Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COMPETITIVE INHIBITORY EFFECT EXERTEDBY BILE SALT MICELLES ON THE MYDROLYSIS OF TFUBUTYRIN BY PANCREATICLIPASE And&

VANDERMEERS,Marie-Claire VANDERMEERS-PIRET,Jean RAT& and Jean CHRISTOPHE Brussels University School of Medicine, 115, B-1000 Brussels, Belgium.

Department of Biochemistry, Waterloo Boulevard Received

February

17,1976

SUMMARY. Colipase was readily adsorbed on tributyrin particles emulsified with sodium taurodeoxycholate used above its critical micellar concentration. The ensuing rate of lipase adsorption on tributyrin depended on the amount of colipase already adsorbed at the interface. A molar excess of colipase over lipase was required to observe lipolysis at maximal velocity. Bile salt micelles competitively inhibited this reaction. High bile lipase

salt

concentrations

(triacylglycerol

lipase

MAYLIE et al (1) this velocity

V and this

colipase,

micelle step for

colipase

ternary

parameter

lipolysis

an alternative

mechanism. First,

Secondly,

the binary

inhibitor

of the reaction.

enzyme-inhibitor for

substrate

colipase-bile

salt

The binding

complex rather

salts

(2).

to represent The present that

colipase

in the presence of bile acting

to

by the presence of

of a lipase-colipase-bile

our study indicates

colipase

According

the group of DESNUELLEhas

This complex was thought

in the presence of bile

occurs in succession,

restored

In addition,

promotes the formation

of pancreatic

to a sharp decrease of maximum

is not entirely

are indeed adsorbed on the substrate this

on triglycerides.

corresponds

cofactor.

complex (2).

reduce the activity

E.C.3.1.1.3)

inhibition

a small protein

shown that

markedly

salts0

as an anchor for lipase

micelle

salt a preliminary data support and lipase However,

binding.

complex is a competitive

of lipase

to this

than an activated

complex yields

an

form of the enzyme required

binding, MATERIALS AND METHODS

Rat pancreatic described

(3).

lipase

Tributyrin

and bovine colipase

were purified

as previously

was purchased from Fluka (Buchs, Switzerland).

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

790

Sodium

BIOCHEMICAL

Vol. 69, No. 3, 1976

taurodeoxycholate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(NaTDC) came from Sigma (St.

the emulsions were prepared with

an Ultra-Turrax

Missouri,

U.S.A.),

a slow but perceptible

All

type TB 10 homogenizer from

Germany) for 1 min at 13,000 rev/min.

Janke and Kiinkel KG (Staufen, emulsion exhibited

Louis,

The

decrease of turbidimetry

However, the standardized

easily

and care was taken not to use these emulsions for more than

one hour after

their

In a first

for quantitative

type of experiment

on tributyrin

emulsified

2 % tributyrin

the rate

particles

emulsion stabilized

and adjusted

to pH 6.0 with

morpholinopropane l-7

preparation

sulfonic

of lipase

with

immediately

increasing

or after

analysis.

and colipase

with 4 mMtaurodeoxycholate

and 150 mMNaCl

Incubation

containing Each mixture

at 25" C. After

lipase

assays. Lipase was determined

at pH 8.0 by an adaptation

dimetric

method of SHIHABI and BISHOP (4) using O-04 % tributyrin

triolein

and 1 mM taurodeoxycholate

dimetric

and 150 mMNaCl. A large

was added per colipase Kinetic xycholate

1.2,

experiments

and colipase

and

of the turbiinstead

of

Colipase was added in excess

to pH 6.5 and contained

5 mM

excess (10 pg) of pure rat

lipase

were performed to evaluate on lipolysis

1.4 and 1.6 %) each at forty concentrations

lipase

assay* the influence

of taurodeo-

at pH 6.0 and at 25O C. The activity

was measured at seven concentrations

colipase

was decanted for

for

Colipase was assayed by the same turbi-

method but the emulsion was adjusted

taurodeoxycholate

lipase

as stabilizer.

(0,s or 5.0 pg)

centrifugation

colipase

emulsion.

0.1 M N-

was centrifuged

supernatant

to the substrate

of a

media were prepared by mixing

amounts of colipase.

1 to 5 min incubation

or to pH 7.0 with

5 min at 10' C and 1000 x g, the clear

(20 pg/ml)

adsorption

consisted

a 0.1 M maleic buffer buffer.

was

was measured. The substrate

ml of the emulsion and 400 ~1 of a solution

together

kinetic

procedure

time

(50 % in 12 hours). reproducible

emulsification

with

different

(see below).

of tributyrin conditions

Tributyrin

791

(0.4,

0.6,

0.8,

of taurodeoxycholate

was emulsified

of rat 1.0, and

in a mixed buffer

BIOCHEMICAL

Vol. 69, No. 3, 1976

made of 2,4 mM acetic fonic

acid,

2,4 mMmaleic acid,

containing

medium was made by mixing

a constant

amount of lipase

in increasing

concentrations.

Were 4, 6, 8, 10 or 12 mM (i.e., concentration

(1).

0.5,

2.4 mMN-morpholinopropanesul-

107 ml emulsion with

400 ~1 water

pg) plus colipase

and taurodeoxy-

taurodeoxycholate

concentrations

(0-l Final

they were all

Lipase concentration

were, respectively,

above the critical

was 1 nM and colipase

of the automated potentiometric

incubation

time was reduced to 2 min and the rate

to measure lipase

micellar concentrations

1, 2, 4, 8, 12, 24 or 48 nMO Lipase was assayed by an

adaptation

60 samples per hour.

RESEARCH COMMUNICATIONS

150 mMNaCl and NaOH to pH 6-O .

2 mM sodium taurodeoxycholate,

Each incubation

cholate

acid,

AND BIOPHYSICAL

The small pH fall

activity

method described

recorded

of analysis

(6) by plotting

WILKINSON

(5).

The

was increased

to

at the end of the incubation

never exceeded 0.15 pH unit.

analysed by the method of

earlier

The results

[S]/v

versus

were

[S].

RESULTSAND DISCUSSION ADSORPTIONEXPERIMENTS. Increasing adsorption cholate. curves

the pH from 6 (Fig.

on tributyrin

emulsified

These observations (7).

It

is clear

lipolysis

increasing

the colipase/lipase

tely

a plateau Fig.

parallel

2 also points that

of lipase,

adsorbed colipase

the rate

at low colipase

of lipase

particles

confirm

that

of initial

1) to 7 (Fig.

ratio

adsorption, out that

obtained

of lipase

adsorption

is the limiting

concentrations,

strongly

suggests that

binding

site

lipase

for

At

to two was nearly i.e.,

pH 6.0,

adsorption

already

on the substrate

rate

792

was started. did not

The percentage

of lipase

tributyrin.

of

adsorption

bound to the interface.

each molecule of bound colipase

on taurodeoxycholate-coated

step

enough to reach immedia-

a more rapid event. the

pH-adsorption

however,

as soon as the incubation

time while

was dependent upon the amount of colipase finding

sodium taurodeoxy-

previously

colipase

with

coated with

and explain

and was in fact

was constant

2) reduced the rate of lipase

This

acted as a The analogy

Vol. 69, No. 3, 1976

0 0J I..-0

BIOCHEMICAL

7

.--7 2

INCUBATION

TIME

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0

3

(MINUTES

1

I 2 3 4 INCUBATION TIME (MINUTES)

5

FIGURE 1, Time study of lipase adsorption on tributyrin in the presence of 4 mMtaurodeoxycholate at pH 6.0 . The colipase/lipase (C/L) ratios in the incubation mixtures were zero ( v-v ), 0.5 ( V-7 ), 1 ( A-A ), 2 ( A-A ), 4 and 8 ( 0-O ). The ordinate indicates the percentage of the total lipase (0.5 kg per assay) adsorbed on tributyrin. Lipase adsorption was estimated by a measurement of its residual activity in the aqueous supernatant after centrifugation of the incubation mixture as detailed under Materials and Methods. FIGURE 2, Time study of lipase and colipase adsorption on tributyrin in the presence of 4 mM taurodeoxycholate at pH 7.0 o Incubation media were prepared by mixing 1,7 ml emulsion with 0.4 ml of a solution containing 5 Pg lipase and colipase in a molar ratio (C/L) of 1 ( A-A ), 2 ( A-A ) and 4 ( 0-0 )Q Each mixture was centrifuged after 0 to 5 min incubation as described under Materials and Methods. Lipase and colipase were assayed in the supernatant-

with

the ternary

lipase

and one bile

Since colipase (Fig.

complex involving

2),

inhibitor

as described

micelle,

did promote lipase

as well

taurodeoxycholate

salt

as on bile

one molecule of colipase,

salt

binding micelles

used at supramicellar

on lipolysis,

The following

by CHARLESet al (2) is obvious,

on tributyrin (2),

it

concentration kinetic

assumption,

793

one molecule of

coated with bile

was logical

to expect

acts as a competitive

experiments

confirmed

this

salt that

Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COLIPASE -6 LIPASE

,5

.OLIPASEeIT LIPASE

1

20

40

60

-77 [s]

‘I.

FIGURE 3. WILKINSON plots (6) of tributyrin hydrolysis at pH 6-O and 25" C. Regression lines for 3 colipase/lipase ratios (C/L) as a function of taurodeoxycholate (NaTDC) concentration. The intercept with the abscissa and the slope of each regression line give the corresponding Km app and V, Each line was calculated from seven points and each point was the mean of two experimental values. Single points are not represented since all correlation coefficients were above 0.99 .

6

4

I 6

I 8 [N~TIJC]

. I IO 12 mM -

FIGURE 4. Effect of taurodeoxycholate on the apparent catalytic constant at kcat was estimated from values of V increasing colipase/lipase (C/L) ratios. calculated as indicated in Fig. 3 and assuming a molecular weight of 48,000 daltons for rat lipase.

794

BIOCHEMICAL

Vol. 69, No. 3, 1976

AND BIOPHYSICAL

Km (app)

RESEARCH COMMUNICATIONS

1 .

40. C/L .2 I . 30. i;

-8

-6

-4

-2

0

2

4

6 I3 [N.~TDC]

,o m.3 -

12

FIGURE 5, Competitive inhibitory effect of taurodeoxycholate on tributyrin hydrolysis for six colipase/lipase ratios (C/L). The values of Km (app) were calculated as indicated in Fig. 3 and the straight lines are regression lines giving

Km (app) = Km (1 + 3.

KINETIC PARAMETERS, Fig. for

3 shows several

the action

of lipase

concentration parallel are valid

for

straight only

series

of typical

on tributyrin

ratios,

lines.

for Km app (the intercepts

The values

deoxycholate

devoted to substrate

clearly

inasmuch as all

the lines

Our results that

increased

These plots

the colipase/lipase

ratio

gave families

on the abscissa)

emulsion utilized area per unit

concentration,

the Km app without

of

at pH 6.0

volume was playing

The presence of tauro-

affecting

V (the slopes)

were parallel.

are at variance

4 mM taurodeoxycholate

(6) obtained

of taurodeoxycholate

3 colipase/lipase

for the present kind of tributyrin

normally

WILKINSON plots

as a function

and at 25' C. This is because the interfacial the role

linear

with

those of MAYLI; et al (1) who observed

decreases V by 98 % and 52 %, respectively, increases

from 0 to 3. On the contrary,

795

Fig.

when 4 shows

Vol. 69, No. 3, 1976

that

BIOCHEMICAL

the relatively

large

high catalytic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

constant

(2 to 48) range of colipase/lipase

decreased when the colipase/lipase at least

partly

conditions

1) with

analysed.

It

inhibition

actual

competitive

inhibitor

tributyrin-bile

salt salt

linear

increasing

relationships

remained constant concentrations

(Le.,

lipase.

lines)

high proportions

of lipase

of colipase

the

similar

salt

inhibited

a result,

the colipase/lipase

of tributyrin

to postulate on its

on the abscissa)

was increased

As

was increased

decreased from

(intercepts

further

concentra-

to still

higher

affected,

while

the K,/Ki

ratios

decreased from 2.75 to O-27 . More

why raising

improved the hydrolysis

improved the binding

The

for the ternary

on the ordinate)

from 2*64 to 9.25 mM.

is tempting

induce a

at steady state.

(Fig0 5). When colipase

(24 or 48mM), the Km values were not

However, it

micelles

versus taurodeoxycholate

(intercepts

the slopes of the straight

still

salt

when

complex.

the Ki values of bile

data are necessary to explain unity

at the steady state

bile

around 2.5 mM, When colipase

the Ki values increased

apparently

complex (2) was probably

were obtained

from 2 to 12 nM, the Km values

by a

observed under these

the K of lipase m

monolayer-colipase

7e64 to 2.53 mM, while

adsorption

when tested

When the values of Km app were plotted tion,

was unaffected

However kcat

not yet

system

of lipolysis

micelle-colipase

-1

was as low as 1.0 or 0.5 . This was

may be concluded that

competitive

bile

ratio

a reaction

purely

binary

ratios,

due to the slow rate of lipase

(Fig,

kinetically

of 5100 set

bile

that

catalyzed

well

above

by pancreatic

a moderate excess of colipase

salt-coated

the binding

ratio

substrate

of lipase

whereas very

on bile

salt

micelles.

ACKNOWLEDGMENTS Supported by Grant 20403 from the Fonds de la Recherche Scientifique Mddicale (Belgium) and Grant RO-IAMfrom the National Institutes of Health (USA).

796

Vol. 69, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1. 2.

3. 4. 5. 6, 7.

MAYLIE, M.F., CHARLES, M., ASTIER, MI, and DESNUELLE, P. Biophys. Res. Commun. 52, 291-297. B.,and DESNUELLE, CHARLES, Me, SARI, H., ENTRESSANGLES, Biochem. Biophys. Res. Commun. 65, 740-745. MoCe, RATHi, J,,and VANDERMEERS, AO, VANDERMEERS-PIRET, (1974) Biochim. Biophys. Acta 370, 257-268. SHIHABI, Z.Ko, and BISHOP, C. (1971) Clinical Chemistry VANDERMEERS, A., VANDERMEERS-PIRET, M.C,, RATHE, J., and (1974) Clin, Chim. Acta 52, 271-276. WILKINSON, G.N., (1961) Biochem. Ja 80, 324-332: VANDERMEERS, A,, VANDERMEERS-PIRET, M.C., RATHE, J., and (1975) FEBS Letters 49, 334-337,

797

(1973) P,

Biochem.

(1975)

CHRISTOPHE,

J.

l7, 1150-1153, CHRISTOPHE,

J.

CHRISTOPHE,

J.