Effects of group-selective reagents on monoamine oxidase and cholinesterase in guinea pig ileum

Pharmacological

Research

EFFECTS

Communications,

OF GROUP-SELECTIVE

REAGENTS

CHOLINESTERASE Paolo

Dal

Letizia

Pra, Department

IN

De of

327

Vol. 3. No. 4. 1971

ON MONOAMINE

GUINEA

Paolis,

PIG

Luigi

Pharmacology,

OXIDASE

AND

ILEUM

Rossini

and

LJniversity

of

Giorgio

Segre

Siena,

Italy Received

22 October

1971

SUMMARY

A series

activities

of

(AchthE)

in

ileum.

tyramine various

The

residues

of

reagents

of

aminoacid

residues

oxidase

(MAO)

and

acetylthiocholinesterase

tissue

fractions

MAO inhibition

were

was

tested,

whereas

occurredwhenaromatic-OH

residues

The

effect

of

isolated

pig

ileum

and

guinea

and

acetylcholine

(Arezzini

et

receptors

are

(Ach)

al.,

1969).

more

preferentially receptors

are

the

present

work

MAO and

AchthE

activities

tissue.(O)

(")

Part of (Rossini,

reagents

-NH2

of

group

the

ED

studied

conclusion affected

by

free

-NH2

of

AchthE

involved.

on

been

terminal for

blockade

a series hence

pig

the

reached

the

reagents

that

the

H

that

whereas

by

on the

for histamine 50 previously

was

residues,

inhibited

reagents

the

reagents

Ach of

the

ring.

the

same

The

preferentially

tyrosine-phenolic In

the

when

were

have

probably

block

guinea

preferential

INTRODUCTION

(H)

higher

from

inhibit

the

experimental 1970).

the

same

inhibitors

of

subcellular

procedure

were

studied

fractions

has

already

on from

been

the

presented

Pharmacological

328

MATERIALS

AND METHODS

phenol 1,

(DNP)

(Merk);

2,

acid

(Pierce

fluoborate

the

MAO assay,

Iodoacetic

acid,

same

5,

products

and

in

crystalline

Erba

(BA),

(DNFA),

4,

5-Trinitrobenzene (CDNP)

p-Nitrobenzene

(Sigma).

AchthE

assay.

and

(AI) DNP

(Eastman);

and

TNBS

interfere

N-Ethyl-maleimide,

Diketene

were

discarded

for

S-Acetylthiocholine

(Merck),

the

terminal

sia

with

measured

bovine

serum

was

a solution

xg

EDTA,

and

frozen.

The

according

to

and

of

Ellman

(0.1

scratched

(19.51)

with

g were

used;

and

Porteous at

by

the

inhibitors.

was

estimated (Ellman

et

2.5

Standard

"mitochondrial", were above

medium,

equivalent/ml)

the

underlying,

epithelial

borders

isolated

(1968).

pH 7.40

using the

for

blank

at

pH 8.0 al.,

Tris-HCl

(w/v).

xg

the

anaesthe-

mM,

l/10

in

measuring

corrections

ether

fractions

from the

estimated

EDTA

g tissue

and

(1961),

reagent

M,

800-8.000

washed

150

ethyl

homogenized

nuclear",

was

Haughton

activity

Other

a standard.

under

0.18

suspended

was

for

situ

and

(1967)

with

AchthE

and

used.

al.

about

twice

Eichholz

interference

in

layers,

phenylhydrazone,

weighing

was

mucosa

MAO activity Green

were

et

as

enriched

then

muscular

Lowry

"microsomal"

fraction

without

prevalently

and xg

but

pigs

removed

O°C,

8.000-105.000 Each

(Aldrich),

(Sigma)

(Sigma)

saccharose

"membraneous

prepared.

(DTNB)

I

to

albumin

washed

of

at

according

guinea

ileum

7.0

Type

iodide

RP reagents.

female

pH

) acid

were

Fasting

with

4-Dinitro-

I-Acetylimidazole

cholinesterase

were

36 mM,

2,

(DCCD) the

hydrochloride

Proteins

line

acid

Iodoacetamide,

erythrocyte

0-800

AI

2,

5-dinitropyridine

5'-Dithiobis-(2-nitrobenzoic

bovine

were:

reason.

Tyramine and

and

Vol. 3, No. 4, 1971

aniline

2-Chloro-3,

(NBDFB),

in

the

and Bromoacetic

N'-Dicyclohexylcarbodiimide

used

(DFDNB),

Co.);

N,

Communications,

4-Dinitro-5-fluoro

(TNBS),

Chem.

diazonium

of

compounds

5-Difluoro-2,4-dinitrobenzene

sulphonic

and

The

Research

the 2,

method

4-dinitro-

readings

by

1961)

and

oxidizing and

thiocho-

continuously

Pharmacological

Research

monitoring

the

reaction

solutions

of

The 0.08

Communications,

M phosphate

reagents

in the

the

at

final

preparations

for

15 min

30°C.

Assays

with

control

except

when

otherwise

enzyme

and

found the

to

Fig.

pig et

and

the

the

assays

DK-2A

at

previously

room and

the

ID

ileum

1969). were

Four

50 was to

repeated

prepared

were

added

were

the

same

inhibiting

four

to

incubating

at

performed, as

SO%), by

doses

the

the

previously

stimulated six

to

the

concentration

a concentration (cont.

in

pH;

before

reagents at

spectrophotometer

indicated

temperature

at

the

recording freshly

concentration)

terminal al.,

the

inhibitor to

329

were

indicated,

correspond

guinea

(Arezzini assay

with

a Beckman inhibitors

buffers

(twice

Vol. 3, No. 4. 7971

were six

H and used

when Ach

for

each

assay

times.

1 - An example of the Eadie-Woolf plot for the oxidation of tyramine by the 0-800xg "nuclear" fraction of the guinea pig ileum (2.0 mg protein equivalent per ml assay mixture). Control, and (hatched line) inhibition kinetics in the presence of 0.40 mM DFDNB. The points are the measured values and the lines represent the v = pmoles tyramine oxidized per min per 1; calculated equations. s = mmoles tyramine per 1 assay mixture. From the straight lines, and their the values of intercepts, i.e. the K and the K values standard errors (in brackets) are dEtermined aK equal to 0.208 and to 1.426 (0.432); from the values of the slopes one (0.026), V obtains l/V and l/V , and then = 0.439(0.046),and V = 0.457 of &gxlines, (0.038). Gi%% the par !i llelism by applying th& formula K. = i / [(Ko/K,)-111, Ki is estimof the competitive inhibition t i ated to be equal to 0.088 mM.

Pharmacological

330

ANALYSIS

OF DATA

by

the

using

Dowd

and

Riggs,

plot

196.5).

The

procedure

Olivetti

kinetic

kinetics

Eadie-Woolf

least-squares an

The

101

pseudoconstants

obtained.

The

slopes,

intercepts,

of (s/v

data

and

4 test

was and

parallelism

-4.

-1

/

/Ol

versus

s)

fitted

of

1

1942);

a weighted (1961),

Final

using

values

errors

toassessthe

evaluated

(Eadie, by

standard

Vol. 3. No. 4, 1971

was

Wilkinson

computer.

their

used

enzymes

to

desk

Communications,

the

were

according

Programma

Research

for

(s.e.)

significance control

and

2

the

5.10

were of

group-reagent

M

of the Eadie-Woolf plot for the hydrolysis of 2 - An example Achth by the BOO-8,Oooxg "mitochondrial" fraction of the guinea pig ileum (2.0 mg protein equivalent per ml assay mixture). Control, and (hatched line) inhibition kinetics in the presence of 0.030 mM DFDNB. The points are the measured values, and the lines represent the calculated equations. v = pmoles Achth hydrolyzed per min per 1 assay mixture. s = Pmoles Achth per 1 assay mixture. From the straight lines, the values of intercepts, i.e. the K and the K values and their standard errors (in brackets) are m determine8 as equal to 0.366(0.088), and to 0.209(0.065); from the values of the slopes one obtains I/V and I/V , and then = 1.396(0.130), and V = 0.451(0.043T?xSince th& intercept La%! he same for both lines! by applying the formula of the noncompetitive inhibition = i / [(Vmax/Vp)-l]], Ki is estimated IKi to be equal to 0.014 mM. Fig.

Pharmacological

Research

assays.

When

identical, It

2).

values

of

(1968);

mixed by

sites

e,

as

plotting

the

increase

into

and

and

calculated

(Fig.1). identical

of

the

when

apparent

calculating

competitive

was

by

intercepts

were

account

kinetics,

studied

using

Inturrisi

and

according

to

varying

both

the Titus

the

s and

of

results The

equation

the

of

correlated

intercept

half

of

per

and

Stedman

have

been

the

enzyme from

ml

the

at

the

only

e concentration,

of

the the

of

of

e,

found

ID

50 a straight equal

s against line to

used

one

by

- o(),

i

In

concentration

to

which

Easson and

this

constant

the

(Myers,

were

applied.

applying

each

fractions

(1

Michaelis

v = pmoles

enzyme

the

Straus

activities

for

the

gave

where

also

one by

derivation

molecules

A plot enzyme

'i = 1-M defined, was

results

of

i,

K., and a slope 1 ion. The equation

and

the

experiments

the

"true"

of

log

the

activity

obtained

i;

of

binding

and

were

inhibitor

amounts

of

the

(1936)

- o()

against

mixtures,

concentration

same

1)

(Ae),

tissue

ID5Gs (1

control

concentra

previously

the

of

per

the

to

(1936):*

the

of

concentration

assay

enzyme

following ratios

Stedman

to

equal

the

1913)

and

concentration

with

the

Easson

min

velocities

relative

by

presence

a single

tested

absolute

values

+ y

of

the

the

at

linearly

concentration

inhibition

per

= Ki

and/or

The

in

1 consumed

the

IDSO

i,

fractional

/v;v.=v

(1944):

A,

defined

the i

1952).

the

and

Goldstein

(1944).

substrate

At

slopes

(1967),

inhibitor

Goldstein

mined

Powell

calculate

the

enzyme

the

the

(1944),

was

of

to

for

were

when max taken

al.

the

non-competitive

inhibition

et

equation

(withoC=v

be

considered

non-competitive

inhibition and

applied

set

V

but

concentrations. The

by

the

Ebersole

Hein

inhibitor

and

of

Uncompetitive

equation

of

was

Km were

for

respectively.

used

different,

competitive

constant

Ki

were

decrease

331

Vol. 3. No. 4. 1971

inhibition

The

Michaelis

was

slopes

considered

(Fig.

the

the the

was

Communications,

were of

the

Hill

plot

of

Loftfield

and

Eigner

the

inhibitor

over

the

K

i instance deterenzyme.

(1910, (1969), enzyme

332

Pharmacological

sites

were The

H as

Research

Communications,

Vol. 3, No. 4. 1971

calculated.

values

of

agonist)

the

over

K.1 MAO (MAO; , acetylthiocholine had

I,

between

as

been

Ki

(guinea

Ach

tyramine

and

inhibitor

K.

ratios

as

pig

H (guinea ileum;

substrate)

substrate)

over

for

indicated

as

Ach

zr

ileum;

as

agonist),

Ki

AchthE , antagonist

a given

z and

Pig

(AchthE; and/or

respectively.

RESULTS Control

The

conditions

tial

fractions

using

are

fractions

stable

it

0-800,

was

800-8.000,

and

but

associated

appreciable in

1952;

De Duve

(O-800

of xg)

a mixed amine and

high

et

al.,

to

study

been

the

the

not as

Popov

the

reactivity

through

nuclear"

fraction

llmicrosomalt'

al., of

a crude

enriched

in

the

inhibition

on

the

"mitochondriall'

plasma

to

fraction studies.

For and

1963).

on

and

forms

of

multiple

our

main

study

comparison, a soluble

mono-

the

morphology pattern

al.,

1967;

interest

the

even

and AchthE

some AchthE

Collins

being

"membraneous

MAO activity study

the

membranes, The

measured

substrate et

The

nuclear

we actually

cellular

to

different

Strittmatter,

that

purification.

used

in

(Hawkins,

(Goedde

1970),

MAO

membrane,

characterize

the

fractions

et

was

to

as

of

fractions

multiple

well

were

portion

membraneous

fact

attempt

various

1970;

the

several

did

isoenzymatic of

to

By

activities

reported

the

-15°C.

were

mitochondrial

and

differen.

g tissue

fractions

major

Oswald

points

(0.1

AchthE

have

at

the

MAO activities

xg

outer

in

storage

relative

relative

MAO activity

of

obtained

included.

the

the

1960;

of

volume

particulate

al.,

oxidases.We

behaviour

same

the

with

fraction

activity

the

the

non-mitochondrial et

of

8.000-105.000

activities

tissues

presence

the

Intracellularly, is

activities

months

that

and

0.09:0.07:1.00. activity

in found

roughly1.00:0.$:0.19,

AchthE

after

suspended

equivalent/ml) of

MAO and

and the

activity experiments

activity

were

if

Pharmacological

TABLE

Research

l-

measured

0-800

AchthE

1 shows fractions

protein

reagent tested

in

cont.

inhibited

the

them not

mM);

the

and

an uncompetitive 2 and

inhibition

inhibitor the

for K

i

values

(0.093)

0.201 0.142 0.459

(0.020) (0.092) (0.047)

(AchthE) 15 ng at of

show

of

the

same

the practically

not the

reagents

inhibited

both

AchthE

competitive,

three obtained.

inhibi-

incompathe

CDNP at

enzymatic

strongly saturating

enzymes. whereas

which and

the

of

1 mM;

on

DFDNR

all

chemical

no effect

all

0.683

total

at

AchthE

at

competitively, DNP,TNBS,

and

mentioned,

the

were

mg

(s.e.)

max

fractions.

with

had

0.68

layers

manifested

reagents

by

3 show

muscular

already

but

these

measured

the

of

activity

inhibited

When

because

MAO inhibitors AchthE

ml.

tissue

any

of

mixture,

used,

show

other

and

MAO and

total

were

MAO activity, (0.24

All

of

the

As

vivo

DCCD did

inhibited

reaction per

effects

some

assays.

of

)

(0.158) (0.055) (0.089)

values

2 mg 720)

mucosa as

V

at

both

kinetics

tibility

the

cholinesterase

borders,

same

tors

Tables

ml

concentration,

Group

titively

per

erythrocyte

epithelial

and

measurzd

equivalent

protein

K

V

(74.

0.869 0.293 0.290

the

and V (pmoles of &ZXMAO and of 0-800,800pig ileum. as the means and Methods 3.

(s.e.)

m

163

bovine ery-throcytes 800-8.000 xg 8.000-105.000 xg

tissue

the

K

xg

Table

the

333

Vol. 3, No. 4. 1971

Means and standard errors (s.e.) of K (FM) per 1 per min) values for the controlmassays and AchthE activities of bovine erythrocytes 8.000,&000-105.000 xg fractions of the guinea In each case the parameters were determined as indicated in the Material of 40 estimates, section, and in the Legends of Tables 2 and

Activity

MAO

Communications.

was

NBDFB.BA AchthE Brush

only

DNFA

non-compebehaved

preparations. borders,

as

Pharmacological

334

TABLE

2-

Research

Communications.

Means and standard errors (s.e.) for K s (mM) (obtained by 4 to 6 determinations) of the inhibitoss of the MAO-activity of the O-800 xg fraction of the guinea pig ileum. Enzyme suspensions were incubated 30 min at 30°C in 0.08 M phosphate buffer, together with tyramine 0.1-10 mM, semicarbazide 0.0125 M and the inhibitor.

Inhibitors

K.

DNFA CDNP BA DFDNB NBDFB AI

TABLE

3-

Inhibitors

DNP DNFA TNBS BA DFDNB NBDFB

Vol. 3, No. 4. 1971

(s.e.)

1

0.129 0.013 8.397 0.074 0.053 2.997

(0.014) (0.079) (0.290) (0.026) (0.017) (0.097)

Means and standard errors (s.e.) for K.s (mM) (obtained by 4 to 6 determinations) of the inhibitors of the AchthE-activity of the bovine erythrocytes and of the 800-8.000 and 8.000-105.000 xg fractionsofguinea pig Enzyme suspensions were incubated at a minimum ileum. of 5 min at 30°C in 0.08 phosphate buffer, together with acetylthiocholine l-100.10-6 M, dithiobisnitroM and the inhibitor. benzoate 0.33x10-3

Ki Bovine

0.930 0.024 21.490 13.053 0.026 0.022

Eryth.

(0.078) (0.013) (2.030) (1.223) (0.012) (0.011)

(s.e.)

800-8.000 1.667 0.088 34.789 14.123 0.062 0.086

xg (0.876) (0.005) (2.150) (1.810) (0.022) (0.010)

8.000-105.000 0.925 0.081 8.196 13.269 0.040 0.031

(0.758) (0.007) (1.675) (0.980) (0.015) (0.009)

xg

Pharmacological

Research

mucosa, tion

and and

muscular

the

Having values

of

Figs. for

sensitivity

proved

the

high

affinity

MAO and

AchthE

equal

to

(AchthE) the to 0.91

MAO and

the

and

(s.e.

= 0.002)

(MAO)

Pmoles

per

molarity to

the

mixture.

obtained

when

The of number

slope the of

latter

at

different

from

for inhibitor

enzyme

were

inhibi-

was

tested.

reagent,

and

10 to

results the

that 95%.

of

DFDNB

The

absolute

0.34

to (s.e.

mola-

be = 0.22)

8 show

are

replotted

MAO inhibition

centres;

inhibition

7 and

binding

sites for

the

enzyme

found

Figs. the

of

concentrations

activities.

0.0039

1 assay

the

behaviour

sites

the

correspond

the

(microsomal)

equation.

DFDNB

inhibitor

binding

Hill

when

activities

6 illustrate

lines

pattern

both

inhibitor

straight the

AchthE

same

of

s were estimated 50 and at a range of

5 and

of

the

ID

4,

rities

showed

of

the 3,

layers order

the

335

Vol. 3. No. 4, 1971

same

concentrations inhibited

Communications,

AchthE

according equals

should the

number

l-d

Fig.

3 - Effects of DFDNB on 0-800xg "nuclear" MAO activity of guinea pig ileum. Plots of (1 - d)vs log i were drawn at one concentration of substrate (tyramine, -25 mM)and at one concentration of enzyme. Velocities studied at the amounts of l'nuclearlt protein per ml assay mixture indicated on each curve. Each point represents the mean of four estimates.

Pharmacological

336

Research

Communications,

Vol. 3, No. 4. 1971

IOWXIOA 500 .-

I

/ -

-1

I 1

0

p molar

1 2 tyrrmine

I 3 ox prr min per I

-6 for DFDNB (x10 M) inhibition of the values of ID 4 - Plot 50 vs "nuclear" MAO activity (pmoles tyramine oxidized per were calculated by min per l
1-d 1.0 -

as

-

,.,/-!y,

( 0.6

1.5

2.5

log1

of DFDNB on 8.000-iO5.OOOxg "microsomal" AchthE Fig. 5 - Effects activity of guinea pig ileum. Plots of fractional inhibition (1 -4)vs log i. Each S shaped curve is drawn on the experimental data (zans of four estimates) at one concentration of substrate (Achth 2.27 PM) and at the amount of enzyme indicated as mg protein equivalent per ml assay mixture. The hatched line represents the results obtained at the enzyme concentration adopted to obtain the data shown on Table 2.

Pharmacological

Research

Communications,

Vol. 3, No.

4. 1971

337

-6

Plot of the values of ID for DFDNB (x10 M) inhibition vs "microsomal" AchthE ac%vity (Pmoles Achth hydrolyzed per min per like). Values of ID calculated by linearizing the curves of Fig. 5. From the straig . 5R t line, one obtains the values of the intercept, i.e. the "true" K. value (see. Analysis of data) as equal to 14.80 (s.e. = 3.25) Pmoies, and of the slope as equal to 1.49 = 0.50/A; the absolute enzyme molarity A is then calculated to be equal to 0.34 (s.e. 0.22) micromoles per liter.

6 -

Fig.

of

inhibitor

1.67.

The

site and

molecules

is

constancy

of

involved

Eigner,

in

1969).

trationwere equal

that the

the K

i

46.6

(AchthE).

slope

values

suggests

Figs.

=22.22)

each

centre

that

only

range

extrapolated

from

(s.e.

with

concentration

calculated to

combines

(MAO),

single

(Loftfield

zero

6 and

14.8

and

one

studied at

4 and

equals

enzyme

found

to

(s.e.

concenbe

= 3.25)

lO-(jM.

Values results

calculated

similar

by

(within

the

10%)

Easson to

and

those

Stedman

obtained

method by

the

gave Myers

method.

DISCUSSION

Labelling

aminoacid

residues

buried, chains some cannot

and of

can

agents that

are

sometimes

proteins.

be

analyzed

selective

hyperreactive,

They

can

by

be

used

affinity whether

discriminate

chemicalfunctionsof yet

with

to

the

pharmacological

any

other

can exposed

between

different

identify

and

approach.

bind

receptors

or side

to

locate that

338

Pharmacological

0 Fig.

0.5

7 - Hill plot of DFDNB activity of guinea ml assay mixture; v and

per

Research

1.0

Communications,

Vof. 3. No.

4. 1971

1+bg1

(x10e3M) for 0-800xg "r~uclear~~ MAO pig ileum. Enzyme : 2.0 mg protein vi as indicated in the Legend of Fig.

z+te(+1)

2.5 -

21) -

1.5 -

1.0 -

0.5

-

I 0

Fig.

8

-1 0.5

protein

Hill plot of DFDNB AchthE activity of per ml assay mixture;

of

5.

-

Fig. The

but

this

cases referred

inhibitions

of

conclusion

must

previously to

accepted an

allosteric

the

I

I

1.0

1.5

-I 2.0

(x10 -3 M) for 8.000-105.OOOxg "microsomal" guinea pig ileum. Enzyme : 1.36 mg v and v as indicated in the Legend i MAO activity

be as

2*bgt

taken

with

purely

change

were

reservations,

competitive due

only

to

the

competitive, since

have flexibility

many

been of

the

3.

Pharmacological

Research

enzyme

and

AchthE

is

eserine

to

possible

preincubated in

from

Communications,

the

absence

competitive

The

enzymes

reagents

to were

15

time

and

been selective

Ach-esterase

site and

to

(Main In

a time

as

of

short

a substrate,

to

before

other

only

for

react of

the

to

overcome an

Hastings,

standard

of the

procedure,

DNFA,

CDNP,

inhibited

mainly

by

K.

s are

shown

by

DNFA,

which

does

not

inhibit

1, AchthE from CDNP,

concentrations,

we looked

the

absolute

(z)

previously

ileum, the

data

the shown

were shown

needs

AchthE,

to

be

still

used,

by

a mixture

and

of

and

of

reversible antagonism 1966). has

the

3.

activity

more

,

H/Ki,

Ach

on

(z') Ki the

guinea

enzymes,

to but

pig from

values

for

similar

the

sensitivity

preparations,

respect

than ratios

calculated

Average

lowest

Apart

saturating

Ki

of

the

at

experiments ratios

been

and

NBDFB.

even

relative

different

of

centre

NBDFB,

AchthE

the

with

lapse

organophosphates

Iverson,

DFDNB

because

the

stressed

and

4 reports

i,MAO /K i,AchthE in Tables 2 and

a non-

This

MAO activity

the

for

and

phase

DFDNB

K

activities

inhibitors

that

Table

calculated

and

AchthE to

potency.

1969).

Lewis,

non-competitive

and

for

and

serine-active

Main

assay

with changes

for

first

when

inhibition

found.

irreversible 1966;

6 min

substrates

enough the

instance,

group-affinity

usually

long

reagents

display

and

be

the

the

was

to

as

the

with

addition

found

For

(Stein

inhibition

has

binding

changes.

allowed

min

339

conformation

non-competitive

competitivetypeof of

Vol. 3, No. 4, 197 1

a point

the

erythrocyte

thousands

of

times

can

be

residues

(BA

and

and

compounds

purified. By examining that

compounds

itself)

the that

prevalently

last

column,

react

with

affect

covalently

bind

aromatic

free

-NH2

the

bifunctional

more

effective

isDNFA.

The

blockers evidence

of is

-NH2-free

MAO activity,

that

(i.e.

the

hydroxyl reagents the

somewhat

AchthE tenuous,

conclusion

that

residues DFDNB activity. nevertheless

other

reached CDNP

than

and

NBDFB)

One

exception the

are

340

Pharmacological

pattern

agrees

Ach

receptors

Table

4),

TNBS,

were

TABLE

4-

with in

even

that

the if

not

found

intact

for

(see

such

"group

with

can

reagents"

as

a suggestive be

in

the

AchthE,

Wilson,

1963;

peptide

(Jansz

but

AI

such

case

of

which

labelling agents AchthE activities.

et

al.,

and

of

and

Baker,

1967),

Shaw,

reagents.Interestingly

centre,

in

which

a carboxyl

binding

the

1969)

of

(1953);

the

should choline

nor

is

response been

pointed kind

whereas and

they AchthE.

(Kremzer the is

and

active-site not

be moiety,

the

the

a quantitative

enough,

residue

between

has

receptors crystallized

1959;

head

correspondence

been

these

reagent

the

cholinergic

for

cationic

from

a correspondence

action

Khorana

"co.01 0.622 1.72 1.15 CT

a correspondence

had

Lenzinger

and

= 2.00 =

conclusions

from

in

Z’

on MAO activity such

one;

the

drawn

action

ileum,

reached

valid

be

H and

Methods).

1.043 6. 0.001

out

the

those

=

pig

by

column

3.644 =

guinea

In

other

21.106 =

of

seem

as

to

Z

No conclusion

Vol. 3, No. 4. 1977

sensitivity

z and z1 ratios calculated for the affinity in guinea pig ileum, and for the MAO and z=K /K .z'=K i'H i'Ach' i,MAO 'Ki , AchthE

DNP DNFA TNBS CDNP BA DFDNB NBDFB AI

cannot

Communications,

(cfr.

tests,

Inhibitors

the

the

tissue

crucial

possible

Research

same

a site the

of anionic

involved is reagent

in not

inhibited (DCCD)

Pharmacological

Research

effective

in

site, of

blocking

another action

plax

membrane

site

is It

and

the

i.e.

the

point

sites"

to

the

and

which,

Tani,

the

that

point

absolute

binding

at

and

of lower

0.68-2

100

protein

concentration,

activities, values

86 mM for

Kis

for "true"

AchthE; by

towards

recalled

(:

that

3.9

moles nmoles fraction

ml,

that

than

in

the

absolute

from per

reagent

the

g tissue, per

as

recepcontribute

enzyme

the

in

(Belleau

values the ratio is

standard

at

a concentration

intact

tissue.

of

the

tissue

6x10 -12

1 = 3.9x10 -11 = 10 moles/g

in

50 vitro

from

Ach the

the

to

fresh

assay of At

the

system. of

"nuclear tissue),

the

MAO and

Again,

a higher

in

latter

values

of

be

this

actual

is

receptor

moles/g

three

inhibitor

activities

zero

S vs enzymatic data, one should

assays,

AchthE

12

to

5 mM for

favour

experiments or

ID

of

far

in

at

the

of

for

this

calculated

order

H and

We found

in

the

are

relation

two

slopes

the

the

are

is

the

be

in

may

constants.

enzyme,

from

to

DFDNB

the

calculated -11

neous"

i these

which

accessory

is

vitro,

measured

extrapolation K

e&erase

data,

the

inhibition

the

from

i,MAO'Ki, AchthE unity (= 3.1), and

tivity

are

in

lower

for

shown

values

K

than

10

times

by

vivo

per

located,

site,

attention

in

values

mg protein

AchthE

choline

our

anionic of

Changeux,

1970).

by

the

protein

expect

the

i inhibitor

than

al., deserves

K

the

the

of

"adjacent

of

receptor

closely

by

properties

et

that

vitro

the

times

Kitz

Ach

electro-

and

centre

spectrum

the

that

are

supported

with

In

assumption

presence

allosteric

1966;

in

the

the

(Podleski

anionic

not

together

regulatory

Another of

to

the

the

the is

1967),

electricus AchE

esteratic

affects DNFA).

receptor

is

the

for

of

cholinergic

1965,

z1

that

of that

(cfr.

concluded

receptor

nevertheless

uncertainty

site

341

residue

Electrophorus

muscarin.ic

(ZGpanEiG,

the

esteratic be

the

glutamic

reagents

from

may

that

tor

of

these

not

Vol. 3, No. 4, 1971

the

proof

of

1970).

Communications.

higher sensi-

It

may

the

enzymes

order

centres and and

be

of for

MAO

membra0.34x10

-10

342

Pharmacological

moles/g for

tissue,

AchthE

or

(:

fraction,

0.34

whose

tissue).

The

figure

for

sites

(2.1/n

= 1.67)

pmoles

per

activity

is

MAO figure

AchthE

determined

through

compared the

pig

of

guinea

pig

ileum

receptor

(Paton

and

Rang,

1965))

uptake

(Ehrenpreis

per

g,

respectively.

and

of

Acetylcholinesterase

same

range

been

reached

et

the

moles

presence

of

of

the

receptor

and/or

of

1970).

Most

number

of

active

estimated toxin

to

were These

in

vitro

must

be

of

the

the

not

identical.

to the

However,

inhibition

studies (for

an

the

of

the receptors,

the

scanty

to exception,

the

within

recently

see

but the

activity differences

al.,

1971)

tissue has 131 IU-bungaro-

been

and

the

receptor

number

of

enzyme

does

not

even

if of

occuring Nakar

need

they

relevance

events

number

the

eel,

et

mediator

of

the

noteworthy

enzyme

the

intact

as

electric

same

that

number

the

has

(Miledi

number

inactivate

is

showed

active

indicate

of

concentrations

in

but

clearly

stressed

AchE

esteratic

Ach-receptors,

weight) the

recently

sites,

entities.

g wet

enzyme

as

exceed

different

the

of

the

by atropine 12 13 10 and 10

being

which

inhibitor

same

necessary

greatly

same

sites

calculations

molecules

values

fresh

of

well

determined

from

be the

binding

protein

to

al.,

the

as

number

electroplax

the

et

sites

per

whole and

number

1969),

the

of

activity

molecules -6

(1-2.5x10 for

the

al,,

An estimated

in

(Karlin

to

that

centres "microsomal"

precedent,

enzymatic

guinea Ach

ileum

-9 moles/g

10 times any

Vol. 3. No. 4, 1971

13 receptor

1 = 0.34x10

without

be

Communications,

= 1.26x10

about

is

can

Research

the in

and

are

vivo

Mannering,

1970).

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