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
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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Fig.
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
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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