Vol. 65, No. 1, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CHARACTERIZATION

AND PARTIAL

PURIFICATION

OF AN I N T E S T I N A L

LIPASE

G. Serrero, Centre

de B i o c h i m i e

R. N~grel,

- Facult~ 06034

Received

April

and G. A i l h a u d

des S c i e n c e s

- NICE

- Parc V a l r o s e

- France

28,1975

S U M M A R Y A n i n t e s t i n a l l i p a s e has b e e n c h a r a c t e r i z e d in d i f f e r e n t specie, i n c l u d i n g man. A b a c t e r i a l o r i g i n for the rat e n z y m e and a p a n c r e a t i c o r i g i n for the pig or the h u m a n e n z y m e have b e e n e x c l u d e d by the use r e s p e c t i v e l y of g e r m - f r e e rats and of immun o s e r a d i r e c t e d a g a i n s t p i g or h u m a n p a n c r e a t i c lipases. The pig e n z y m e has b e e n p u r i f i e d 242 fold; it is m a i n l y a c t i v e a g a i n s t short- and m e d i u m - c h a i n t r i g l y c e r i d e s . A role in the a b s o r p t i o n of n e u t r a l lipids, m a i n l y in p a t h o l o g i c a l s i t u a t i o n s , is d i s c u s s e d .

Different

enzymes

lysis o f c a r b o x y l i c mucosa was

described

glycerides was

found

mucosa

not directly

likely

from

of h u m a n

lingual

pancreatic

medium-chain

intestinal

on mono-, active

and p a r t i a l l y

excluded

for t h e s e

origin

di-,

and/or

from c h i c k e n

a bacterial

activities.

(5), has b e e n

and tri-

on m o n o g l y c e r i d e s

purified

a pancreatic

that p a t i e n t s function

lipase

ori-

A lipase,

triglycerides,

found in the s t o m a c h

in the m u c o s a l

cells

is shown

lipases

(7) or w i t h

against

In the p r e s e n t from i n t e s t i n e

to be d i f f e r e n t

the p i g short-

in a g e n e r a l

a specific

absorb

work

involand

loca-

specie

gastric

and

origin.

After

lipase

and m e d i u m - c h a i n

exo-

short-

a lipase,

of different

from

intestinal

defect

efficiently

a n d not to be of b a c t e r i a l

242 fold p u r i f i c a t i o n , active

with a defect

(8,9), w i l l

triglycerides.

i n c l u d i n g man,

mainly

the h y d r o -

from pig

(6).

ving pancreatic

pancreatic

catalyze

a lipase

pH on short- a n d m e d i u m - c h a i n

It is k n o w n

lized

intestine

active

lipase mainly

In b o t h cases

at a c i d i c

crine

as e q u a l l y

(2), a n o t h e r

active

small

(i) . W h i l e

in rat i n t e s t i n e

(3,4).

gin w a s

of the

esters

appears

a

to be

triglycerides.

MATERIAL AND METHODS Intestinal cells obtained after careful w a s h e s and s c r a p i n g o f the m u c o s a w e r e h o m o g e n e i z e d as d e s c r i -

Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form reserved.

89

Vol. 65, No. 1, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

bed in Table I; under these c o n d i t i o n s the a c t i v i t y due to pancreatic lipase represents r e s p e c t i v e l y 0%, 3% and 0% of the total lipase a c t i v i t y p r e s e n t in the intestinal cells of human, m a l e W i s t a r rat (180 to 230 g) and pig. Human b i o p s i e s of jejunum and human ileum w e r e o b t a i n e d t h r o u g h the c o u r t e s y of Prof. D e l m o n t and a s s o c i a t e s (Centre de G a s t r o e n t ~ r o l o g i e - U n i v e r s i t ~ de Nice). Two d i f f e r e n t assays of initial rates of h y d r o l y s i s w e r e used for intestinal lipase : first a t i t r i m e t r i c a s s a y w i t h trib u t y r i n (at pH 8.3) or t r i o l e i n (at pH 8.5) as substrates (i0,ii) and r e f e r r e d as to t r i g l y c e r i d e h y d r o l a s e a c t i v i t y (E.C.3.1.I.3.), second a s p e c t r o p h o t o m e t r i c assay w i t h p a l m i t y l - C o A as s u b s t r a t e a n d r e f e r r e d as to t h i o l e s t e r a s e a c t i v i t y (E.C.3.1.2.2.) . In this last assay the m e d i u m c o n t a i n e d (in m i l l i m o l a r c o n c e n t r a t i o n s ; final volume 0.9 ml) : T r i s - C l b u f f e r pH 7.8, 33; NaCl, 150; 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), 0.83 and p a l m i t y l -COA, 0.08. The h y d r o l y s i s was f o l l o w e d at 412 nm for at least 2 min. a n d good p r o p o r t i o n n a l i t y up to 20 mU per assay was o b s e r ved. p - n i t r o p h e n y l a c e t a t e h y d r o l a s e was d e t e r m i n e d by o m i t t i n g DTNB and r e p l a c i n g p a l m i t y l - C o A by 0.5 m M p - n i t r o p h e n y l a c e t a t e . All specific activities are e x p r e s s e d in units (~moles/min)or m i l l i u n i t s (nmoles/min) per m g o f p r o t e i n (12). A typical p u r i f i c a t i o n of the pig enzyme was as followed : 15 jejunums of pig i n t e s t i n e w e r e r e m o v e d and t h o r o u g h l y w a s h e d w i t h cold 10 m M Tris-Cl b u f f e r pH 7.3 c o n t a i n i n g 154 m M N a C I and i0 m M M g C I 2. A f t e r gentle scraping of the mucosa, the cells were h o m o g e n e i z e d at 4°C in 4 volumes (w/w) of i0 m M TrisC1 b u f f e r pH 7.3 c o n t a i n i n g 1 m M Ca C12, i0 m M MgCI2, 0.25 M Sucrose and 1 m M DFP. The starting m a t e r i a l was o b t a i n e d t h r o u g h the f o l l o w i n g scheme : 3000g x i0 m i n 10000g x 20 m i n Homogenate • ) Supernatant I ) i05000g x 45 m i n a l k a l i n e t r e a t m e n t at pHZ8 S u p e r n a t a n t II ) Pellet I ) S u p e r n a t a n t IV then i05000g x 45 m i n S u p e r n a n t a n t III d i s c a r d e d S u p e r n a t a n t IV (1400 ml; 1.67 g of proteins) was d i a l y z e d against 30 volumes of 10 m M T r i s - C l b u f f e r pH 7.3 and then p l a c e d on a D E A E - c e l l u l o s e column (3.5 x 30 cm) e q u i l i b r a t e d w i t h the same buffer. The e l u t i o n was p e r f o r m e d w i t h 3000 ml of a linear g r a d i e n t from 0 to 0.5 M NaCI in the same Tris-C1 b u f f e r as above. The a c t i v i t y e m e r g e d b e t w e e n 0 . 1 M and 0.3 M w i t h a p e a k at 0.2 M. The active fractions w e r e pooled, d i a l y z e d and c o n c e n t r a t e d batchw i s e on a second D E A E - c e l l u l o s e column (2.5 x 5 cm). The concentrated f r a c t i o n (25 ml, 137 mg) was p r e c i p i t a t e d at 0.5 s a t u r a t i o n with ammoni~ sulfate. The p e l l e t thus o b t a i n e d (77 mg) was dissolved in 10 m M Tris-Cl b u f f e r pH 7.3 and p u t onto a column of S e p h a d e x G-200 (3 x 91 cm) e q u i l i b r a t e d w i t h 0.2 M NaCI b u f f e r e d as above. The active fractions (130 ml; 23 mg) w e r e pooled, dialyzed as usual and further p u r i f i e d on a second D E A E - c e l l u l o s e column (0.9 x 12 cm) e q u i l i b r a t e d w i t h i0 mM Tris-Cl b u f f e r pH 8.5, using a linear g r a d i e n t from 0 to 0.3 M NaC1 (Tot&l v o l u m e : 400 ml). The a c t i v i t y e m e r g e d as a symetrical peak b e t w e e n 0.06 M and 0.14 M w i t h a m a x i m u m at O.i M; the p o o l e d fractions were d i a l y z e d against i0 m M Tris-Cl b u f f e r pH 7.3 and stored at 0°C. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s was p e r f o r m e d using the T r i s - g l y c i n e system n u m b e r 1 a c c o r d i n g to M a u r e r (13).

90

Vol. 65, No. I, 1975

BIOCHEMICAL AND BiOPHYSiCAL RESEARCH COMMUNICATIONS

C o A was o b t a i n e d from P-L L a b o r a t o r i e s and the a c y l - C o A s y n t h e t i z e d a c c o r d i n g to A i l h a u d et al. (14). The d i f f e r e n t tri= g l y c e r i d e s w e r e p r o v i d e d by F l u k a as w e l l as DFP and N - e t h y l m a -

ZPECIFIC ACTIVITIES OF THE HOMOGENATE SPECIES

UTYRIN

TRIOLEIN

(mu/mg) WITH

PALMITYL-CoA

1500

131

19,5

GUINEA-PIG

II0

206

11,2

CONVENTIONAL RATS

500 950

CHICKEN

GERM-FREE RATS

22,3 21,5

RABBIT JEJUNUM ILEUM

480 940

90 218

31,9 30,4

400 900

850 1130 57O

32,5 55,2 55

786 080

585 -

i0 34,2

PIG DUODENUM JEJUNUM ILEUM

MAN JEJUNUM ILEUM

Table

I : I n t e s t i n a l !ipase a c t i v i t y in d i f f e r e n t specie The h o m o g e n a t e s w e r e o b t a i n e d a f t e r t h o r o u g h w a s h i n g of the i n t e s t i n e w i t h cold 0.154 M NaCl. A f t e r g e n t l e s c r a p i n g of the mucosa, the cells were h o m o g e n e i z e d in I0 m M T r i s - C l b u f f e r pH 7.3 c o n t a i n i n g 1 m M CaCI 2, i0 m M MgCI 2 and 0.25 M S u c r o s e and f i l t e r e d t h r o u g h g a u z e to r e m o v e mucus.

91

Vol. 65, No. 1, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

leimide. I o d o a c e t a m i d e and E600 w e r e p u r c h a s e d from Sigma, m o n o and d i g l y c e r i d e s from H o r m e l / ~ l ~ 4 o t h e r c h e m i c a l s were o b t a i n e d from Boehringer. G l y c e r y l t r i (i- C octanoate) was a p r o d u c t of N e w - E n g l a n d Nuclear. I m m u n o s e r a d i r e c t e d a g a i n s t h o m o g e n e o u s pig p a n c r e a t i c lipase (kindly s u p p l i e d by Dr. M. Charles, CBM-CNRS, Marseille) and a g a i n s t human and rat p a n c r e a t i c juices (obtained t h r o u g h the c o u r t e s y of Dr. C. Figarella, INSERM, Marseille) w e r e u s e d after p r e c i p i t a t i o n of the i m m u n o g l o b u l i n fraction b e t w e e n 0 and 0.4 s a t u r a t i o n in a m m o n i u m sulfate and d i a l y s i s a g a i n s t 20 m M p h o s p h a t e b u f f e r pH 7.2 c o n t a i n i n g 150 m M NaCI.

RESULTS

Table

ding man,

contain

or t r i o l e i n It was

I shows

as w e l l

on b o t h

Of i n t e r e s t with germ-free cates

an h y d r o l y t i c as a g a i n s t

shown w i t h pig

is active

that the h o m o g e n a t e s

parts

tributyrin

used

(see below)

incluand/

as substrates.

that a single

enzyme

substrates. is the fact that

rats

as c o m p a r e

for rabbit,

palmityl-CoA

similar

levels

to c o n v e n t i o n a l

is not of b a c t e r i a l

pig and man,

of the intestine.

versus

against

palmityl-CoA

intestine

that the a c t i v i t y

As shown

activity

of all specie

varies

origin

it is p r e s e n t

The a c t i v i t y

ratio

approximately

were o b t a i n e d

rats,

which

indi-

in this species.

in the d i f f e r e n t

against

tributyrin

from 25 for the pig to

300 for the rat. In Table Supernatant rial o n l y

II are given

IV as starting

contained

in the homogenate,

the total

units)

purification Fig.

genate

or a c y l - C o A

as substrates. fraction

as that of fraction

phadex

G-200

the p u r i f i c a t i o n

However

The curves

V)

molecular

and

Moreover

(last co-

in the pig homo-

triglycerides

is still Gel

of

thiolesterase

procedure

using

VI (not shown).

lipase

versus

the h e t e r o g e n e i t y

(fraction

gave an a p p a r e n t

48 000 for p a n c r e a t i c

An o v e r a l l

hydrolase

the absence

activities

matepresent

the use

From the diagrams

hydrolase

suggests

hydrolytic

154 fold p u r i f i e d well

along

II), w h i c h

of o t h e r

since

are due to the same enzyme.

of t r i g l y c e r i d e

similar

lumn of Table

units

but up to 59% of

poor p u r i f i c a t i o n .

that b o t h t r i g l y c e r i d e

activities

values

very

variable

using

the s t a r t i n g

of the total

for p u r i f i c a t i o n

(which c o n t a i n e d led to rather

1 it is a p p a r e n t

remain

chosen

of a p u r i f i c a t i o n ,

Although

percentage

of 242 fold was obtained.

thiolesterase the ratio

material.

a small it was

of S u p e r n a h a n t ~ I I I

the results

of the

obvious,

filtration

weight

and/

as

on Se-

of 70 000 a g a i n s t

(15).

of i m m u n o p r e c i p i t a t i o n

92

are p r e s e n t e d

in Fig.

2.

Vol.

65,

No.

1, 1975

BIOCHEMICAL

AND

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RESEARCH

COMMUNICATIONS

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1

Gel f i l t r a t i o n of pig i n t e s t i n a l lipase on S e p h a d e x G-200 (see M a t e r i a l and M e t h o d s for details) The t r i g l y c e r i d e h y d r o l a s e was m e a s u r e d w i t h tributyrin as substrate. The inset r e p r e s e n t s the a c t i v i t y per slice of p o l y a c r y l a m i d e gel (2 m m t h i c k n e ~ ) ¢ O : a c t i v i t y m e a s u r e d w i t h g l y c e r y l t r i (|i - ~-C| octanoate) as substrate; ~ : a c t i v i t y ~ e a s u r e d w i t h p a l m i t y l - C o A as substrate. After c u t t i n g of the gel, slices w e r e i n c u b a t e d for 48 hours at 4°C in i0 m M • ris-Cl buffer (pH 7.3) and the d i f f e r e n t s u p e r n a t a n t s a s s a y e d for activity. The 100% value c o r r e s p o n d s to the band w i t h an Rf of 0.43.

:

The absence nal

lipase

of any c r o s s - r e a c t i o n and the i m m u n o s e r u m

pancreatic

enzyme

intestinal

and p a n c r e a t i c

nal

lipase,

clearly

lipases;

the p r e c i p i t a t i o n

0.i ml of immunoserum),

cells

purified

against

of intesti-

lipase

is normal

(16 lipase

from i s o l a t e d

pig

between

units

lack of p r e c i p i t a t i o n

prepared

intesti-

homogeneous

in the p r e s e n c e

of p a n c r e a t i c

a similar

pig

the n o n - i d e n t i t y

of i m m u n o s e r u m

with homogenates

per

was

pig i n t e s t i n a l

(not shown). Moreover

tinal

directed

indicates

(curve B). W i t h an excess

observed

between

identical

and p a n c r e a t i c

results

lipases

ted a g a i n s t

rat p a n c r e a t i c

unpublished

experiments).

The absence lipases

is s u b s t a n t i a t e d

(half-life

juice

of identity

of 27 min.

were

found w h e n

comparing

from rat w i t h an i m m u n o s e r u m (V. F e r n a n d e z

between

by the rates

and 68 min.

94

- Lopez

intestinal

et al.;

and p a n c r e a t i c

of d e s a c t i v a t i o n

respectively)

intesdirec-

as w e l l

at pH 6.2 as by

Vol. 65, No. 1, 1975

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

~>'80

p.

"E 4o E

~2o

\

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Fi@.

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4

6

8

10 f2 14 T i m e [hours)

16

N o n - i d e n t i t y between intestinal and p a n c r e a t i c lipases from pig 25 units of hog i n t e s t i n a l lipase ( O a n d • ) , 140 units ( A a n d A ) , 70 units ( [ ] a n d m ) , 35 units ( v a n d •) of pig p a n c r e a t i c lipase and a m i x t u r e of 25 units of i n t e s t i n a l and 35 units of p a n c r e a t i c lipases (Qand,) were i n c u b a t e d in i0 m M p o t a s s i u m p h o s p h a t e buffer pH 7.3 c o n t a i n i n g 0.15 M NaCI in the p r e s e n c e of 0.1 ml of rabbit control serum (open symbols) or 0.i ml of rabbit serum d i r e c t e d against homogeneous hog p a n c r e a t i c lipase (solid symbols). At the time indicated aliquots w e r e w i t h d r a w n and dirictly assaYed for t r i b u t y r i n a s e activity; the values o b t a i n e d at zero time in the control e x p e r i m e n t s were taken as 100%. The e q u i v a l e n c e p o i n t was found to be 0.i ml of i m m u n o s e r u m per I00 units of pig p a n c r e a t i c lipase.

the i n h i b i t i o n with E600 under conditions lipase remains by E 6 0 0 o c c u r s

fully active

through a p s e u d o - f i r s t

life at 30°C and pH 8.0 of 130 min. concentration

o r d e r rate kinetic and i0 min.

of 0.1 m M and 1 m M r e s p e c t i v e l y ) .

N-ethyl-maleimide, tration)

where the p a n c r e a t i c

(16). I n h i b i t i o n of intestinal

DFP, FNa and D e o x y c h o l a t e

lipase

(half-

at an inhibitor Iodoacetamide,

(i m M final concen-

had no effect.

An o p t i m u m pH was found at pH 7.2 and at pH 8.2 w h e n using palmityl-CoA different gastric

and t r i b u t y r i n

and lingual

lipases.

in Table III. A l t h o Q g h acyl-thioesters and m e d i u m - c h a i n triesters

as substrate

respectively,

from the o p t i m u m pH of a c t i v i t y The substrate

very

around 5 found for specificity

is given

the enzyme is active on long chain fatty

above C12,

it catalyzes

triglycerides

such as t r i o l e i n

the h y d r o l y s i s

preferentially.

Both i n s o l u b l e

and soluble m o n o e s t e r s

95

of short-

such as ethyl

Vol. 65, No. 1 , 1 9 7 5

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C)

LO

0.4

C-4

~

~

LID

',...0

0

O0

f'~

~

z

Z O

w u z O

O

u cO W I---

m E

Q

w

O 0

I-GO

0

~

J

oo I

0

~

~

J

96

J

U

~

~

~

d

0

Vol. 65, No. 1, 1975

acetate

are c l e a v e d

positional f r o m pig olein

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by the p u r i f i e d

specificity

is g i v e n

and i n a c t i v e

l-monoolein

is g r e a t e r enzyme

pancreatic

for the

with

and

The

levels

of

active

the a c t i v i t y lipase

than

since

were

lipases

on l-monotoward for the

400

intestinal

as substrate)

of the

intestinal

are

30 fold)

of i n t e s t i n a l

of m a g n i t u d e

6000 m U / m g

with

Units lipase

used

respec-

bring

direct

evidence

from the p a n c r e a t i c and p o s i t i o n a l ferentiate Di N e l l a

The

di-

work).

from human

Although

could

described

lipase

are v e r y

lipase

is d i f f e r e n t

the studies

of s u b s t r a t e

III and

allow

lipase

IV)

to dif-

from that d e s c r i b e d

homogenates,

which

is c o n c e n t r a t e d

sensitive

is e q u a l l y

enzyme

lipids

in p a t i e n t s

ferent

origins

congenital

affected

(7-9).

(G. Serrero

is unclear.

et al., cells

A possible

by

active

by p a n c r e a t i c

of p a n c r e a t i c

lipase

sub-

partially

unpublished

of intestine,

of n e u t r a l

deficiencies

for i n s t a n c e

tip

role in human,

in the a b s o r p t i o n

It is known

absence

lysis w i t h

and has b e e n

in the a b s o r p t i v e

be an i n v o l v m e n t

in the villus

to s p o n t a n e o u s

from the cells

intestine

present

the role of this if any,

(Tables

(2) on crude

loss of a c t i v i t y

purified

Moreover

as

by E600,

and t r i g l y c e r i d e s .

intestinal

which

sequent

enzyme.

as well

pH and of i n h i b i t i o n

that the i n t e s t i n a l

specificities

et al.

on mono-,

are in the

(from ii00 to

of i m m u n o p r e c i p i t a t i o n ,

at acidic

the h i t h e r t o

activity

specie

as substrate).

In pig the e x p e r i m e n t s of d e s a c t i v a t i o n

lipase

for the d i f f e r e n t

tributyrin

those

Table

However

Units

tributyrin

same o r d e r

with

and

enzymes

intestinal

31

A comparison

in the assays.

DISCUSSION

cells

Both

(approximately

lipase

(as d e t e r m i n e d tively

pancreatic

IV.

on 2-monoolein.

pancreatic of

between

in T a b l e

enzyme.

that

absorb

of dif-

infants 30 to 50%

I I I : S u b s t r a t e s p e c i f i c i t y of p u r i f i e d pig i n t e s t i n a l lipase The i n t e s t i n a l rates of h y d r o l y s i s are r e l a t i v e to that of t r i o l e i n taken as 100% (21800mU/mg). The h y d r o l y s i s of a c y l - C o A was f o l l o w e d at pH 7.8 and 37°C, and that of the d i f f e r e n t esters at pH 8.5 and 25°C. T r i g l y c e r i d e s (triacetin excepted) and e m u l s i f i e d m o n o e s t e r s w e r e a s s a y e d in the p r e s e n c e of M e t h o c e l l (ii), soluble m o n o e s t e r s , p h o s p h o l i p i d s and T w e e n 20 in the a b s e n c e of detergent. The h y d r o l y s i s of l - m o n o o l e i n w a s f o l l o w e d t i t r i m e t r i c a l l y at pH 8.5 and 25°C in the p r e s e n c e of (final c o n c e n t r a t i o n s ) : d e o x y c h o l a t e 0.75%; CaCl 2, 0.4 m M and NaCI, 150 mM.

97

VoI. 65, No. 1,197:5

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ENZYME

SUBSTRATE

I-MONOOLEIN

2-MONOOLEIN

INCUBATION TIME (MIN,)

2

5

i0

15

PANCREATIC LIPASE

4,4

6,7

12,5

25

INTESTINAL LIPASE

5,5

17

25

37,5

PANCREATIC LIPASE

0

0

0

0

INTESTINAL LIPASE

0

0

0

0

Table IV: Comparison of positional specificity of intestinal and pancreatic lipases The incubation media (25°C; 0.5 ml total volume) contained (final concentrations) : potassium phosphate buffer 50 mM pH 6.0, sodium taurocholate 1 mM, monoolein 25 mM, and 400 Units of homogeneous pig pancreatic lipase or 31 Units of 150 fold purified intestinal lipase (as measured with tributyrin as substrate). At the times indicated, 0.i ml aliq~ots were remove~ and unhydrolyzed monoolein was extracted according to Bligh a~d Dyer (17). Glycerol was assayed in the aqueous phase after periodate oxidation and titration of the liberated formaldehyde by chromotropic acid (18). Control experiments were performed in the absence of enzyme; under these conditions no chemical isomerization of 2-monoolein occured (18) and no glycerol was released, but the monoolein still remaining in the aqueous phase (4% at all times) was substracted from the reported values. The results (from duplicate experiments) are expressed in percentage of hydrolysis of the substrate initially present.

of ingested fat. The presence of a gastric lipase in adult man, with an acidic optimum pH, has been described and characterized by Cohen et al.

(6); this lipase is active against short- and

medium-chain triglycerides. lipase,

I% is proposed that, besides gastric

the intestinal lipase could also contribute to the

hydrolysis of these neutral lipids.

98

In man the quantitative

Vol. 65, No. 1, 1975

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

contribution of gastric and intestinal lipases is difficult to evaluate as compare to pancreatic lipase

(9). In rat r e c e n t

experiments indicate that the intestinal lipase may represent up to 20% of the total lipase activity present in the intestinal contents. for man,

Thus,

if the situation for rat could be extrapolated

the contribution of the intestinal

lipase to the ab-

sorption of neutral lipids could become significant, in pathological

particularly

situations.

ACKNOWLEDGMENTS The authors wish to thank Dr. M. Balerna for help concerning polyacrylamide gel electrophoresis. This work was supported by grants from the "D~l~gation G~n~rale ~ la Recherche Scientifique" (Convention n ° 73-7-1205), from the "Fondation pour la Recherche M~dicale Franqaise) and from the "Centre National pour la Recherche Scientifique" (Laboratoire Associ~ 201).

R E F E R E N C E S

i- Desnuelle,P. (1972) in The Enzymes (Boyer,P.ED), vol. 7 575-616, Academic Press, New York and London. 2- Di NelIa,R.R., Meng,H.C. and Park,C.R. (1960), J. Biol. Chem. 235, 3076-3081. 3- Senior,J.R. and Isselbacher,K.J. (1963), J. Clin. Invest. 42, 187-195. 4- Pope,J.L., Mc Pherson,J.C. and Tidwell,H.C. (1966), J. Biol. Chem. 241, 2306-2310. 5- Hamosh,M. and Scow,R.O. (1973), J. Clin. Invest. 52, 88-95. 6- Cohen,M., Reginald,G.H., Morgan,M.B. and Hofman,A.F. (1971), Gastroenterology 60, 1-15. 7- Fernandez,J., Van de Kamer,J.H. and Weijers,H.A. (1962), J. Clin. Invest. 41, 488-495. 8- Sheldon,W. (1964), Arch. Dis. Child. 39, 268-271. 9- Figarella,C., N~gri,G.A. and Sarles,H. (1972), Biochim. Biophys. Acta 280, 205-210. i0- S~m~riva,M., Benzonana,G. and Desnuelle,P. (196~), Biochim. Biophys. Acta 144, 703-709. ii- S~m~riva,M., Dufour,C. and Desnuelle,P. (1971) Biochemistry I0, 2143-2149. 12- Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951), J. Biol. Chem. 193, 265-275. 13- Maurer,H.R. (1971) in Disc Electrophoresis and related techniques of polyacrylamide gel electrophoresis, p. 44, W. de Gruyter, Berlin and New York. 14- Ailhaud,G.P., Vagelos,P.R. and Goldfine,H. (1967) J. Biol. Chem. 242, 4459-4465. 15- Verger,R., DeHaas,G.H., Sarda,L. and Desnuelle,P. (1969) Biochim. Biophys. Acta 188, 272-282. 16- Mayli~,M.F., Charles,M. and Desnuelle,P. (1972) Biochim. Biophys. Acta 276, 162-175. 17- Bligh, E. and Dyer,W. (1959) Canad. J. Biochem. 37, 911 18- Ailhaud,G., Samuel,D., Lazdunski,M. and Desnuelle,P. (1964) Biochim. Biophys. Acta 84, 643-664. 99

Characterization and partial purification of an intestinal lipase.

Vol. 65, No. 1, 1975 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS CHARACTERIZATION AND PARTIAL PURIFICATION OF AN I N T E S T I N A L LIP...
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