INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

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.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

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 GenBank^TM (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.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

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.

View larger version (100K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE of PLI. Lane 1, molecular weight standards; 2, untreated PLI; 3, PLI digested with PNGase F; 4, PLI digested with PNGase F after the O-glycanase and N-acetyl neuraminidase digestion; 5, PLI digested with the combination of O-glycanase and N-acetyl neuraminidase. The molecular masses (in kDa) of the standard proteins are indicated.

View larger version (56K): [in this window] [in a new window]   Fig. 2.   Chemical cross-linking of PLI. Aliquots of the inhibitor were treated with bis(sulfosuccinimidyl)subrate: lane 1, 0 mM; 2, 0.08 mM; 3, 0.16 mM; 4, 0.31 mM; 5, 0.63 mM; 6, 1.25 mM; 7, 2.5 mM; 8, 5 mM; 9, 10 mM. The samples were analyzed on 7.5% SDS-polyacrylamide gel, followed by Coomassie staining. The molecular masses (in kDa) of the standard proteins are indicated.

View this table: [in this window] [in a new window]   Table I Sulfhydryl titration of PLI The thiol content was determined as described under "Experimental Procedures" in the presence or absence of 6 M guanidine HCl.

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.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Gel filtration of the mixture of PLA2 and PLI on a Superose 12 column. The column had been equilibrated with Hepes buffer containing 0.05% (w/v) Tween 20 (pH 7.5, µ = 0.2). (a) 0.23 nmol of PLI, (b) 0.7 nmol of PLA2, (c) 0.23 nmol of PLI and 0.7 nmol of PLA2, (d) 0.23 nmol of PLI and 2.1 nmol of PLA2.

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.

View larger version (70K): [in this window] [in a new window]   Fig. 4.   cDNA sequence and deduced protein sequence of PLI. The nucleotide sequence (upper) and the deduced amino acid sequence (lower) are numbered. Possible polyadenylation sites are wavy-underlined, and four potential N-glycosylation sites are shown in bold face. The N-terminal and internal sequences elucidated by sequencing of the lysyl endopeptidase digests are underlined.

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.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Northern blot analysis of PLI. 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 positive-charged nylon membrane. The membrane was hybridized with the biotinylated PCR fragment corresponding to nucleotides 112-656. RNA size markers are indicated on the right. The arrow indicates the position of the PLI transcript.

View larger version (72K): [in this window] [in a new window]   Fig. 6.   Southern blot analysis of PLI. Chinese mamushi genomic DNA was digested with various restriction endonucleases, electrophoresed on a 0.7% agarose gel, and transferred to a nylon membrane. The membrane was hybridized with the peroxidase-labeled probe of the portion of the coding region corresponding to nucleotides 112-656 (Panel A) or with that of the portion of the 3'-noncoding region corresponding to nucleotides 1132-2256 (Panel B). Lanes 1, PvuII; 2, KpnI; 3, EcoRV; 4, PstI; 5, EcoRI; 6, HindIII; 7, BamHI. DNA size markers are indicated.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

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.

View larger version (44K): [in this window] [in a new window]   Fig. 7.   Comparison of the amino acid sequences of A. blomhoffii siniticus PLI and human LRG. The amino acid sequence of A. blomhoffii siniticus PLI was compared with that of human LRG (22). Identical residues are shown in stippled boxes.

View larger version (43K): [in this window] [in a new window]   Fig. 8.   Alignment of the deduced amino acid sequence of A. blomhoffii siniticus PLI. The sequence of A. blomhoffii siniticus PLI was divided into signal sequence, N-flanking, leucine-rich tandem repeats, and C-flanking regions. Conserved residues in leucine-rich repeats are shown in stippled boxes. Secondary structures of the repeating unit predicted on the basis of the three-dimensional structure of ribonuclease inhibitor (23) are indicated. Cysteine residues in N-flanking and C-flanking regions are also shown in stippled boxes.

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 (K_i) of 5.9 × 10¹⁴ M (28). The inhibitory constant of PLI for A. blomhoffii siniticus basic PLA₂ was 7.5 × 10¹⁰ 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.