Vol. 188, No. 3, 1992 November
BIOCHEMICAL
Molecular
cloning
and characterization
A. Aleman-Gomez+,
Jose
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 964-971
16, 1992
Geriatric Division Received
Niall,
Research of Geriatric
September
S. Colwell",
Education Medicine
21,
of rabbit pancreatic T.
and Clinical St. Louis
Sasser,
Center, University,
triglyceride and Vijaya
lipase” B. Kumar@
VA Medical Center and St. Louis, MO 63125
1992
SUMMARY: Pancreatic lipase (triacylgycerol acylhydrolase, EC 3.1.1.3) has been cloned from a gtll cDNA library made from poly Af RNA of adult rabbit Pancreatic lipase (PL) assits the absorption of dietary triglycerides pancreas. by hydrolyzing them at 1 and 3 positions to free fatty acids and 2monoacylglycerol in the presence of bile acids and colipase in the intestinal Since rabbits are classically used for the study of the diet induced lumen (1). changes in the lipid metabolism, as a prelude to studying the diet and age dependent changes in the expression of this enzyme, a full length PL cDNA clone was obtained from its pancreas. The coding region of rabbit pancreatic lipase cDNA consists of 1407 base pairs contained in an open reading frame encoding 469 Northern blot amino acids including the 16 that constitute the signal peptide. When rabbit enzyme is compared to other analysis revealed a band around 1.5 kb. an over all homology of 70-80% was observed at the nucleotide level. species, in the amino acid sequence and composition is also apparent High homology between rabbit and other species like dog (65%), pig (76%) and rat (63%). The regions of Highest homology is found to be around active-site serine. sites of interaction of lipase homology with other species may help to define with co-lipase. 0 1992 Academic PIXSS, Inc.
Among
the
the
digestive
principal
enzymes lipolytic
triglycerides
at
brush
border
insoluble
the
membranes
both
duodenal
lingual lumen,
of
enterocytes.
substrates
compared and
of Cardiology, University School
@To whom correspondence 0006-291X/92 Copyright All rights
should
assists
the
hydrolysis
more
polar,
enzyme
the It
to
gastric
*Sequence data from this article have GenBank Data Libraries under Accession pancreatic lipase. +Division Washington
by
helps
the
pancreatic
secreted
into
hydrolysis
lipase
and
It
3 positions
Such
emulsified
Although
enzyme.
1 and
monoacylglycerides.
synthesized
been No.
soluble
be addressed.
$4.00 964
of fatty
of
ones
activities
affinity
St. MO
Louis 63110.
and
2-
across for
(2). are
for
PL is
long-chain
acids
glycerides
also
efficient
deposited with EMBL/ M99365 for rabbit of Louis,
pancreas,
considerable
indispensable
Jewish hospital of Medicine, St.
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
transport
lipase is
free
shows
the
the
present digestion
in
Vol.
188,
of dietary and
Accumulation particles
which
inhibits
is
Colipase
is adsorbed
colipase
allows
spectral
analysis,
function
relationship
research
in this
cloned
characterized
studies
of
pancreatic
area (12,
gtll
exogenous
at
binding
reported of
interface
the
lipase
another
interfaces
(6).
acids
enzyme (7-11).
the
is
lipase
activity protein,
chemical
still
(3).
of emulsified
and binding
Using amino
substitution
the
to
of fats
(4,
5).
colipase. of lipase
modifications
to and
or regions
of
However,
structure-
lacking
despite
lipase
intensive
13).
to understand
the
salts
covered
to the substrate
site
we have undertaken from
by its
active
the
and abolishes
to amphiphile
several
may constitute
In order
countered
access
by
such as bile adsorption
RESEARCH COMMUNICATIONS
is shown to cause malabsorption
overcome
may be
lipase
AND BIOPHYSICAL
deficiency
of amphiphiles
Such an inhibition
level
Its
glycerides.
steatorrhea
that
BIOCHEMICAL
No. 3, 1992
the mechanism of to obtain
library
by sequencing,
total
constructed northern
MATERIALS
action
of
cDNA structure with
analysis
lipase
at
the molecular
of the enzyme.
mRNA from and comparison
rabbit
cDNA is
pancreas
to other
and
species.
AND METHODS
Isolation
of pancreatic RNA: Total RNA was obtained from 1.5 g of lyophilized rabbit pancreas by guanidinium isothiocyanate (Bethesda Research Labs., MD) method of Chirgwin et al (14). Approximately 5.3 mg of total RNA was obtained by this method. 4 mg of this RNA was subjected to two rounds of oligo-dT cellulose (Sigma chemical Co., St. Louis) chromatography (15) which yielded 22 ug of urified poly A' RNA as assessed by absorbance at 260 nm. 5 ug of the poly A P RNA was used to construct cDNA library. of cDNA library: cDNA library was generated by oligo dT priming followed by first strand synthesis using reverse trascriptase and the second strand synthesis using E. coli DNA polymerase supplied by Pharmacia biochelaicals CA. NotI/EcoRI linkers were ligated to the double stranded DNA and size selected using 1% agarose gel in Tris-acetate buffer. Size selected DNA was then ligated to lambda gtll arms supplied by Stratagene company and packaged using packaging extracts of Gigapack gold extracts (Vector Cloning Systems, CA). The library was titrated using Y 1090 r- bacterial cells. Construction
Isolftion of lipase specific clones: cDNA library was plated at a density of 30 x 10 plaques/l50 mm plate. Six such plates were lifted in duplicate onto nitro cellulose filters (Mid West Scientific, St. Louis), denatured, neutralized and baked at 80' for two hours. Screening of the filters was accomplished by an end labeled synthetic oligo nucleotide, (5'ACGAAAGACTCGGCTGCTTCAGTGAT), derived from :~~eJipw;;h s="s~~~T,'"e~h~ar~~~r ,'f') bol~u;l~~;f~ ng~~ide,p;-;;~
biochemicals) were plaque
and purified by Sephadex G-25 spin column. Six positive clones purified, DNA was isolated and the insert sizes were estimated. A 965
Vol.
188, No. 3, 1992
BIOCHEMICAL
1.5 kb insert from further analysis.
one of
the
AND BIOPHYSICAL
clones
RESEARCH COMMUNICATIONS
was subcloned
into
pUC 19 vector
for
Southern blotting: Southern blotting to analyze clones was essentially done as described before
the DNA inserts of the positive (16). After digesting the DNA with specified restriction enzyme, the digest has been subjected to electrophoresis on 1% agarose gel in Tris-acetate buffer, denatured, neutralized, transferred to a nylon membrane and probed with end labeled probe at 56' in 6 x SSC, 5 x Denhardt and lOOug/ml tRNA. After hybridization, the filter was washed with 2 x SSC at 56' and exposed to X-Omat XR-5 autoradiographic film for 24 hrs.
blot analysis: Twenty micrograms of total RNA was subjected to electrophoresis on a 0.9% agarose gel under denaturing conditions (16). After electrophoresis, RNA was de-purinated using O.lN HCl, neutralized and transferred to nylon membrane as described before (16). Full length cDNA for lipase was excised from the vector (pUC 19), random primer labeled using primer labeling kit supplied by Bohringer Mannheim, IN, according to the instructions of the manufacturer. Northern blot was hybridized with the labeled cDNA at 65' in 3 x SSC for 12 hours, washed at 56' with 0.2 x SSC and exposed to X-Omat XR 5 film for 12 hours to localize lipase specific mRNA.
Northern
Bidirectional sequencing of the double stranded DNA was done by Initial sequencing was done dideosynucleotide chain termination method (17). using universal forward and reverse primers. Total sequence was completed by synthetic primers derived from the universal primers.
DNA sequencing:
RESULTS AND DISCUSSION Phage DNA was isolated
from six positive
blotted
onto nitrocellulose
results
showed that
However,
paper
the
inserts
RTGL-5K
varied
(18) and digested
was
subcloned
into
with
to the end labeled
the clones
hybridized
bacterial
size
plasmid
The
to the primer.
insert
pUC 19
Eco Rl,
probe.
from 0.8 kb to 1.5 kb (data
RTGL-4K and RTGL-5K showed the largest
bidirectional
not shown).
of 1.5 kb each.
and
subjected
to
in
1.
sequencing.
The complete
nucleotide
and amino acid
There is a 1407 nucleotide 16 constitute
the leader
open reading peptide.
assigned
based on the
in rabbit
is modified
to
preceding
ATG.
immediately lipase
and hybridized from all
the size of the inserts
Two clones,
clones
to that
consensus
from other
is
frame encoding
Tentative sequence,
CCACCATGwith A comparison species
sequence
protein
CCACGATGreported a change of
966
figure
469 amino acids start
from
‘G'
the nucleotide
showed 83% homology
66% to rat.
presented
of which
codon 'ATG' could earlier to
'C'
(19) at
be
which
the base
sequence of
rabbit
to human, 70% to dog and
Vol.
188,
BIOCHEMICAL
No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
10 20 30 40 50 AWCOCOOCCOCAA~CCACOAT~~OCT~AC~CCT~~CTGWGGCCOTOGCAOGA ~tWo~T~l~~.ProL~n~uL~lyAl~V~lAlSGly
60
80 90 100 110 120 TCSMTCCCCTWTCMCCWhUAAA CTGQAAOTCTGCTACGAGAGACTCOLsuOluV~1Cy~y~lu*~~Ol~P~ly~~I1eProTr~.r0ly0l~hrLauOluA 150
160
70
130
140
190
200
210
260
270
260
340
360
410
420
IS0
490
550
660
600 t 610 620 AGGGTTG0ATCCAOCTGAACCTTACTTTCAGGGTAC
630
100
170
GACC~AOCAW~COC~ACCCCCAAAGAT~CMCAWCWWC~CCTCTACACTAATGAOAA r$Pro~SSrThr~roQ~~rProLy~~V~lA~QThrA~QPh~~U~~Ty~ThrA~~QlUAa 220
230
240
250
CCCAMCU~CAAOu*TC~QWAGffiWA~MCCA~A~A~TCCAATWCAOAACAGATAOQ
nProA~nPh~l~luIl.S.rAl~pAl~S~rThrIlaArgOlySar~nPh~AArQfh~A6pArg 290 300 310 320 AAAACTCGWTCATTATCCACGGATTCAC0WCAWWAOMQAGAACTGOCTGT~MCTTQ?GCGAGA
330
LyBfhrArgPh~Il~Il~R1,OlyPh~~~A~L~QlyOl~OluA~nTrpLeUS~~A~nLeUCysOlUA 360 370 300 390 ACCZCrWCAGGT0QAMCCOMCTGCA~T~TOOACTGGAAG~GCTCCCWA~ACATACCC
400
snL~uPh~lnV~lGluThrV~l~n~Il~~V8lA~pT~pL~QlyOlySe~Arg~rThrTyrPr 430 440 450 460 470 OCAOGCCACOCAGAACATCCGGA~~GQQOCGQM~QGCGTACWAQWOOCACTCTACAGTCQTCG
oQlnC11~Thr01~~nIIwArgl:l~V~1QlyAl~O1uV~lAl~lytL~uV~lOlylhrL~uOlnScrSer 500 510 620 5303 CTOGCOTACTCTCCWCCAACA~CA~A~WCCACAQC~WG~ACW~C~OQGAQOTA~
540
L~~Ol~yrS~rPr~S~rA~nIl~Hi~V~lIl~lySi~S~rLeuOlyAl~Si~Al~Al~OlyQluV~lQ 570 580 GACGGAGOACCAACGOQACTAWGOACOAATCAC
590
lyArgArpThrA~nGlyThrIl~GlyA~gIl~Th~ly~~~pPrOAl~OlUPro~rPh~O~nOlyTh 640 650 660 670 ACCTGAAATAGTCCGGWQGATCCCAQWA~~CAA~TQGATGTAA7TCACACG~ATGCTGCTCCC
BBO
690
700
rProGIuIleV~lArgL~UA~pP~oS~rA~pAl~GlnPheV~lA~pV8l~l~Sl~ThrAspAl~Al~Pr~ 710 720 730 740 ATGG~CCCAACTTOG~~GOAA~AGCCAM~OTTQOTCACC?AGAWTCTWWAAATGGAOGAA ~etV~1Pto~n~nQlyPh~Ol~tSe~lnThrV~lQ~ySl~L~~~pPhePh~Pr~A~nQlyQlyL
750
760
770
780 790 000 Ill0 AAGAAATOCCCGQAMfCA~MGAAT~~~~AQA~WGACATAAATGGQQTCTGQGAAOGAAC
620
830
840
970
980
Y~G~U~tP~OOlYC~GJQL~A~~V~1~~~r(fl~IlSV~1~pIl~A~~OlyValTrpOluGlyTh
920 930 940 950 Q0CmO-~TCW01CCCrCCTACACWC~A~CAMCAAQTGCTTCCCCTGTTCCAAT~
660
OlYPheAl~GlYPh~Se~CAl~~~~rTh~Al~PheS~rAl~~nLy~Cy~PheP~~Cy~S~~A~~Q 990
1000
1010
1020 1030 1040 AO0AAAACAGATGOAQTOG0CCAGACATTCTA
GATWCCGCAGAMGOACATTATOCTOATAWWTTCT
1050
1yCysPr~Jn~tGlyS~slyrAl~S~irgPh~~rA~L~ThrA~~1yV~lGlyGlnThrPheTy 1060 1070 1oeo 1090 TCTCMCACTGOTGAWCCA~AA~OCTCOTT~GOTATCAG~~CTO~ACACT~CT~GAAOG
1100
1110
1120
11eo
1190
rLe~~oTh~lyA~pSsrS~~A~aPheAl8A~T~A~rQlnV~lAl~V~lThrL~~Se~QlyA~g 1130 1140 1150 1160 1170 AWGTTACAOOACATGTGCTGGTGTCTWGTATGGAAGTAAAWGAACTCTMOCAOTAC~AAATTTTCA
ArgV~lThrOlyHisV~l~V~lS~rL~u~l~~rL~Oly~nSsrLyyrGluIlcPhcT 1200 1210 COOOCC-AAACCAGGTGATACTCAWTAAATG
1220
1230 1240 1250 AGTT’NACTCTOATGT00ATGTCGGOOATGTGCA
1260
hrOlyLcuInnL~PrOlyA~Th~Si~~~D~lUPh~A~pSe~AapV~lA~pValOlyA~pV~lGl 1290
1270 1210 GA~WMO~OmOCTATMCAA~ATCMCCCTIC
1300
,310
1320
1330
1390
1400
nLyV8lLysPhtV~lTrp~yrA9nA8nV~lIle~nProThrLcuProLysV~lGlyAlaScrGlnIle 1340 1350 1360 1370 ACAOMGAACMAAMATWAA~?~CA~~OCAQCACAGACACCOT~AGGGAGGACATCCTGC
13so
ThrV.l01t1QlnA~~.pGlyA~gV~lPh~Ly8PhcCyshrV~lA~gQluAspIlcLauL ,410 1420 1430 TCACCCTCACCCCGTGWAGGAGGCTCCTQQAAATQACCACC cuThrLeuThrProCy&nd
1440
Nucleotide and amino acid sequence of rabbit psncreatic lipase. nucleotide sequence of RTGL-5 was obtained as described in the text. histidine and aspartste that constitute the active triad are indicated Serfne, by asterisks. Putative glycosylation site and the primer wed for isotatfon of the positive clones from the cDNA library are over and underlined respectively. E-E?
A comparison
(20) 1 canine homology
between
of (21)
the and rabbit
amino
acid
porcine enzyme
sequence (22)
is
and
other
of
rabbit
shown species
in
lipase figure is
with 2.
observed.
human 70-85% There
(1))
rat
over
all
is
81%
Vol.
188, No. 3, 1992
BIOCHEMICAL
MLLLWALPIXLLGAVAG P TS T TVSF AQ VSI TIA F AKA ____________.____
AND BIOPHYSICAL
Rabbit HUUlall Rat C.%lil-le Porcine
Signal
RESEARCH COMMUNICATIONS
peptide
LEVCYERLGCFGNRIPWSGGTLERPFSTLPSTPKDVNTRFLLYTNENPNNF9EISADAST K SDDS I X LHI ws VA S S K DN SDAE A X AI LKL WS EKI TA TLQLSDPL L K QI SDAE A,X AI LKV WS ERIG K TLLPSDPS S FP SDDA A IVXQRPLKI PDXK D QQ Y LV P
60
IRGXSNFRTDRKTRFIIHGFTDKGEENWLSNLCENLFQVETVNCICVDUKGGSRTTYPQA i AVK K s SX KN GT TI A i WDHKMK E K QGT QVA Q K. T IN LDMKHK E K QST TIEA TNX RI% I D IK KS GT * * TQNIRIVGAEVAYLVETLQSSLGYSPSNIH~GEVGRRT~IGRITGLDP S F EF AF V A ANVV Q QHIDI VKNYS KV L V A ANVV V Q QMLSM SANYS AS PXL Q'JQL S F EV K V S A E AEPYFQGTPEIVRLDPSDAQNDVIHTDAAPWPNLGFCHSPPGC C L K c I V VANE E D G LI F TN MS T D V AS E LI F T QM C L K PI1
120
180
240 is E Q
QKNVLSQIVDINCVVEGTRDNACN;?LRSYKWADSIVNPNGFAGFSCASYTAFSANKCF K I DI A T D P NVT K A DIS A LE L D AYP KD ES SE L D SYP K A NLD I R ES I DI i D PD NVT
300
PCXSNGCPQMGHYADRFSRD~GQT~TGDSSNFARWRYQVAVTLSGRRVTGHVLV PG YPG ND K D A KS KK KYAG SGDEP K F D EAK RSLI KM PDP K AV SDET KYF G SI KA PDQ PE PG N SVF A KS lx
360 Q:K
QAK I
SLYGSKGNSKQYEI~GWPGDTHLNEFDSDVDVGDVQKVKFVWYNNXVINPTLPKVGA KT DS S LM I x R FN AF TR D R II DA SS AKL TIEN L N QX SF R AF THFN KI S S AKL TIE LN XV F FNE R YKTQDN SD E L I N R SQITVEQNDGRVXFKFCSTDTVREDILLTLTPCU KI TVKQXN PE EV L AK I QKGEEKTEYN EE ET AK QKGEEKTVHS ES V K R K XYD QE EV N
420
Rabbit Human Rat Canine Porcine
U ETSATVU U U
Figure 2. Comparison of emino acid sequence of rabbit pancreatic lipase with that of different species. Amino acid sequence of PLs from rat (ZO), canine (21) and porcine (22) are aligned with the sequence of rabbit PL. Spaces between the amino acids indicate the conservation. X- deletion. Asterisks indicate amino acids involved in the active site. Putative glycosylation site is overlined. Conserved possible lipid binding site is boxed.
conservation to
canine
in
signal
lipase.
conserved
in
the
sequence
IGHSLG,
including
RPL.
similar
the
to
other
peptide
The rabbit
when
catalytic
triad,
at
positions
a probable
lipid
Only
one
species
putative except
compared
153,
serine, 178 and
binding
site,
N-glycosylation the
dog PL which 968
to
human
and only
aspartate
and
histidine
266 respectively. is site does
conserved could not
38% compared
Hydrophobic in
all
be detected
have
are
this
site
species in (1).
RPL
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
.6
Figure 3. Northern blot RNA from rabbit pancreas transferred to nitrocellulose as described in the text.
blot
Northern
corresponding
to
broad extending of
multiple
reported Table
analysis the
size
of
the pig
of
molecule. Table
Amino number Signal
3)
full
length
1.
acid
seq.
lipase. 2opg of on 0.9% agarose random primer labeled RPL mRNA.
revealed
comparable
a
cDNA.
size
broad
band
at
The band obtained
This
suggests
coding
for
possible
several
total gel, RPL
1.5 is
kb,
rather
existence
isoenzymes
as
(23).
1 shows the percent
the lipase
(Fig.
from 1.4 kb to 1.6 kb in size.
RNA species
for
analysis of rabbit pancreatic was subjected to electrophoresis membrane and probed with Arrow indicates the hybridized
conservation
of amino acids
Maximum conservation Percentage
conservation regions Rabbit human
between of
of Rabbit rat
amino RPL
from various
species acids
from
Rabbit: canine
exists various
Rabbit porcine
81
63
38
l-60
73
52
50
53
61-120
82
68
67
60
121-180
87
67
58
85
180-240
80
73
78
88
240-300
82
72
73
83
301-420
80
46
66
72
421-432
65
44
53
68
969
not
available
regions
between
of
amino
Vol.
188,
No. 3, 1992
to 300.
acids
120
Least
homology
the
This
is
cholesterol
these
BIOCHEMICAL region
observed
termini
carries
at
carboxyl
the
not
very
of
acidic,
both
C-and
esterase
are
AND BIOPHYSICAL active-site
N-termini
(24)
it
important
RESEARCH COMMUNICATIONS
is
serine
of
the
molecule.
probable
for
that
the
enzyme
amino
acids
and histidine. As shown
the
in
amino
acids
or
co-factor
activity
in
binding. A comparison rat
pig,
and
hydrophobic from
amino
six
in
species
Results
enzyme
in both
active-site
serine
modification
(25,
265
177 respectively. the
triad,
activation
of
lipase
(28,
Such
this In
lipase
Experiments
important
of for
other
in
the
of
of
lysines
canine.
dog,
number
of
varied
As lysines
may
heparin
may
to intestinal
the the
by The
may not
site co-factor
functional
are
its high
may not
activity
the
loop,
during
the
structure of
be
vital
of
the
the
of
primary with
to
of enzyme
the
of at
function to
locate
amino
acids
these It
is
interfacial the
C-terminus activity of
other the of
of rabbit
species. C-terminus
the
the that
enzyme. sites
of
may not
be
are underway.
Acknowledgment: Authors wish to acknowledge Drs. John Morley, and Terry Zenser for their help in conducting this investigation. Assistance provided by Mrs. Padmaja Satram and the secretarial Cole and Carol McCleary is greatly appreciated.
970
that
structure that
of
positions
(27).
functional
dissimilarity
mutagenesis stretches
structure
for
site
shown triad
the
chemical
RPL at
have
moves
by
active
a catalytic
the
presented
directed and
in
be necessary
regions
in
studies
a surface
the
is
retained
shown
in
conserved
lipase
It
been
involved
forming
primary
pancreatic
levels. has
also
change
we have
rabbit
acid
acids
serine
29).
region
that
crystallographic
covered
revealed this
amino
aspartate-176
conclusion
involving
interaction
lipolysis
and compared
that
show
for
portion
a comparison
suggesting
in
of binding
importance
active-site
this
pancreatic
thirty
amino
X-ray
that
protein.
as
extent
enzymes
The number
and
The
suggested
the
as many
the
conservation
nucleotide
and
with
that
to
investigation
at
26).
histidine-263
suggests
of
and histidines.
(24),
this
whose
lipases
bond
degree
in
to species.
conserved
hydrogen
a high
binding
presented
and
and neutral
cysteines
rabbit
in heparin
from
highly
shows
acids,
the
participate vary
human
basic
Steve help
Giddings Technical of Carolyn
Vol.
188,rNo.
3,
1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES Lowe,
M.E.,
Rosenblum,
J.L.
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Adv. Lee. G.
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6. 7.
Borgstrom, B. and Erlanson, C. (1973) Eur. J. Biochem. Maylie, M.F., Charles, M., Gache, C. and Desnulle, Biophys. Acta 229, 286-289. De Garo, J.D., Rouimi, P. and Rovery, M. (1986) Eur.
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15. 16. 17. 18. 19. 20. 21. 22. 23. 24 25. 26. 27.
28 29.
Chapus, C. and Semeriva, M. (1976) Biochemistry 15, 4988-4991. Guidoni, A., Benkouka, F., De Caro, J and Rovery, M. (1981) Biochim. Biophys. Acta @O, 148-150. Semeriva, M?, Dufour, C. and Desnulle, P. (1971) Biochemistry, l& 21432419. Granon, S. Rahmani-Jourdheuil, D., Desnulle, P. and Chapus, C. (1981) Biochem. Biophys. Res. Commun. 2, 114-119. Semeriva, M. and Desnulle, P. (1979) Adv. Enzymol. &&, 319-370. Borgstrom, B and Erlanson-Albersson, C. (1984) in Lipases (Borgstrom, B., and Brockman, H.L. eds) pp 152-183. Elsever-N.Y. Chirgwin, J.M., Przybyla, A.E., McDonald, R.J. and Rutter, W.J. (1979) Biochemistry l8, 5294-5299. Nat1 Acad Sci, U.S.A @, 1408-1412. Aviv, H. and Leder, P. (1972) Proc. Maniatis, T., Fritsch, F.F. and Sambrook, J. (1982) Molecular Cloning : A Laboratory Manual. Cold Spring Harnbor Laboratory. Cold Spring Harbor, NY. Sanger, G. Nickelen, S. and Coulson, A.R. (1977) Proc. Nat1 Acad. Sci. U.S.A Helms, C., Graham', M.Y., Dutchik, J.E. and Olson, M.V. (1985) DNA, 4, 3949. Kozak, M. 1980) Nucleic Acids Res. 8, 127-142. Wicker-Planquart, C. and Puigserver, A. (1992) FEBS, 296, 61-66. Puigserver, A. and Scheele, G. (1986) Kerfelec, B., LaRorge, K.S., Pancreas, 5, 430-437. Decaro, J., Boudouard, M., Bonical, J., Guidoni, A., Denuelle, D. and Rovery, M. (1981) Biochim. Biophys. Acta, 671, 129-138. Bianchetta, J. Bidaud, J. Guidoni, A., Bonicel, J. and Rovery, M. (1979) Eur. J. Biochem. 97, 395-405. Colwell, N.S., Aleman-Gomez, A. and Kumar, B.V. (1992) Biochim. Biophys Acta, Submitted. Guidoni, A., Benkouka, G., Decaro, J. and Rovery, M. (1981) Biochim. Biophys Acta, 660, M-150. Maylie, M.G., Charles, M. and Desnulle, P. (1972) Biochim. Biophys Acta, 276, 162-175. Moreau, H. Moulin, A., Gargouri, Y., Noel, J.P. and Verger, R. (1991) Biochemisty, 30, 1037-1041. Winkeler, F.K., D'Arcy, A. and Hunzike (1990), Nature, 343, 771-774. Brady, L., Brzozowski, A.M., Derewenda, Z.S.,. Dodson, E., Dodson, G., Tolley, S. and Trukenburg, J. (1990) Nature, 343, 767-770.
971
BIOCHEMICAL
Molecular
cloning
and characterization
A. Aleman-Gomez+,
Jose
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 964-971
16, 1992
Geriatric Division Received
Niall,
Research of Geriatric
September
S. Colwell",
Education Medicine
21,
of rabbit pancreatic T.
and Clinical St. Louis
Sasser,
Center, University,
triglyceride and Vijaya
lipase” B. Kumar@
VA Medical Center and St. Louis, MO 63125
1992
SUMMARY: Pancreatic lipase (triacylgycerol acylhydrolase, EC 3.1.1.3) has been cloned from a gtll cDNA library made from poly Af RNA of adult rabbit Pancreatic lipase (PL) assits the absorption of dietary triglycerides pancreas. by hydrolyzing them at 1 and 3 positions to free fatty acids and 2monoacylglycerol in the presence of bile acids and colipase in the intestinal Since rabbits are classically used for the study of the diet induced lumen (1). changes in the lipid metabolism, as a prelude to studying the diet and age dependent changes in the expression of this enzyme, a full length PL cDNA clone was obtained from its pancreas. The coding region of rabbit pancreatic lipase cDNA consists of 1407 base pairs contained in an open reading frame encoding 469 Northern blot amino acids including the 16 that constitute the signal peptide. When rabbit enzyme is compared to other analysis revealed a band around 1.5 kb. an over all homology of 70-80% was observed at the nucleotide level. species, in the amino acid sequence and composition is also apparent High homology between rabbit and other species like dog (65%), pig (76%) and rat (63%). The regions of Highest homology is found to be around active-site serine. sites of interaction of lipase homology with other species may help to define with co-lipase. 0 1992 Academic PIXSS, Inc.
Among
the
the
digestive
principal
enzymes lipolytic
triglycerides
at
brush
border
insoluble
the
membranes
both
duodenal
lingual lumen,
of
enterocytes.
substrates
compared and
of Cardiology, University School
@To whom correspondence 0006-291X/92 Copyright All rights
should
assists
the
hydrolysis
more
polar,
enzyme
the It
to
gastric
*Sequence data from this article have GenBank Data Libraries under Accession pancreatic lipase. +Division Washington
by
helps
the
pancreatic
secreted
into
hydrolysis
lipase
and
It
3 positions
Such
emulsified
Although
enzyme.
1 and
monoacylglycerides.
synthesized
been No.
soluble
be addressed.
$4.00 964
of fatty
of
ones
activities
affinity
St. MO
Louis 63110.
and
2-
across for
(2). are
for
PL is
long-chain
acids
glycerides
also
efficient
deposited with EMBL/ M99365 for rabbit of Louis,
pancreas,
considerable
indispensable
Jewish hospital of Medicine, St.
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
transport
lipase is
free
shows
the
the
present digestion
in
Vol.
188,
of dietary and
Accumulation particles
which
inhibits
is
Colipase
is adsorbed
colipase
allows
spectral
analysis,
function
relationship
research
in this
cloned
characterized
studies
of
pancreatic
area (12,
gtll
exogenous
at
binding
reported of
interface
the
lipase
another
interfaces
(6).
acids
enzyme (7-11).
the
is
lipase
activity protein,
chemical
still
(3).
of emulsified
and binding
Using amino
substitution
the
to
of fats
(4,
5).
colipase. of lipase
modifications
to and
or regions
of
However,
structure-
lacking
despite
lipase
intensive
13).
to understand
the
salts
covered
to the substrate
site
we have undertaken from
by its
active
the
and abolishes
to amphiphile
several
may constitute
In order
countered
access
by
such as bile adsorption
RESEARCH COMMUNICATIONS
is shown to cause malabsorption
overcome
may be
lipase
AND BIOPHYSICAL
deficiency
of amphiphiles
Such an inhibition
level
Its
glycerides.
steatorrhea
that
BIOCHEMICAL
No. 3, 1992
the mechanism of to obtain
library
by sequencing,
total
constructed northern
MATERIALS
action
of
cDNA structure with
analysis
lipase
at
the molecular
of the enzyme.
mRNA from and comparison
rabbit
cDNA is
pancreas
to other
and
species.
AND METHODS
Isolation
of pancreatic RNA: Total RNA was obtained from 1.5 g of lyophilized rabbit pancreas by guanidinium isothiocyanate (Bethesda Research Labs., MD) method of Chirgwin et al (14). Approximately 5.3 mg of total RNA was obtained by this method. 4 mg of this RNA was subjected to two rounds of oligo-dT cellulose (Sigma chemical Co., St. Louis) chromatography (15) which yielded 22 ug of urified poly A' RNA as assessed by absorbance at 260 nm. 5 ug of the poly A P RNA was used to construct cDNA library. of cDNA library: cDNA library was generated by oligo dT priming followed by first strand synthesis using reverse trascriptase and the second strand synthesis using E. coli DNA polymerase supplied by Pharmacia biochelaicals CA. NotI/EcoRI linkers were ligated to the double stranded DNA and size selected using 1% agarose gel in Tris-acetate buffer. Size selected DNA was then ligated to lambda gtll arms supplied by Stratagene company and packaged using packaging extracts of Gigapack gold extracts (Vector Cloning Systems, CA). The library was titrated using Y 1090 r- bacterial cells. Construction
Isolftion of lipase specific clones: cDNA library was plated at a density of 30 x 10 plaques/l50 mm plate. Six such plates were lifted in duplicate onto nitro cellulose filters (Mid West Scientific, St. Louis), denatured, neutralized and baked at 80' for two hours. Screening of the filters was accomplished by an end labeled synthetic oligo nucleotide, (5'ACGAAAGACTCGGCTGCTTCAGTGAT), derived from :~~eJipw;;h s="s~~~T,'"e~h~ar~~~r ,'f') bol~u;l~~;f~ ng~~ide,p;-;;~
biochemicals) were plaque
and purified by Sephadex G-25 spin column. Six positive clones purified, DNA was isolated and the insert sizes were estimated. A 965
Vol.
188, No. 3, 1992
BIOCHEMICAL
1.5 kb insert from further analysis.
one of
the
AND BIOPHYSICAL
clones
RESEARCH COMMUNICATIONS
was subcloned
into
pUC 19 vector
for
Southern blotting: Southern blotting to analyze clones was essentially done as described before
the DNA inserts of the positive (16). After digesting the DNA with specified restriction enzyme, the digest has been subjected to electrophoresis on 1% agarose gel in Tris-acetate buffer, denatured, neutralized, transferred to a nylon membrane and probed with end labeled probe at 56' in 6 x SSC, 5 x Denhardt and lOOug/ml tRNA. After hybridization, the filter was washed with 2 x SSC at 56' and exposed to X-Omat XR-5 autoradiographic film for 24 hrs.
blot analysis: Twenty micrograms of total RNA was subjected to electrophoresis on a 0.9% agarose gel under denaturing conditions (16). After electrophoresis, RNA was de-purinated using O.lN HCl, neutralized and transferred to nylon membrane as described before (16). Full length cDNA for lipase was excised from the vector (pUC 19), random primer labeled using primer labeling kit supplied by Bohringer Mannheim, IN, according to the instructions of the manufacturer. Northern blot was hybridized with the labeled cDNA at 65' in 3 x SSC for 12 hours, washed at 56' with 0.2 x SSC and exposed to X-Omat XR 5 film for 12 hours to localize lipase specific mRNA.
Northern
Bidirectional sequencing of the double stranded DNA was done by Initial sequencing was done dideosynucleotide chain termination method (17). using universal forward and reverse primers. Total sequence was completed by synthetic primers derived from the universal primers.
DNA sequencing:
RESULTS AND DISCUSSION Phage DNA was isolated
from six positive
blotted
onto nitrocellulose
results
showed that
However,
paper
the
inserts
RTGL-5K
varied
(18) and digested
was
subcloned
into
with
to the end labeled
the clones
hybridized
bacterial
size
plasmid
The
to the primer.
insert
pUC 19
Eco Rl,
probe.
from 0.8 kb to 1.5 kb (data
RTGL-4K and RTGL-5K showed the largest
bidirectional
not shown).
of 1.5 kb each.
and
subjected
to
in
1.
sequencing.
The complete
nucleotide
and amino acid
There is a 1407 nucleotide 16 constitute
the leader
open reading peptide.
assigned
based on the
in rabbit
is modified
to
preceding
ATG.
immediately lipase
and hybridized from all
the size of the inserts
Two clones,
clones
to that
consensus
from other
is
frame encoding
Tentative sequence,
CCACCATGwith A comparison species
sequence
protein
CCACGATGreported a change of
966
figure
469 amino acids start
from
‘G'
the nucleotide
showed 83% homology
66% to rat.
presented
of which
codon 'ATG' could earlier to
'C'
(19) at
be
which
the base
sequence of
rabbit
to human, 70% to dog and
Vol.
188,
BIOCHEMICAL
No. 3, 1992
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
10 20 30 40 50 AWCOCOOCCOCAA~CCACOAT~~OCT~AC~CCT~~CTGWGGCCOTOGCAOGA ~tWo~T~l~~.ProL~n~uL~lyAl~V~lAlSGly
60
80 90 100 110 120 TCSMTCCCCTWTCMCCWhUAAA CTGQAAOTCTGCTACGAGAGACTCOLsuOluV~1Cy~y~lu*~~Ol~P~ly~~I1eProTr~.r0ly0l~hrLauOluA 150
160
70
130
140
190
200
210
260
270
260
340
360
410
420
IS0
490
550
660
600 t 610 620 AGGGTTG0ATCCAOCTGAACCTTACTTTCAGGGTAC
630
100
170
GACC~AOCAW~COC~ACCCCCAAAGAT~CMCAWCWWC~CCTCTACACTAATGAOAA r$Pro~SSrThr~roQ~~rProLy~~V~lA~QThrA~QPh~~U~~Ty~ThrA~~QlUAa 220
230
240
250
CCCAMCU~CAAOu*TC~QWAGffiWA~MCCA~A~A~TCCAATWCAOAACAGATAOQ
nProA~nPh~l~luIl.S.rAl~pAl~S~rThrIlaArgOlySar~nPh~AArQfh~A6pArg 290 300 310 320 AAAACTCGWTCATTATCCACGGATTCAC0WCAWWAOMQAGAACTGOCTGT~MCTTQ?GCGAGA
330
LyBfhrArgPh~Il~Il~R1,OlyPh~~~A~L~QlyOl~OluA~nTrpLeUS~~A~nLeUCysOlUA 360 370 300 390 ACCZCrWCAGGT0QAMCCOMCTGCA~T~TOOACTGGAAG~GCTCCCWA~ACATACCC
400
snL~uPh~lnV~lGluThrV~l~n~Il~~V8lA~pT~pL~QlyOlySe~Arg~rThrTyrPr 430 440 450 460 470 OCAOGCCACOCAGAACATCCGGA~~GQQOCGQM~QGCGTACWAQWOOCACTCTACAGTCQTCG
oQlnC11~Thr01~~nIIwArgl:l~V~1QlyAl~O1uV~lAl~lytL~uV~lOlylhrL~uOlnScrSer 500 510 620 5303 CTOGCOTACTCTCCWCCAACA~CA~A~WCCACAQC~WG~ACW~C~OQGAQOTA~
540
L~~Ol~yrS~rPr~S~rA~nIl~Hi~V~lIl~lySi~S~rLeuOlyAl~Si~Al~Al~OlyQluV~lQ 570 580 GACGGAGOACCAACGOQACTAWGOACOAATCAC
590
lyArgArpThrA~nGlyThrIl~GlyA~gIl~Th~ly~~~pPrOAl~OlUPro~rPh~O~nOlyTh 640 650 660 670 ACCTGAAATAGTCCGGWQGATCCCAQWA~~CAA~TQGATGTAA7TCACACG~ATGCTGCTCCC
BBO
690
700
rProGIuIleV~lArgL~UA~pP~oS~rA~pAl~GlnPheV~lA~pV8l~l~Sl~ThrAspAl~Al~Pr~ 710 720 730 740 ATGG~CCCAACTTOG~~GOAA~AGCCAM~OTTQOTCACC?AGAWTCTWWAAATGGAOGAA ~etV~1Pto~n~nQlyPh~Ol~tSe~lnThrV~lQ~ySl~L~~~pPhePh~Pr~A~nQlyQlyL
750
760
770
780 790 000 Ill0 AAGAAATOCCCGQAMfCA~MGAAT~~~~AQA~WGACATAAATGGQQTCTGQGAAOGAAC
620
830
840
970
980
Y~G~U~tP~OOlYC~GJQL~A~~V~1~~~r(fl~IlSV~1~pIl~A~~OlyValTrpOluGlyTh
920 930 940 950 Q0CmO-~TCW01CCCrCCTACACWC~A~CAMCAAQTGCTTCCCCTGTTCCAAT~
660
OlYPheAl~GlYPh~Se~CAl~~~~rTh~Al~PheS~rAl~~nLy~Cy~PheP~~Cy~S~~A~~Q 990
1000
1010
1020 1030 1040 AO0AAAACAGATGOAQTOG0CCAGACATTCTA
GATWCCGCAGAMGOACATTATOCTOATAWWTTCT
1050
1yCysPr~Jn~tGlyS~slyrAl~S~irgPh~~rA~L~ThrA~~1yV~lGlyGlnThrPheTy 1060 1070 1oeo 1090 TCTCMCACTGOTGAWCCA~AA~OCTCOTT~GOTATCAG~~CTO~ACACT~CT~GAAOG
1100
1110
1120
11eo
1190
rLe~~oTh~lyA~pSsrS~~A~aPheAl8A~T~A~rQlnV~lAl~V~lThrL~~Se~QlyA~g 1130 1140 1150 1160 1170 AWGTTACAOOACATGTGCTGGTGTCTWGTATGGAAGTAAAWGAACTCTMOCAOTAC~AAATTTTCA
ArgV~lThrOlyHisV~l~V~lS~rL~u~l~~rL~Oly~nSsrLyyrGluIlcPhcT 1200 1210 COOOCC-AAACCAGGTGATACTCAWTAAATG
1220
1230 1240 1250 AGTT’NACTCTOATGT00ATGTCGGOOATGTGCA
1260
hrOlyLcuInnL~PrOlyA~Th~Si~~~D~lUPh~A~pSe~AapV~lA~pValOlyA~pV~lGl 1290
1270 1210 GA~WMO~OmOCTATMCAA~ATCMCCCTIC
1300
,310
1320
1330
1390
1400
nLyV8lLysPhtV~lTrp~yrA9nA8nV~lIle~nProThrLcuProLysV~lGlyAlaScrGlnIle 1340 1350 1360 1370 ACAOMGAACMAAMATWAA~?~CA~~OCAQCACAGACACCOT~AGGGAGGACATCCTGC
13so
ThrV.l01t1QlnA~~.pGlyA~gV~lPh~Ly8PhcCyshrV~lA~gQluAspIlcLauL ,410 1420 1430 TCACCCTCACCCCGTGWAGGAGGCTCCTQQAAATQACCACC cuThrLeuThrProCy&nd
1440
Nucleotide and amino acid sequence of rabbit psncreatic lipase. nucleotide sequence of RTGL-5 was obtained as described in the text. histidine and aspartste that constitute the active triad are indicated Serfne, by asterisks. Putative glycosylation site and the primer wed for isotatfon of the positive clones from the cDNA library are over and underlined respectively. E-E?
A comparison
(20) 1 canine homology
between
of (21)
the and rabbit
amino
acid
porcine enzyme
sequence (22)
is
and
other
of
rabbit
shown species
in
lipase figure is
with 2.
observed.
human 70-85% There
(1))
rat
over
all
is
81%
Vol.
188, No. 3, 1992
BIOCHEMICAL
MLLLWALPIXLLGAVAG P TS T TVSF AQ VSI TIA F AKA ____________.____
AND BIOPHYSICAL
Rabbit HUUlall Rat C.%lil-le Porcine
Signal
RESEARCH COMMUNICATIONS
peptide
LEVCYERLGCFGNRIPWSGGTLERPFSTLPSTPKDVNTRFLLYTNENPNNF9EISADAST K SDDS I X LHI ws VA S S K DN SDAE A X AI LKL WS EKI TA TLQLSDPL L K QI SDAE A,X AI LKV WS ERIG K TLLPSDPS S FP SDDA A IVXQRPLKI PDXK D QQ Y LV P
60
IRGXSNFRTDRKTRFIIHGFTDKGEENWLSNLCENLFQVETVNCICVDUKGGSRTTYPQA i AVK K s SX KN GT TI A i WDHKMK E K QGT QVA Q K. T IN LDMKHK E K QST TIEA TNX RI% I D IK KS GT * * TQNIRIVGAEVAYLVETLQSSLGYSPSNIH~GEVGRRT~IGRITGLDP S F EF AF V A ANVV Q QHIDI VKNYS KV L V A ANVV V Q QMLSM SANYS AS PXL Q'JQL S F EV K V S A E AEPYFQGTPEIVRLDPSDAQNDVIHTDAAPWPNLGFCHSPPGC C L K c I V VANE E D G LI F TN MS T D V AS E LI F T QM C L K PI1
120
180
240 is E Q
QKNVLSQIVDINCVVEGTRDNACN;?LRSYKWADSIVNPNGFAGFSCASYTAFSANKCF K I DI A T D P NVT K A DIS A LE L D AYP KD ES SE L D SYP K A NLD I R ES I DI i D PD NVT
300
PCXSNGCPQMGHYADRFSRD~GQT~TGDSSNFARWRYQVAVTLSGRRVTGHVLV PG YPG ND K D A KS KK KYAG SGDEP K F D EAK RSLI KM PDP K AV SDET KYF G SI KA PDQ PE PG N SVF A KS lx
360 Q:K
QAK I
SLYGSKGNSKQYEI~GWPGDTHLNEFDSDVDVGDVQKVKFVWYNNXVINPTLPKVGA KT DS S LM I x R FN AF TR D R II DA SS AKL TIEN L N QX SF R AF THFN KI S S AKL TIE LN XV F FNE R YKTQDN SD E L I N R SQITVEQNDGRVXFKFCSTDTVREDILLTLTPCU KI TVKQXN PE EV L AK I QKGEEKTEYN EE ET AK QKGEEKTVHS ES V K R K XYD QE EV N
420
Rabbit Human Rat Canine Porcine
U ETSATVU U U
Figure 2. Comparison of emino acid sequence of rabbit pancreatic lipase with that of different species. Amino acid sequence of PLs from rat (ZO), canine (21) and porcine (22) are aligned with the sequence of rabbit PL. Spaces between the amino acids indicate the conservation. X- deletion. Asterisks indicate amino acids involved in the active site. Putative glycosylation site is overlined. Conserved possible lipid binding site is boxed.
conservation to
canine
in
signal
lipase.
conserved
in
the
sequence
IGHSLG,
including
RPL.
similar
the
to
other
peptide
The rabbit
when
catalytic
triad,
at
positions
a probable
lipid
Only
one
species
putative except
compared
153,
serine, 178 and
binding
site,
N-glycosylation the
dog PL which 968
to
human
and only
aspartate
and
histidine
266 respectively. is site does
conserved could not
38% compared
Hydrophobic in
all
be detected
have
are
this
site
species in (1).
RPL
Vol.
188, No. 3, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
.6
Figure 3. Northern blot RNA from rabbit pancreas transferred to nitrocellulose as described in the text.
blot
Northern
corresponding
to
broad extending of
multiple
reported Table
analysis the
size
of
the pig
of
molecule. Table
Amino number Signal
3)
full
length
1.
acid
seq.
lipase. 2opg of on 0.9% agarose random primer labeled RPL mRNA.
revealed
comparable
a
cDNA.
size
broad
band
at
The band obtained
This
suggests
coding
for
possible
several
total gel, RPL
1.5 is
kb,
rather
existence
isoenzymes
as
(23).
1 shows the percent
the lipase
(Fig.
from 1.4 kb to 1.6 kb in size.
RNA species
for
analysis of rabbit pancreatic was subjected to electrophoresis membrane and probed with Arrow indicates the hybridized
conservation
of amino acids
Maximum conservation Percentage
conservation regions Rabbit human
between of
of Rabbit rat
amino RPL
from various
species acids
from
Rabbit: canine
exists various
Rabbit porcine
81
63
38
l-60
73
52
50
53
61-120
82
68
67
60
121-180
87
67
58
85
180-240
80
73
78
88
240-300
82
72
73
83
301-420
80
46
66
72
421-432
65
44
53
68
969
not
available
regions
between
of
amino
Vol.
188,
No. 3, 1992
to 300.
acids
120
Least
homology
the
This
is
cholesterol
these
BIOCHEMICAL region
observed
termini
carries
at
carboxyl
the
not
very
of
acidic,
both
C-and
esterase
are
AND BIOPHYSICAL active-site
N-termini
(24)
it
important
RESEARCH COMMUNICATIONS
is
serine
of
the
molecule.
probable
for
that
the
enzyme
amino
acids
and histidine. As shown
the
in
amino
acids
or
co-factor
activity
in
binding. A comparison rat
pig,
and
hydrophobic from
amino
six
in
species
Results
enzyme
in both
active-site
serine
modification
(25,
265
177 respectively. the
triad,
activation
of
lipase
(28,
Such
this In
lipase
Experiments
important
of for
other
in
the
of
of
lysines
canine.
dog,
number
of
varied
As lysines
may
heparin
may
to intestinal
the the
by The
may not
site co-factor
functional
are
its high
may not
activity
the
loop,
during
the
structure of
be
vital
of
the
the
of
primary with
to
of enzyme
the
of at
function to
locate
amino
acids
these It
is
interfacial the
C-terminus activity of
other the of
of rabbit
species. C-terminus
the
the that
enzyme. sites
of
may not
be
are underway.
Acknowledgment: Authors wish to acknowledge Drs. John Morley, and Terry Zenser for their help in conducting this investigation. Assistance provided by Mrs. Padmaja Satram and the secretarial Cole and Carol McCleary is greatly appreciated.
970
that
structure that
of
positions
(27).
functional
dissimilarity
mutagenesis stretches
structure
for
site
shown triad
the
chemical
RPL at
have
moves
by
active
a catalytic
the
presented
directed and
in
be necessary
regions
in
studies
a surface
the
is
retained
shown
in
conserved
lipase
It
been
involved
forming
primary
pancreatic
levels. has
also
change
we have
rabbit
acid
acids
serine
29).
region
that
crystallographic
covered
revealed this
amino
aspartate-176
conclusion
involving
interaction
lipolysis
and compared
that
show
for
portion
a comparison
suggesting
in
of binding
importance
active-site
this
pancreatic
thirty
amino
X-ray
that
protein.
as
extent
enzymes
The number
and
The
suggested
the
as many
the
conservation
nucleotide
and
with
that
to
investigation
at
26).
histidine-263
suggests
of
and histidines.
(24),
this
whose
lipases
bond
degree
in
to species.
conserved
hydrogen
a high
binding
presented
and
and neutral
cysteines
rabbit
in heparin
from
highly
shows
acids,
the
participate vary
human
basic
Steve help
Giddings Technical of Carolyn
Vol.
188,rNo.
3,
1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
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