufacturer : Shell Oil company

8. Biological Properties.:

Dieldrin is an organochlorine base

insecticide. It is stable compound having low

vapor pressure. Which readily kills insect by

contact. In contrast to earlier insecticide

offered the possibility of proloned control,

from a single application, of insects on plant.

In the following years chemical research into

organochlorine compound led to the

development of a range of insecticides,

including aldrin, heptachlor, chlordane and

endrin. The direction of development was

towards increased insecticidal activity, which

was unfortunately often associated with higher

mammalian toxicity and increased persistence

in the environment. These insecticides all

produce their action by affecting the

functioning of the nervous system. The

pesticide molecules interfere with the ionic

permeability of nerve cell membrans and so

produce an unstable state in which spurious

nerve impulses induce uncontrolled activity in

the whole organism. The effect appears to

depend not upon a chemical interference with

any enzyme system but up on a spatial

disruption of the cell membrane, related to

the shape of the organochlorine molecule.

i Material And Methods 48

2.4 Experimental design

1. Experimental animals: Mice

2.Pesticide used:Malathion(organophosphate)

and Dieldrin (organochlorine)

3. Total period of experimentation : 30 days.

4. Organs and tissue tested: Plasma,Liver and

Brain,

5. Parameters estimated:

a ) Acetylcholinesterase,

b) Carboxylesterase

c) Polymorphic forms of

acetylcholinesterases

Pathogen free-6-week old mice were

purchased from Dr. Punjabrao Medical

College Amravati. Animals are individually

housed in polycarbonate cages with filler tops,

corn cob bedding and provided with water

and feed ad libitum.

Mice of either sex each weighing

between 30 to 40 gm. were divided into three

groups. Animals in each group were

maintained on specific diet.( Table-2.1.1) The

animal of group A were fed a stock diet used

as control. Animal from group B were given

orally malathion (80.6 mg/kg) body weight/

day. a suspension made in distilled water.

however, mice form group 'C ' were given

orally dieldrin. (0.25 LDj ,) body weight / day,

a suspension made in distilled water. The

composition of diet is presented in Table -1 .

Animals were given water and food ad

libitum..

2.5 Pesticide treatment

For pesticide treatment, the mice were

collected transported and acclimated as stated

earlier. The specimens were divided into 3

groups. Mature healthy mice were selected

for experimentation.

Group I : - Served as a control for sublethal

group exposed to only control diet and

sacrified at the end of the experimental period

of thirty days (20 mice)

Group II :- Mice were exposed the sub­

lethal concentration of malathion ic. 1/3 of

Lc^J 96 h.

(20 mice)

Group III:- Mice were exposed to sublethal

concentration of Dieldrin ie. 1/3 of Lc ^ / 96

h. (20 mice).

Group 11 and III were further divided into five

subgroups each subgroup of 4 mice as under

: GII 2 days, GII4 days. GII 8 days, Gil 15

• Material And Methods 49

days, GI 30 days . for GrouplI"'^ and for

Group IlF" division was as GUI 2 days, GUI

4 days, GUI 8 days G III 15 days, GUI 30

days.

After the start of experiment each

sub-group at respective treatment period was

sacrificed and was used for enzyme analysis.

Sublethal concentration for malathion and

dieldrin was evalulated as per formula.

2.6 Exploratory test

Before performing full scale experime

nsts acute toxicity tests were performed. The

test animals were exposed to widely spaced

concentrations of pesticides malathion and

dieldrin for 24 hours. Based on this data,

detailed toxicity tests were performed.

2.7 Lethal Toxicity test

Lethal toxicity tests were carried out for

four different concentrations of the pesticides.

Animals were divided into four sets.

a) in the first set of solution 50 % mice were

killed till 24 hours of exposure.

b) in the second set of solution 50 % mice

were killed till 48 hours of exposure.

c) in the third set of solution 50% mice were

killed till 72 hours of exposure.

d) in the fourth set of solution 50 % mice

were killed till 96 hours of exposure.

Twenty mice were exposed to each

concentration mortality percentage noted for

different hours of exposure.

2.8 Characterization of Acetylcholine -

sterase

2.8.1 Enzyme Extraction

2.8.1.1 For Plasma AChE

The groups having 4 mature mice in

each were kept in separate cages.

During the dissection of mice, thoracic

cage was opened ,the blood withdrawn

directly from heart with syring,transferred to

heparinised tubes(graduated) and centrifused

at 1 OOOg for 10 minutes.

The plasma was removed with a pipett.

The AChE was measured in plasma using the

method of Ellman (1961).

2.8.1.2 For Liver AChE

Pesticide treated mice were killed by

cervical disslocation, were dissected and liver

was taken out, washed with 0.9% NaCl,

• Material And Methods 50

weighed and homogenized in 0.9% NaCl and

was estimated for acetylcholinesterase by using

the method of Ellman (1961).

2.8.1.3 For Brain AChE

Brains were dissected out quickly and

washed with 0.9% NaCl. (0.15 M).,

containing 0.1% EDTA to remove blood from

brains. Blood clots and overlying membranes

were removed from the brains if any. Brains

of mouse of a group were pooled together and

homogenized in an electrical homogenizer to

prepare 1:10 % o.9 % NaCl homogenate.

Homogenates were immediately transfered to

separate tightly capped glass containers

(coming 20 ml culture tubes), kept in ice filled

thermos flasks and transported to the

laboratory within 30 to 40 minutes and was

estimated for acetylcholinesterase by using the

method of Ellman (1961).

2.8.2: Enzyme Assay for AChE

The enzyme were assayed by the

method of Ellaman et al., (1961).

Principle: The enzyme acetylcholine esterase

(AChE) catalyses the hydrolysis of

acetylcholine as under

Acetylcholine + water — > Thiocholine +

Acetic acid.

Thiocholine reacts with DTNB. ( 3:5

dithiobis (2- nitrobenzoic acid) to give a yellow

coloured derivative 5-thio-2-nitrobenzoic

acid,which is measured spectroph -

otometrically at 912 nm.

Reagents:

a) Potassium Phosphate buffer -0.1 M

(pH8)

b) Acetyl-choline iodide- (50nm)-14.5

mg in 1 ml of distilled water.

c)DTNB reagent. -(0.01 M)- 39.6 mg

of 15 ml of potassium phosphate buffer (pH

7) and 15 mg sodium bicarbonate.

Procedure: The reaction mixture prepared for

the assays of enzyme activity consisted of 2.33

ml phosphate buffer, 0.1 ml DTNB reagent

and 0.05 ml enzyme extract. The mixture was

allowed to equlibrate for 20 minutes and then

absorbance was read at 912 nm on a Elico

spectrophotometer. The substrate acetyl­

choline iodide (0.02 ml) was then added to

the reaction mixture to make total volume to

2.5 ml. The increase in absorbance was

recorded exactly after 5 minutes.

The specific activity was calculated as

A A/0.1 mg protein/minute. The protein content

•Material And Methods 51

of the enzyme extracts was estimated by the

method of Lowery et al., (1951) using bovin

serum albumin (fraction v) from Sigma

Chemical Co. U.S.A. as standard.

2.8.3 Standardization of optimum

parameters for ACIiE

Experiments were carried out to

estimate the optimum values of paramerters

such as:

Hydrogen ion concentration (pH)

Assay tempereature

Protein concentration.

Incubation period.

Substrate concentration.

For the activity of soluble and

membrane bound acetylcholinesterase from

the brain and from liver and blood of mice.

The reaction mixture consisted of the

components as described under the subtitle

enzyme assay except for the variable

parameters. Standardization of the above

parameters is necessary to conduct the toxicity

experiments and experiments performed for

the effect of the pesticides on the

acetylcholinesterase activity.

2.8.3.1 H- ion concentration for AChE :

The potassium phosphate buffer (0.1 M)

of pH 5 to 9 were used for varying the H-ion

concentration in the reaction mixture. In order

to compensate for the effect of pH on non

enzymatic hydrolysis of the substrate and

corresponding increase in colour with DTNB,

a corresponding set of enzymes blank was

maintained with respective set of tubes of each

pH.

2.8.3.2: Protein concentration :

To standardize the protein concentrtion

in enzyme extract, different concentrations of

protein from 0.01 to 0.19 mg were used in

reaction mixture.

2.8.3.4 :Assay temperature :

To evaluate the effect of assay

temperature on AChE activity, the reactions

were carried out at six different temperatures

viz. 10,20,28,30,40 and 50 , for both plasma

and liver and soluble and membrane bound

fractions of brain AChE of experimental mice.

2.8.3.5 Substrate Concentrations :

It is necessary that substrate

Material And Methods 52

concentrations in reaction mixture must be

optimum. Therefore effect of varying

concentrations viz. 0.2, 0.4,0.8,1.2,1.6,2,3

and 4mm of substate acetylthiocholine iodine

(AChEI) on the enzyme activity liver and

blood and soluble and membrane-bound

fractions of AchEfrom the brain of mice.

2.8.4 Response To Inhibitor

In order to further ascertain the

assigment of the activity of the enzyme

fractions of acetylcholinesterase (AChE-

3.1.1.7) and to show that the recorded activity

is not due to psuedocholinesterase (PChE-

3.1.1.8) an experiment was conducted where

0.1 mm ethopropazine hydrochloride (10-(2-

diethylaminoporpyl)-phenothiozine

hydrochloride), a known specific inhibiter of

PchE was used in the assay mixture.

The final confirmation of the presence

of AchE in enzyme extract was done by using

BW 248 (1,5 bis (4- allyldimethyl ammoniam

phenyl) pentane -3-one bromide) a specific

inhibitor of AChE, at a concentration of 0.1

mm in the reaction mixture.

2.8.5 Thermal stability

The thermal stability of the enzyme was

studied by preincubating small aliquots. (about

1 ml) of each extrat at 20°C ±\°C (room temp

a/c room) 28°C ±1°C (room temp) and 35°C

±\°C for varying length of time upto 15

minutes, The activity of incubated enzymes

was assayed immmediately at pH 7.5 (for

AChE), The specific activity computed in

terms of AA/ 0.1 mg. protein /minute.

2.9 Characterization of Carboxyl -

esterase.

2.9.1 Enzyme Extraction.

Enzyme extraction was carried out by

the method of ( Mendoza and et al, 1971).

2.9.1.1: For plasma CE

For the enzyme extraction of plasma in

mice, blood was withdrawn from posterior

inferior vena cava into a heparinized tube.

Heparinised blood, 1 ml was mixed with 5 ml

of 0.15 mm. NaCl solution and the cells were

sedimented at 15 000 g for 10 min precipitates

were discarded and supernants assayed

immediately for the enzyme or frozen for

further use.

•Material And Methods 53

2.9.1.2 For liver and brain CE:

Mice were dissected and liver, brain

tissue were taken out and weighted Tissue

were rinsed in an ice-cold 0.8 % Nacl soluton

immediately after rinsing. A 20% extract (w/

v) was prepared in ice-cold distilled water with

an all g lass grinder. One to two gm of tissues

were ground into fine particulates for 0.5 to

1.0 min and then homogenate for 2 min with

the grinder motor at miximum safe speed.

Homogenates were centrifused at 2000

g with a refrigerated centrifuge for 5 minutes.

Precipitates were discarded and supernants

assayed immediately or frozen for future use.

2.9.2 Enzyme Assay

2.9.2.1 For plasma CE:

The assay mixture contained enzyme

(0.1 ml plasma). 0.05 M Tris HCL. pH 8.2;

and 0.05 ml of 0.02 MIPA in absolute ethanol

in a total volumeof 3.0 ml. The hydrolysis

of IPA to indophenol was monitored at 522

mm in specrophotometer.

2.9.2.2 For liver and brain CE :

Thawed extracts were diluted with Tris

buffer and portions were centrifuged at

20,000 g. stocks were diluted 100 times for

liver and brain, stock extracts were diluted

further with buffer to give tissue

concentrations (mg of wet tissue/ ml of assay

medium).

To each 5 ml of enzyme solution, 50 ml

of 0.02 M I PA was added by means of a

Hamilton syringe. T he solutions were mixed

immediately, and were scanned at 522 mu

wavelength in spectrophotometer, (equipped

with on automatic cell changer and

programmer). The control solution contained

all regents but no enzyme. The cell holder

was kept at 37°C. Unless indicated enzyme

activities were calculated based on the amount

of indophenol formed per mg of protein in 15

mia

Protein concentrations in all assay

solutions were determined by the method

Lowery et al., (1951) . Crystallized and

lyophillized albumin from bovine serum was

used as a protein standard.

2.9.3 Standardization of optimum

parameters forCE.

Experiments were carried out to

estimate the optimum values of parameters

i Material And Methods 54

such as

Hydrogen ion concentration (pH)

Assay temperature

Protein concentration

Incubation period

Substrate concentration.

For the activity of carboxylesterase from

plasma, hver and brain of mice. The reaction

mixture consisted of the components as

discribed under the subtitle enzyme assay

except for the variable parameters.

Standardization of the above parameters is

done. Then experiments performed for the

effect of pesticides in the activity of

carboxylesterases.

2.9.3.1 Ion Concentration

Tris HCL (0.05m) of buffers of pH 5 to

9 were used for varying the H-ion

conentration in the reaction mixture for a

corresponding set of enzymes blank was

maintained with reepective set of tubes of each

pH.

2.9.3.2 Protein Concentration

To standardize the protein concentration

in enzyme extract, different concentrations of

protein from 0.01 t o 0.19mg were used in

reaction mixture.

2.9.3.3 Assay Temperature

To evaluate the effect of assay

temperature on carboxylesterase activity the

reactions were carried out at six different

temperatures viz. 10,20,28,30,40 and 50

C . For carboxylesterase act ivity in plasma,

liver and brain of mice.

2.9.3.4 Substrate Concentration

It is necessary that Substrate

concentration in rection mixture must be

optimum. Therefore effect of varying

concentration viz 0.2,0.4,0.8,01.2, 1.6,2.3,

and 4 mm of substrate indophenylacetate

(IPA) on the enzyme activity of

carboxylesterase in serum, liver and brain of

mice.

2.10 Thermal stability

The Thermal stability of the enzyme was

studied by preinabating small, aliquots. (about

1 ml) of each extract at 20''C ±PC (room

temp A/C room) 28° C t l^C (room

temp.)and 35°C±1''C (incubator) for varying

length of time upto 15 minutes. The activity of

incubated enzymem was assayed immediately

atpH 8.5. The specific activity computed in

•Material And Methods 55

terms of A A/0.1 mg protein / minute.

2.11 : Po lymorphic forms of

Acetylcholinesterase.

2.11.1: Enzyme Extraction .

2.11.1.1: For Plasma AChE

Blood was collected in a hepatic tube

and homogenized in 10mm. Tris-HCl,

containing 1.0 M NaCl and 50 mM

MgCl^ The ratio of buffer to blood was 3:1.

After stirring ,the homogenate was centriflised

at 10,0000av. for an hour. The resulting

supernant was designated" salt extract". The

pellet was resuspended in 3 vol. of 10 mM

Tris-HCl, pH 7.4 containing 144 mM NaCl

and 1 % Triton x-100. After stirring for another

2hrs, the homogenate ws centrifuged as above

the resulting supernant was designated a

"detergent extract."

2.11.1.2 For Liver and Brain AChE

All procedures were performed on ice,

with pre-cooled buffers.Salt soluble AChE

(SS. AChE) was extracted by homogenization

of liver and brain of mice in 10 mm. Tris-

HCL , pH 7.4, containing 1. M Nacl and 50

niM Mg clj. The ratio of buffer to tissue was

3:l(w/w ) After stirring for 2 hr., the

homogenate was centrifuged at 10,0000 av.

for one hour. The resulting supernatant was

designated " salt extract". The pellet was

resuspended in 3 vol. of 10 mM Tris- HCL,

pH 7.4 contaning 144 mM Nacl and 1 %

Triton x -100. After stirring for another 2

hrs., the homogenate was centrifuged as

above. The resulting supernantant was

designated" detergent extract".

2.11.2 Enzyme assay.

2.11.2.1 For salt extractable form

Homogenate of brain and plasma, liver

were applied to a 5-30 % linear sucrose

gradient made in extracting buffer and

centrifuged at 195, 000 g av for 16 hr. at 4

°C, using a 6 X 14 ml swingout roter (MSE)

Fractions were collected from t he bottom

and assayed for AChE activity by the Ellman

assay as stated earlier.

2.11.2.2 For detergent extractable

AChE form.

Homogenate of plasma,liver and brain

were applied to a 5.30 % linear sucrose

gradient made in extracting buffer which

contained 1% Triton x -100 . and centrifused

• Material And Methods 56

at 195, 000 g av. for 16 hrs. at 4 °C using 4 x

14 ml swing out rotor (MES) Fractions were

collected and assayed for AChE activity by

the Ellman assay (1961).

2.12 Enzyme kinetics

The data obtained from any experment

on enzyme kinetic are analysed by plotting

them in the form of various plots. The earliest

effort was that of Michaelis Menten (1913)

in which the initial velocity (v)is plotted against

the substrate concentration (s). Lineweaver-

Burk (1934) developed a linear transformaton

of the Michaelis-Menten equations by taking

receprocal of V and S and plotting 1 A' verses

1/S Later many modifications like Eadieplot

(1942) using V versus V/S and Hofstee (1952)

plot with S/V against S were proposed. Till

sixties it was conventional to use any one of

the above plots while analysing the data on

enzyme kinetics.

Robert Eisenthal and Anthel

Cornish-Bowden.(1974) introduced a new

graphic procedure for estimating enzyme

kinetic parameters called the direct linear plot

in which Michaelis Menten- Equation.

V.S V =

Km+S.

is obeyed.

Where V and Km are constants known

as the maximum velocity and the Michaelis

constant respectively. According to them, the

dependance of v and S shown in Michalies

Menten equation can be represented by an

equation

V max = V x v/s x Km.

There fore for each set of values of S

and V it is possible to plot v against Km as a

straight line with slope v/s intercept S on Km

. axis and intercept V max on v axis.

Observations are plotted as lines in

parameter space, instead of points in

observation space. With appropriate

modification this direct linear plot is

applicable to most problems of interest to the

enzyme kinetics. It has the following

advantages over t raditional methods plotting

kinetic results.

f) It is very simple to construct, because it

is composed entirely of straight lines and

requires no calculations or mathematical

tables,

ii) The kinetic constants are read off the

•Material And Methods 57

plot directly, again without calculations.

iii) It may be used during the course of

an experiment to judge the success of the

experiment to and to modify the expermental

design.

iv) It provides clear and accurate

information about the quality of the

observation, and identifies aberrant

observations.

v) It provides a clear indication of the

precision of the kinetic constant.

vi) Constructed with care, it provides

unbiased estimates of the kinetic constants, the

same as those as those provided by a

computer program.

vii) It may be used to stimulate result for

illustrative purposes very rapidly and simply.

In our experment, the kinetic data

obtained were analysed by all the two

methods, however the final calculations of

constants were made employing the Eisenthal

Cornish-Bowden plot.

i Material And Methods 58