From the Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Osaka 569-1094, Japan
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ABSTRACT |
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The phospholipase A2
(PLA2) inhibitor PLI, purified from the blood plasma of
Chinese mamushi snake (Agkistrodon blomhoffii siniticus),
is a 160-kDa trimer with three 50-kDa subunits; and it inhibits
specifically the enzymatic activity of the basic PLA2 from
its own venom (Ohkura, N., Okuhara, H., Inoue, S., Ikeda, K., and
Hayashi, K. (1997) Biochem. J. 325, 527-531). In the
present study, the 50-kDa subunit was found to be glycosylated with
N-linked carbohydrate, and enzymatic deglycosylation
decreased the molecular mass of the 50-kDa subunit to 39-kDa. One
160-kDa trimer of PLI
was found to form a stable complex with three
basic PLA2 molecules, indicating that one basic
PLA2 molecule would bind stoichiometrically to one subunit
of PLI
. A cDNA encoding PLI
was isolated from a Chinese
mamushi liver cDNA library by use of a probe prepared by a
polymerase chain reaction on the basis of the partially determined amino acid sequence of the subunit. The cDNA contained an open reading frame encoding a 23-residue signal sequence followed by a
308-residue protein, which contained the sequences of all the peptides
derived by lysyl endopeptidase digestion of the subunit. The molecular
mass of the mature protein was calculated to be 34,594 Da, and the
deduced amino acid sequence contained four potential
N-glycosylation sites. The sequence of PLI
showed no significant homology with that of the known PLA2
inhibitors. But, interestingly, it exhibited 33% identity with that of
human leucine-rich
2-glycoprotein, a serum protein of
unknown function. The most striking feature of the sequence is that it
contained nine leucine-rich repeats (LRRs), each of 24 amino acid
residues and thus encompassing over two-thirds of the molecule. LRRs in
PLI
might be responsible for the specific binding to basic
PLA2, since LRRs are considered as the motifs involved in
protein-protein interactions.
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INTRODUCTION |
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Phospholipases A2 (PLA2s,1 EC
3.1.1.4) catalyze the hydrolysis of the
acyl-ester bond at the sn-2 position of glycerophospholipids to yield fatty acids and lysophospholipids. Snake venom is one of the
most abundant sources of secretory PLA2s. Secretory
PLA2s have been classified into three groups, I, II, and
III, according to their primary structures (1). Elapidae venom contains
group-I PLA2s; and Viperidae venom, group-II ones. These
snake venom PLA2s exhibit a wide variety of toxicity, such
as neurotoxicity and myotoxicity (2). Venomous snakes have
PLA2 inhibitory proteins (PLIs) in their blood sera in
order to protect them from the leakage of their own venom
PLA2s into the circulatory system. We have purified three
distinct types of PLA2 inhibitors (PLI, PLI
, and
PLI
) from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus (3). PLI
was found to be a 75-kDa
glycoprotein and a trimer of 20-kDa subunits having sequence homology
to the carbohydrate-recognition domain (CRD) of C-type lectins (4). We
have already purified this type of inhibitor from the blood plasma of
the Habu, Trimeresurus flavoviridis (5, 6). These CRD-like
inhibitors inhibited specifically the group II acidic PLA2s, and thus they seemed to recognize a unique aromatic
patch structure on the surface of these acidic PLA2
molecules (7). On the other hand, PLI
was found to be the same type
of inhibitor as those purified from the plasma of Naja
kaouthia (8) and Crotalus durissus terrificus (9, 10).
It is a 100-kDa glycoprotein containing 25- and 20-kDa subunits, and
their amino acid sequences were characterized by two tandem patterns of
cysteine residues found in Ly-6 related proteins (11). This type of
inhibitor exhibited a broad inhibition spectrum against
PLA2s, and it is thought to recognize the
Ca2+-binding loop of PLA2s, which is conserved
among all the secretory PLA2s. PLI
was shown to be a
160-kDa glycoprotein having a trimeric structure composed of 50-kDa
subunits (3). It inhibited only some group II basic PLA2s
and did not inhibit other kinds of PLA2s. The N-terminal
30-amino acid sequence of the 50-kDa subunit was determined, but no
significant homology was found to the known PLA2
inhibitors.
In the present study, to elucidate the complete primary structure of
PLI, we cloned and sequenced the PLI
cDNA and found that
PLI
is a protein with leucine-rich repeats (LRR) and that its
sequence is 33% identical to that of leucine-rich
2-glycoprotein (LRG) from human serum.
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EXPERIMENTAL PROCEDURES |
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Materials--
The blood plasma, venom, and liver of Chinese
mamushi (A. blomhoffii siniticus) were obtained from Ueda
Trading Co. (Gifu, Japan). A. blomhoffii siniticus basic
PLA2 was purified from the venom as described previously
(4). Chinese mamushi PLI was purified to homogeneity from blood
plasma as described previously (3).
Deglycosylation and SDS-PAGE of PLI--
PLI
was
deglycosylated with an enzymatic deglycosylation kit (Bio-Rad).
O-Linked oligosaccharides were cleaved by the combination of
O-glycanase and N-acetylneuraminidase at 37 °C
for 1 h in 50 mM sodium phosphate buffer, pH 6.0. N-Linked oligosaccharides were cleaved by
peptide-N-glycosidase F (PNGase F) at 37 °C for 3 h
in 150 mM sodium phosphate buffer, pH 7.6. PLI
used for
the PNGase F digestion was denatured by treatment with 0.1% SDS and 50 mM
-mercaptoethanol at 100 °C for 5 min prior to the
digestion.
Mass Spectrometry Analysis of Deglycosylated PLI--
Mass
analysis was performed on a Voyager DE STR (PerSeptive Biosystems)
matrix-assisted laser desorption time of flight (MALDI-TOF) mass
spectrometer. The instrument was operated in the linear positive ion
mode with an accelerating potential in the source of 20 kV. Ionization
was accomplished with the 337-nm beam from a nitrogen laser with the
laser energy precisely tuned for an optimal signal to noise ratio.
Calibration was done externally with a mixture of two standard
compounds (bovine serum albumin and horse heart myoglobin).
Chemical Cross-linking--
Aliquots (5 µl) of the purified
PLI (140 µg/ml) in 50 mM Hepes buffer (pH 7.5, ionic
strength 0.2) were treated with bis(sulfosuccinimidyl)-suberate (Pierce) for 3 h at room temperature. The samples were diluted with the double-strength gel sample buffer containing 0.2 M
dithiothreitol, heated to 100 °C for 5 min, and analyzed by
SDS-PAGE, followed by Coomassie Blue staining.
Direct Binding of Basic PLA2 to PLI--
A
0.23-nmol amount of PLI
was incubated for 1 h at room
temperature with 0, 0.7, and 2.1 nmol of basic PLA2 in 100 µl of 50 mM Hepes buffer (pH 7.5, ionic strength 0.2)
containing 0.05% (w/v) Tween 20. The mixture was then applied to a
Superose 12 HR10/30 column (Amersham Pharmacia Biotech) equilibrated
with the same buffer. The fractions containing the
PLI
·PLA2 complex were further analyzed by
reversed-phase HPLC on a Vydac C4 column (The Separations Group).
Amino Acid Sequencing of Peptides Derived from
PLI--
PLI
was pyridylethylated in the presence of 6 M guanidine hydrochloride by the method described
previously (13). The pyridylethylated PLI
was digested with lysyl
endopeptidase (Wako Chemicals), and the resultant fragments were
separated by HPLC on a Vydac C4 column with 0.1% trifluoroacetic acid
containing a linear concentration gradient of acetonitrile from 0 to
48%. The fragments thus obtained were sequenced with an Applied
Biosystems model 477A protein sequencer equipped with a 120A PTH
analyzer.
Titration of Sulfhydryl Groups--
The free thiol group in
PLI was quantified in 50 mM Hepes buffer, pH 7.5, containing 0.6 mM 5,5'-dithiobis(2-nitrobenzoic acid)
(DTNB) in the absence or presence of 6 M guanidine HCl
according to Riddles et al. (14).
Construction of Chinese Mamushi Liver cDNA
Library--
Total RNA was isolated from fresh liver by use of a total
RNA extraction kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Poly(A)+-enriched RNA was
obtained using an oligo(dT)-cellulose column (Amersham Pharmacia
Biotech). Oligo(dT)-primed cDNA synthesis was carried out by means
of a Superscript choice system (Life Technologies, Inc.). cDNAs
were ligated to the MOSElox arms (Amersham Pharmacia
Biotech), and packaged via a
-in vitro
packaging module (Amersham Pharmacia Biotech).
Screening and Isolation of Clones Encoding PLI--
To obtain
the probe for cDNA screening, we performed the reverse
transcriptase polymerase chain reaction (RT-PCR) using a Chinese
mamushi liver total RNA as template. On the basis of the determined
N-terminal and internal peptide sequences of PLI
, two degenerated
oligonucleotide primers, ABSPR5 and ABSPR6, were designed for
amplification of a cDNA internal fragment by PCR. The primer ABSPR5
(5'-GARAAYGTNACNGARTTYGT-3', where N, R, and Y denote G/A/T/C, G/A, and
T/C, respectively) was oriented in the sense direction and corresponded
to the N-terminal residues 11-17 of PLI
. The primer ABSPR6
(5'-ACDATRCAYTGDATNGGRTT-3', where D denotes G/A/T), was oriented in
the antisense direction and corresponded to residues 186-192. The
total RNA was subjected to first-strand cDNA synthesis with
Superscript reverse transcriptase (Life Technologies, Inc.) using an
oligo(dT) primer. The obtained single-strand cDNA was used as a
template for PCR amplification with Taq DNA polymerase
(Takara) under the following thermal cycling conditions: 5 cycles of 1 min at 94 °C, 2 min at 60 °C, and 3 min at 72 °C, followed by
50 cycles of 1 min at 94 °C, 2 min at 55 °C, and 3 min at
72 °C. The amplified 550-bp product was cloned into
pMOSBlue T-vector (Amersham Pharmacia Biotech), cut out, and
labeled with horseradish peroxidase by means of an ECL direct nucleic
acid labeling system (Amersham Pharmacia Biotech).
DNA Sequencing and Analysis-- Both strands of DNA were sequenced with a Thermo Sequenase core sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech) on an SQ-3000 DNA sequencer (Hitachi) after subcloning into pBluescript (Stratagene). Analysis of DNA sequencing data and alignments of protein sequences were performed by use of DNASIS (Hitachi Software Engineering Co. Ltd.) and BLAST (15) software on EMBL (16) and GenBankTM (17) data bases.
RNA and DNA Blotting Analyses--
A 0.23-µg amount of
poly(A)+ RNA prepared from Chinese mamushi liver was
electrophoresed on a 1.2% agarose gel containing 2.2 M
formaldehyde and transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech). The biotinylated probes for RNA blotting analysis were prepared from the amplified 550-bp product with a BioPrime DNA
labeling kit (Life Technologies, Inc.). After prehybridization, the
membrane was incubated with the probes at 42 °C overnight in
SuperSignal NA hybridization buffer (Pierce), washed twice for 5 min
with 2× SSC containing 0.1% SDS at room temperature, twice for 5 min
with 0.2× SSC containing 0.1% SDS at room temperature, and then twice
for 15 min with 0.2× SSC containing 0.1% SDS at 42 °C. After the
final wash, the PLI transcript was chemiluminescently detected with
a SuperSignal NA complete blotting kit for detection of biotinylated
probes (Pierce) according to the manufacturer's instructions.
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RESULTS |
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Chemical Characterization of PLI--
PLI
was purified from
the blood plasma of Chinese mamushi, A. blomhoffii
siniticus, by sequential chromatography on Sephadex G-200,
Q-Sepharose, Hi-Trap Blue, Hi-Trap Phenyl HP, and Mono-Q HR5/5 columns,
as described previously (3). SDS-PAGE of the purified PLI
showed one
protein band with an apparent molecular mass of 50 kDa (Fig.
1). When the PLI
was treated with
N-glycosidase F and a combination of
N-acetylneuraminidase and O-glycanase, the
apparent molecular mass was reduced to 39 and 47 kDa, respectively. Since PLI
treated with N-glycosidase F after the
treatment with the combination of N-acetylneuraminidase and
O-glycanase gave the same molecular mass of 39 kDa as that
treated only with N-glycosidase F, PLI
was suggested to
be a glycoprotein having N-linked, but not
O-linked, carbohydrate. Actually, no sugars in PLI
were
detected after the N-glycosidase F treatment (data not
shown). Since the molecular mass of 39 kDa, estimated from the
migration of the deglycosylated PLI
on SDS-PAGE gel, seemed to be
different from that of 34,594 Da, which was calculated from the
sequence determined in the present study, the molecular mass of the
deglycosylated PLI
was confirmed by means of mass spectrometry. The
observed molecular mass was 34,674 ± 61.8 Da, which was
comparable to the calculated value, indicating that the leucine-rich
structure of PLI
may affect its migration on SDS-PAGE. The molecular
mass of 50 kDa obtained from SDS-PAGE of the intact PLI
subunit was also determined to be 43,857.2 ± 81.9 Da by the mass
spectrometry.
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Direct Interaction of PLI with Basic PLA2--
As
shown in Fig. 3, complex formation of
PLI
with A. blomhoffii siniticus basic PLA2
was investigated by gel filtration on a Superose 12 column. When 0.23 nmol of PLI
and 0.7 nmol of PLA2 were separately applied
to the column, they were eluted with a single peak at 28 min and 42 min, corresponding to their respective apparent molecular masses of 160 and 8 kDa. However, when their mixture was applied to the column, the
latter peak disappeared, and the height of the former peak increased,
suggesting the formation of a stable complex between PLI
and basic
PLA2. The fraction containing the complex of PLI
and
basic PLA2 was obtained by the gel filtration of the
mixture containing an excess amount of the PLA2 over
PLI
. Then, PLI
and basic PLA2 in this fraction were
separately quantified by reverse-phase HPLC. The molar ratio of
PLA2 to PLI
was found to be 3.17, suggesting that each
subunit of PLI
may bind one molecule of basic PLA2.
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Cloning of PLI cDNA--
Previously, we determined the
N-terminal amino acid sequence of PLI
(3). In order to elucidate the
internal sequences of PLI
, we digested reduced and
S-pyridylethylated PLI
with lysyl endopeptidase. Of the
resulting peptides, fourteen peptides were isolated by reverse-phase
HPLC, and their complete or partial amino acid sequences were
determined. On the basis of the determined partial amino acid sequences
of PLI
and its peptides, we synthesized degenerate primers, ABSPR5
and ABSPR6, corresponding to residues 11-17 (ENVTEFV) and 186-192
(NPIQCIV), respectively. Using these degenerate primers for RT-PCR with
mamushi liver total RNA as template, we obtained a cDNA fragment of
550 bp. When this fragment was subcloned and sequenced, the deduced
amino acid sequence coincided with that of some lysyl endopeptidase
peptides, suggesting that the fragment was a part of PLI
cDNA.
Thus this fragment was used as a probe to screen the mamushi liver
MOSElox cDNA library. From the 2 × 106 plaques screened, five positive clones were obtained.
Clone pABS205, which contained the largest insert (2.2 kb), was
selected and sequenced. The complete nucleotide sequence of PLI
cDNA and the deduced amino acid sequence are shown in Fig.
4. It contained 2256 bp with a
5'-noncoding region of 12 bp, followed by an open reading frame region
of 993 bp, and a 3'-noncoding region of 1241 bp and a poly(A) tail. The
open reading frame of this cDNA sequence encoded a protein of 331 amino acids, and its deduced amino acid sequence included the amino
acid sequences determined from the lysyl endopeptidase peptides of the
purified PLI
(Fig. 4, underlined). The first 23 amino
acids following the initiator methionine have the features of a signal
sequence (19). Valine at the residue 1 was regarded as the N-terminal
residue of the mature protein on the basis of the N-terminal sequence
determined from the purified PLI
. Thus the mature PLI
was
predicted to be composed of 308 amino acid residues with a calculated
molecular mass of 34,594 Da, which was consistent with the
corresponding value of the deglycosylated PLI
determined by mass
spectrometry. There were four potential N-linked
glycosylation sites (Asn-X-Ser/Thr) in the deduced sequence (Fig. 4, boldface). One of them, asparagine at residue 12, would actually be glycosylated, since no PTH derivatives of asparagine were detected at the corresponding position on the amino acid sequence
study of the purified PLI
(3). Two potential polyadenylation signals
(AATAAA) occurred at 1503 and 2226 (Fig. 4). Since the latter signal
was present 17 bp upstream of the poly(A) tail, it was used as the
actual signal by the mRNA from which this cDNA clone was
derived.
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RNA and Genomic DNA Blotting Analysis for PLI--
Northern
blot analysis revealed that a single hybridizing band of approximately
2.7 kb was observed on the blot of the poly(A)+ RNA
isolated from Chinese mamushi liver, indicating that the size of the
obtained cDNA is close to that of the full-length copy of mRNA
(Fig. 5). Since no other shorter
transcripts could be detected, no alternative polyadenylation would
occur.
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DISCUSSION |
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Three distinct types of PLA2 inhibitors (PLI,
PLI
, and PLI
) are present in the blood plasma of the Chinese
mamushi, A. blomhoffii siniticus (3). These inhibitors
inhibit three group-II PLA2s (acidic, neutral, and basic
PLA2s) from the venom with different specificity. PLI
,
specifically inhibits the acidic PLA2 by binding of 1 mol
of the PLA2 to 1 mol of the PLI
which is a trimer of 20-kDa subunits (4). PLI
shows a broad inhibition spectrum; i.e. it inhibited all three venom PLA2s almost
equally and, furthermore, it inhibited other groups of
PLA2s, including Elapidae venom PLA2s (group I)
and honeybee PLA2 (group III) (3). PLI
is a specific inhibitor of the venom basic PLA2 of A. blomhoffii
siniticus, and it does not inhibit the other two PLA2s
(3). As shown in Fig. 3, 1 mol of the PLI
, which was composed of
three identical subunits of 50-kDa, was found to bind 3 mol of the
basic PLA2s, suggesting that each subunit would bind one
PLA2 molecule.
In the present study, the complete nucleotide sequence of cDNA
encoding PLI was determined. The mature PLI
was composed of 308 amino acid residues with a calculated molecular weight of 34,594. This
value is consistent with that obtained by mass spectrometry of the
deglycosylated PLI
. PLI
was found to be a heavily glycosylated
protein having N-linked glycosidic chains. Four potential
N-linked glycosylation sites were identified in the deduced
sequence.
The amino acid sequence of PLI showed no significant structural
similarities to those of PLI
and PLI
. PLI
contained neither the CRD sequences, which were found in the sequences of PLI
(6) and
PLA2 receptors (20, 21), nor the two tandem patterns of cysteine residues found in the sequences of the two homologous subunits
of PLI
(11) or in Ly-6-related proteins. Searching for homologies by
use of the BLAST program revealed that PLI
shared 33% identity over
the whole molecule with human leucine-rich
2-glycoprotein (LRG) (Fig.
7). LRG is a human serum protein in which
leucine-rich repeats (LRRs) were first discovered (22). Like LRG,
PLI
contained 9 tandem LRRs of 24 residues each, which encompassed
over two-thirds of the molecule (Fig. 8).
An alignment of LRRs of PLI
showed that they contained the consensus
sequence of
X-L-X-X-L-D-L-S-X-N-X-L-X-X-L-X-X-X-X-F-X-X-L-X
(X denotes any amino acid). Similar LRR consensus sequences
have been found in the primary structure of a large number of proteins,
including proteins that participate in biologically important
processes, such as receptors for hormone, enzymes, enzyme inhibitors,
proteins for cell adhesion, and ribosome-binding proteins (reviewed in Refs. 23 and 24). Although the functions of these proteins are
different, all proteins containing LRRs are thought to be involved in
protein-protein interactions. The functions of LRG remain unknown, and
its physiological ligand remains unidentified. But, both LRG and PLI
are serum proteins, and there is 33% identity between their sequences.
This may raise the possibility that PLI
corresponds to the snake LRG
and, furthermore, that human LRG functions as a PLA2
inhibitor, although evidence on this hypothesis has yet to be
demonstrated.
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Many LRR-containing proteins contain homologous regions flanking the
LRR domains. These regions are characterized by the patterns of
cysteine residues; the consensus sequences can be described as
CP(~2X)CXC(~6X)C for the
amino-flanking and
PXXCXC(~20X)C(~20X)C for the carboxy-flanking regions (23). The carboxy-flanking region of
PLI was nearly identical to the consensus sequence. The
amino-flanking region of PLI
did not conform to the consensus sequence, but contained a related sequence with two cysteine residues. In addition, there was a proline-rich cluster in the amino-flanking region of PLI
. This proline-rich cluster is not present in any other
LRR-containing proteins, and thus might play an important structural or
functional role in PLI
.
Two cysteine residues, Cys-147 and Cys-190, were found in the LRR
domain of PLI, while no cysteine residues were found in that of LRG.
One of the LRR-containing proteins, ribonuclease inhibitor (RI),
contained most of cysteine residues in its LRR domain, and all of the
cysteine residues occur as free thiol groups (25). Of the 10 cysteine
residues present in the PLI
subunit, two were found to occur as free
thiols as shown in Table I. Therefore, the 2 cysteine residues with
free thiol groups might be assigned to be Cys-147 and Cys-190 found in
the LRR domain, because the intrachain disulfide bonding of these
cysteine residues could be expected to be unfavorable geometry for the
conformation of LRR.
The crystal structure of porcine liver RI has been determined (26). RI
was found to be a nonglobular, horseshoe-shaped molecule with a curved
parallel -sheet lining the inner circumference of the horseshoe and
the helices flanking its outer circumference. In this structure, the
individual LRR units have essentially the same conformation of
/
structural units, consisting of a short
-strand and an
-helix
approximately parallel to each other. The crystal structure of the
complex of RI and bovine ribonuclease A (RI-RNase A) has also been
determined (27). RNase A binds to a concave surface of the inhibitor,
mainly comprising its parallel
-sheet and the loops C-terminal to
the
-strands. Therefore, the structure of RI-RNase A could serve as
a model for the interaction between PLI
and PLA2. If the
LRRs in PLI
constitute
/
structural units that occur in RI and
the whole molecule reveals a horseshoe structure just like the molecule
of RI, the PLA2 molecule can be expected to bind to the
concave surface of the horseshoe structure of PLI
. That is, the
concave surface would contain a large excess of negatively charged
residues, especially aspartate at the sixth residue of the consensus
sequence of LRR, and these negative charges could electrostatically
interact with the positively charged residues on the basic
PLA2 molecule. PLI
is a selective inhibitor against the
group II basic PLA2s from Crotalidae venom, and it does not inhibit other kind of PLA2s. The residues His-1, Arg-6,
Glu-17, Trp-70, Lys-111, and Ile-124 are conserved among these group II basic PLA2s, but they are replaced by other residues in all
the PLA2s not inhibited by PLI
. Some of these residues
in the PLA2 molecule are therefore thought to be involved
in the interaction with the PLI
molecule.
The very extensive binding surface of the RI-RNase A complex was
suggested to be in part responsible for the extremely low inhibitory
constant (Ki) of 5.9 × 1014
M (28). The inhibitory constant of PLI
for A. blomhoffii siniticus basic PLA2 was 7.5 × 10
10 M (3). That this interaction is much
weaker than that of RI and RNase suggests that the number of amino acid
residues of PLI
closely contacting with the PLA2
molecule is much smaller than that for the RI-RNase complex and that
they were located scatteringly and specifically on the extensive
concave surface of the horseshoe. The narrow inhibition spectrum of
PLI
may be accounted for by the local distribution of the contact
residues despite the extensive contact area.
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FOOTNOTES |
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* This work was supported in part by Grant 07672400 (to S. I.) from the program Grants-in-Aid for Scientific Research (C) of the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB007198.
To whom correspondence should be addressed: Dept. of Biochemistry,
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. Tel.: 81-726-90-1075; Fax:
81-726-90-1005; E-mail: inoue{at}oysun01.oups.ac.jp.
1
The abbreviations used are: PLA2,
phospholipase A2; PLI, phospholipase A2
inhibitor; PCR, polymerase chain reaction; bp, base pair; kb, kilobase
pair; LRR, leucine-rich repeat; LRG, leucine-rich 2-glycoprotein; PAGE, polyacrylamide gel
electrophoresis; HPLC, high-performance liquid chromatography; PTH,
phenylthiohydantoin; RI, ribonuclease inhibitor.
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REFERENCES |
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