Conus are a group of predatory marine snails that prey on fish, molluscs, and polychaete worms(1) . Venom from cone snails are extraordinarily complex and often contain more than 100 distinct components. Most venom characterizations have focused on small disulfide-rich peptides that target selectively to receptors and ion channels on nerve and muscle cells. Known targets include calcium channels (-conotoxins)(2, 3, 4) , sodium channels (µ- and -conotoxins)(5, 6, 7, 8) , N-methyl-D-aspartate receptors (conantokins)(9, 10, 11) , nicotinic acetylcholine receptors (-conotoxins)(12, 13, 14, 15, 16, 17) , and vasopressin receptors (conopressins) (18, 19) . Small neuroactive molecules, e.g. serotonin, have also been found(20) .

Although larger protein components are present in cone venoms, these are generally less well characterized biochemically. Reports have included a convulsion-inducing peptide from Conus geographus, with an apparent molecular weight of 13,000(21) ; a cardiotonic glycoprotein (approximate molecular weight of 25,000) from Conus striatus, which appears to act on voltage-sensitive sodium channels(22, 23) ; a protein from Conus magus (molecular weight estimated to be between 45,000 and 65,000), which induces powerful rhythmic contractions of guinea pig vas deferens(24) ; and a 25,000 molecular weight protein from Conus distans, which inhibits neurotransmitter release in rat hippocampus(25) .

In this study, we have characterized a 13.6-kDa component from the venom of the fish-eating cone snail, C. magus. Novel - and -conotoxins have been isolated from this species previously(13, 26) . We detail the isolation and characterization of a polypeptidic venom component that we show to be a novel phospholipase A (PLA). ()This is the first biochemical characterization of an enzymatic activity from Conus venoms.

Low molecular mass secreted PLAs (14-18 kDa) have been reported from a wide variety of other venoms (snakes, insects, and lizards) as well as mammalian pancreatic and inflammation fluids (27, 28, 29, 30) . These enzymes hydrolyze the ester at the sn-2 position of phosphoglycerides. Snake venom and pancreatic PLAs are particularly well characterized(31, 32) . All require millimolar concentration of calcium as a catalytic cofactor. In addition to PLA activity, some of these venom proteins show potent neurotoxicity(33, 34, 35) . For instance -bungarotoxin, from the Formosan banded krait (Bungarus multicinctus) (36) and notexin isolated from the Australian tiger snake Notechis scutatus scutatus(37) exert potent neurotoxic action by inhibiting the release of acetylcholine from nerve terminals in neuromuscular junctions(38) . -Bungarotoxin consists of a PLA chain covalently bonded by a disulfide bridge to a polypeptide (molecular weight 20,000)(29) . The PLA chain of -bungarotoxin is homologous to notexin. Enzymes isolated from the venoms of Naja naja atra and Naja niggricollis, however, show more potent PLA activity, have less presynaptic effects, and are much less lethal(39) . More than 50 proteins with PLA activity have been isolated from snake venoms and show substantial sequence homology with each other and with PLA enzymes isolated from mammalian pancreas and inflammatory exudates(40, 41) . Sequence information reveals that the C. magus enzyme reported here differs substantially from any other PLA characterized to date and defines a new group of these enzymes.

EXPERIMENTAL PROCEDURES

Materials

The preparation of 1-O-hexadecyl-2-deoxy-2-S-thiohexadecanoyl-sn-glycero-3-phosphocholine and DMPM has been described previously(43, 44) . [H]DPPC (50 Ci/mmol), [C]PAPC (50 mCi/mmol), and [C]SAPI (50 mCi/mmol) were from DuPont NEN. [H]DPPE (10 mCi/mmol), [H]DPPA (5 mCi/mmol), and [H]SAPS (400 mCi/mmol) were prepared as described(45, 46) . [H]DPG was synthesized by treating 0.15 mg of [H]DPPC (50 mCi/mmol) in 0.2 ml of 200 mM sodium phosphate, 400 µM ZnCl, 1 mM 2-mercaptoethanol, pH 7.0, with 30 µg of phospholipase C (Bacillus cereus, Sigma P-7147) for 2 h at 37 °C with vigorous shaking. The reaction mixture was extracted three times with ether, the extract was dried over anhydrous NaSO, and the residue was dissolved in ether and applied to a silica gel TLC plate. The plate was developed with chloroform:ether (9:1), the product band (visualized with iodine) was scraped from the plate, and the product was eluted with ether. The PLA inhibitors linoleoyl amide and MG14 were prepared as described(47, 48, 49) . Porcine pancreatic PLA was obtained as a generous gift from Professor M. K. Jain (University of Delaware) (50) , bee venom PLA was from Boehringer Mannheim, and pancreatic lipase was from Sigma (L-0382).

Protein Purification and Structural Analysis

Venom Extraction

HPLC Purification

Figure 1: HPLC purification of conodipine-M. Panel A, extracted crude venom (see ``Experimental Procedures'') was applied to a Vydac reversed phase C18 column (10-µm particle size; 22-mm inner diameter 25-cm length) and eluted with a gradient that began at 95% A, 5% B for 5 min and increased to 100% B over 28 min. B was held at 100% for 5 min and then decreased to 5% over 4 min. The flow rate was 4 ml/min. Fractions were collected every 3 min beginning at 12 min. Panel B, material eluting at 34.3 min (arrow in panel A) was run on a Vydac reversed phase C18 column (10-µm particle size, 10-mm inner diameter 25-cm length) and eluted by beginning at 70% A, 30% B for 4 min; increasing to 36% B over 6 min; and then increasing to 56% B over 60 min. The flow rate was 3 ml/min. Fractions were collected every 1.0-1.25 min, beginning at 3 min. Panel C, material eluting at 23.3 min (arrow in panel B) was run on a Microsorb MV reversed phase C18 column (5-µm particle size, 4.6-mm inner diameter 25-cm length). The gradient began at 40% C, 60% buffer D for 1 min; increased to 70% D over 30 min; and increased to 80% D over 60 min. The flow rate was 1 ml/min. Fractions were collected according to visual inspection of the protein absorbance. Panel D, material eluting near the peak at 47.4 min (arrow in panel C) was run again using the same conditions as in panel C. Buffer A was 0.1% trifluoroacetic acid; buffer B was 0.1% trifluoroacetic acid, 90% acetonitrile in panels A and B. Buffer C was 0.1% trifluoroacetic acid, 2% MeOH, 98% HO; buffer D was 0.09% trifluoroacetic acid, 90% MeOH, 10% HO in panels C and D. It should be noted that prolonged use of trifluoroacetic acid/methanol in the Microsorb MV column is not recommended by the manufacturer because of potential hydrolysis. The absorbance was monitored at 237 nm in panel A and at 214 nm in panels B-D.

Protein Reduction and Alkylation

Figure 2: Conodipine-M has two subunits. Native and modified conodipine-M were analyzed by HPLC. HPLC conditions are the same for each panel and are described in Experiment 1 under ``Experimental Procedures.'' Panel A, unmodified conodipine-M, when chromatographed as described below, elutes at 27.4 min. Electrospray mass spectrometry of this material shows a molecular mass of 13,602. Panel B, reduction of conodipine-M (described under ``Experimental Procedures'') produces two primary protein absorbances with migration times (24.7 and 31.9 min) which differ from unmodified material. Mass spectroscopy of these proteins shows molecular masses of 8,571 and 5,036. The arrow indicates the expected migration time of unmodified conodipine-M. The sum of the molecular masses of these two proteins approximates that of the intact protein. Panel C, reduction and alkylation of conodipine-M (described in under ``Experimental Procedures'') also results in two protein absorbances with migration times of 25.7 and 34.9 min.

Enzymatic Digests

Pyroglutamate Aminopeptidase

Endoproteinase Lys-C

Endoproteinase Arg-C

Sequence Analysis

Mass Spectrometry

Membrane Binding

Membrane Preparation

[H]Isradipine Binding

Lipolytic Assays

Hydrolysis of small unilamellar DMPM vesicles was carried out using a pH-stat as described previously(43) , and 4-ml reaction mixtures contained 0.6 mM CaCl, 1 mM NaCl, 0.6 mg of DMPM, and typically 0.3 µg of conodipine-M. For the analysis of inhibitors, assays were carried out under conditions that measure the initial velocity(51) , and 4-ml reaction mixtures contained 2.5 mM CaCl, 1 mM NaCl, 20 µg of polymyxin B sulfate (Sigma), 0.6 mg of DMPM, and typically 0.1 µg of conodipine-M.

In some cases, radiometric assays of PLA activity were carried out. Covesicles of nonlabeled DMPM vesicles containing radiolabeled phospholipids were prepared as described previously(45) . The reaction mixtures were extracted with organic solvent, and the amounts of liberated radioactive fatty acids were analyzed as described previously(45) . In this way the calcium dependence of PLA activity of conodipine-M was measured using reaction mixtures with 0.3 mg of DMPM vesicles containing 120,000 cpm of [H]DPPC in 2 ml of 10 mM HEPES, 100 mM NaCl, 100 mM EGTA, pH 7.4. The mixtures also contained different amounts of 1 M KCaEGTA solution to give the desired concentrations of free calcium(52) . Reactions were initiated by the addition of 0.04 µg of conodipine-M and were quenched with organic solvent after a 10-min incubation at 20 °C. Reactions for determining the pH dependence of conodipine-M-catalyzed vesicle hydrolysis were carried out as above except that the buffer was 10 mM HEPES, 100 mM NaCl, 1 mM CaCl, pH 6-9.

Competitive substrate specificity studies using the double radioisotope method were carried out as described(45) . The reaction mixtures contained 0.6 mg of sonicated DMPM vesicles with 1 10-8 10 cpm of H- and C-labeled phospholipids in 4 ml of 0.6 mM CaCl, 1 mM NaCl, 20 µg of polymyxin B at 21 °C. Reactions were carried out in the pH-stat with 0.07 µg of conodipine-M (added last) and were quenched with organic solvent after 120-130 nmol of titrant was consumed. Reactions with DMPM vesicles containing [C]PAPC and [H]DPG were quenched and extracted with organic solvent as described above. The residue from the extract was dissolved in low boiling petroleum ether:ether:acetic acid (70:30:1), and nonlabeled arachidonic acid and 1,2-dioleoyl-sn-glycerol (Sigma) were added as markers. The mixture was applied to a silica gel TLC plate (4 12 cm), and the plate was developed with the same solvent. Regions corresponding to fatty acid and diacylglycerol were scraped from the plate, and the silica was soaked in scintillation fluid for 1 h followed by scintillation counting. Relative k/K values for the radioactive substrates were calculated using Equation 4 of(45) .

Studies to measure the positional specificity of the phospholipase activity of conodipine-M were carried out with reaction mixtures of 200 µg of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphomethanol in 4 ml of 2.5 mM CaCl, 1 mM NaCl, pH 8.0, containing 20 µg of polymyxin B at 21 °C in the pH-stat. After about 30% of the substrate was hydrolyzed (30 µl of 3 mM NaOH titrant), the reaction was quenched by the addition of 4 ml of 0.1 M EDTA, 2 M NaCl, pH 7.0, followed by 0.56 ml of 88% formic acid and 4 ml of ethyl acetate. After mixing on a vortex mixer, the layers were separated by centrifugation, and the organic layer was transferred to a new tube. The aqueous phase was extracted with a second portion of ethyl acetate, the extracts were combined, and the solvent was removed with a stream of nitrogen. To the residue were added 0.5 ml of ether and 50 µl of methanol, and sufficient diazomethane in ether was added until the yellow color persisted. After 30 min at room temperature, the solvent was removed with a stream of nitrogen. The residue was taken up in ether, and a portion was analyzed by gas chromatography on a DB-5 column (J & W Scientific). Peaks corresponding to methyl oleate and methyl palmitate were integrated, and the peak areas obtained from a control reaction (omission of conodipine-M) were subtracted from those obtained in the presence of enzyme.

RESULTS

Purification of Conodipine-M

Crude venom from C. magus was extracted and initially chromatographed using a preparative HPLC column (see Fig. 1A). Fifteen fractions were collected and tested, and fraction 8 eluting at 34.3 min was found to inhibit [H]isradipine binding to rat brain membranes. This fraction was rechromatographed using a semipreparative column and a shallower gradient. The eluant resolved into two major broad peaks, as well as a number of minor components (see Fig. 1B). Activity (as measured by the isradipine binding assay) was associated with the earlier eluting major peak in Fig. 1B.

Further purification was carried out using an analytical HPLC column (4.6-mm inner diameter 25-cm length). Resolution was difficult because of the presence of at least three closely migrating components (Fig. 1C). The trifluoroacetic acid/acetonitrile buffer system used in the initial steps of purification did not fully resolve these components (data not shown). The best resolution was obtained with a very slow gradient of trifluoroacetic acid/methanol buffer (see Fig. 1C for buffer conditions) using a reversed phase C18 Microsorb MV column. Using these conditions, a center cut of the middle peak reliably gave a homogeneous appearing peak on rechromatography (see Fig. 1D). Although the material in Fig. 1C did not appear chromatographically homogeneous, the amount of [H]isradipine binding activity was proportional to the protein absorbance across all peaks, suggesting that the active material might be present as multiple isoforms. The major component was purified (see Fig. 1D) and was the material that was further characterized below, which we have called conodipine-M.

Biochemical Characterization of Conodipine-M

Initial attempts at amino acid sequencing revealed that the NH terminus of the -chain is blocked. The -chain was reduced, alkylated (pyridylethylated), and purified by reversed phase HPLC. The alkylated -chain was unblocked by digestion with pyroglutamate aminopeptidase, repurified, and sequenced successfully through the first 34 residues. To analyze the remainder of the chain, digests with endoproteinase Lys-C (cleaves peptide bonds at the COOH-terminal side of Lys residues) and endoproteinase Arg-C (cleaves peptide bonds COOH-terminally at Arg residues) were performed. Fragments were isolated by reversed phase HPLC and sequenced. The -chain of conodipine-M was reduced, pyridylethylated, and sequenced. Results of the sequence analysis are summarized in Table 1. It is noteworthy that the sequence of the -chain of conodipine-M is considerably shorter than the -chain of other two-subunit PLAs (e.g. compare -bungarotoxin in Table 2). Mass spectrometry measurements of conodipine-M, however, confirmed the size of the indicated sequences of the - and -chains (see below).

In the conodipine-M sequences, a few residues could not positively be assigned (indicated by X in the tables). In several cases, these residues appear as blanks in the sequencing run. These blanks appeared in multiple sequencer runs, always in the same position. Peptides from Conus contain a number of unusual amino acids and post-translational modifications which appear as blanks during sequencing(9) . It is likely that the unassigned residues in the middle portions of the sequence represent such modifications (1 or 2 residues at the COOH terminus could not be assigned with certainty because of a diminishing signal). The size of any post-translational modification is small, however. By comparing the observed mass (measured by mass spectrometry) with the calculated mass of the sequence shown in Table 1, the average mass for the unassigned residues in the -chain is 132.2 ((8,571 - 7,910)/5) and in the -chain 137.0 ((5,036 - 4,625)/3).

Although conodipine-M potently inhibits the binding of isradipine to L-type calcium channels, several lines of evidence suggest that this might not be the result of direct competition. As shown in Fig. 3the inhibition of isradipine binding depends on the time of preincubation of conodipine-M with cortical membranes. These results raise the possibility that the inhibition may be indirect.

Figure 3: Time dependence of conodipine-M inhibition of [H]isradipine binding. Cortical rat brain membrane was incubated in the presence or absence of 2 nM conodipine-M at 37 °C for the indicated lengths of time. Specific binding was defined as the difference between [H]isradipine counts bound in the absence (total binding) and presence (nonspecific binding) of 10 µM nifedipine (see ``Experimental Procedures''). Data were acquired in duplicate and the results averaged.

The larger size of conodipine-M and the time course of isradipine binding led us to investigate whether conodipine-M had any enzymatic activity. Since many venoms have been reported to contain PLAs, we tested conodipine-M for its ability to hydrolyze phospholipids. As shown in the following section, conodipine-M is indeed a PLA.

The sequence of conodipine-M can be compared to other known PLAs. The sequence of the -chain of conodipine-M and group I-III PLAs is shown in Table 2. Although conodipine-M shows little homology to any known PLA, certain catalytic residues may be similar (see Table 2, Table 4and ``Discussion'').

Conodipine-M Has PLA Activity

Vesicles of the anionic phospholipid DMPM have been used extensively to detect and characterize the kinetics of hydrolysis of the sn-2 ester of phospholipids by PLAs(43, 56, 57) , and a brief overview of this system is given here as it relates to the action of conodipine-M on DMPM vesicles. Secreted PLAs (molecular masses of 14 kDa) bind irreversibly to DMPM vesicles and hydrolyze all of the substrate in the outer layer in a processive fashion termed scooting mode hydrolysis (enzyme does not leave the surface). The reaction progress stops when all of the outer layer substrate in enzyme-containing vesicles has been hydrolyzed. At this point the reaction products remain in enzyme-containing vesicles, and the vesicles remain structurally intact. As shown in Fig. 4, the reaction ceases after a limited amount of total DMPM is hydrolyzed by porcine pancreatic PLA. This is because the inner layer DMPM is not hydrolyzed, and there are more vesicles than enzyme, and the enzyme does not hop from one vesicle to another. Thus, the total mol of product formed is equal to the mol of catalytically active enzyme times the mol of substrate in the outer layer of a DMPM vesicle(58) .

Figure 4: Reaction progress curves for the hydrolysis of small unilamellar vesicles by 0.3 µg of porcine pancreatic PLA (solid line) and 0.3 µg of conodipine-M (dashed line). Other conditions are given under ``Experimental Procedures.''

As shown in Fig. 4, the addition of conodipine-M to DMPM vesicles leads to the immediate onset of lipolysis. With conodipine-M, the hydrolysis of DMPM vesicles does not stop abruptly, but a steady-state velocity is seen later in the reaction progress (Fig. 4). This continued reaction could be caused either by slow dissociation of conodipine-M from vesicles followed by rebinding to different vesicles (slow hopping) and/or by conodipine-M-promoted fusion of vesicles; such fusion would bring substrate into substrate-depleted, enzyme-containing vesicles. In any case, the fact that both conodipine-M- and porcine pancreatic-catalyzed reactions produce similar amounts of products before the reaction ceases (or slows) indicates that a large fraction, perhaps all, of the conodipine-M mass has lipolytic activity, and thus this activity is not due to a minor contaminant.

The addition of the cationic peptide polymyxin B leads to rapid intervesicle exchange of DMPM, and this replenishes the substrate in enzyme-containing vesicles. As a result, the initial reaction velocity (when the mol fraction of substrate in the vesicle is close to 1) is prolonged, and the progress curves are linear for several min(51) . Under such conditions conodipine-M displays a specific activity of 200 s, which is similar to that of porcine pancreatic PLA (320 s)(59) . Further support that conodipine-M has lipolytic activity comes from an additional experiment. Conodipine-M was chromatographed on an HPLC column. The lipolytic activity of each fraction was measured using the DMPM/polymyxin B assay, and as shown in Fig. 5, the profile of lipolytic activity exactly matches the peak of protein eluting from the column. Further indication of the purity of conodipine-M is evident from HPLC analysis of reduced and alkylated material. As shown in Fig. 2C, when alkylated conodipine-M (43.5 µg) is chromatographed, two sharp protein absorbances are seen (A and B chains). Contaminant peaks (if any) represent no more than 0.1% of the total protein absorbance at 220 nM.

Figure 5: Conodipine-M (23 µg) was run on a HPLC column using conditions described in panel C of Fig. 1. Fractions were collected every 30 s as indicated. Absorbance was monitored at 220 nM (solid line). One percent of each fraction was tested for PLA activity using the DMPM/polymyxin B assay described under ``Experimental Procedures.'' Activity (nmol of NaOH titrant used per min in the pH-stat) is normalized such that the maximum activity (10.6 nmol/min) is plotted at the same ordinate value as the UV absorbance. Note that the PLA activity profile (open circles) also corresponds to the protein absorbance profile.

Vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphomethanol were used to test the positional specificity of the esterase activity of conodipine-M. Such vesicles were readily hydrolyzed, and gas chromatographic analysis of the extracted fatty acid fraction shows that the major product is oleic acid (<5% of the fatty acids formed is palmitic acid). Thus, conodipine-M has little, if any, PLA activity.

Conodipine-M Is a Calcium-dependent PLA

Figure 6: Calcium dependence of the conodipine-M-catalyzed hydrolysis of [H]DPPC in DMPM vesicles. Conditions are given under ``Experimental Procedures.''

Substrate Specificity of Conodipine-M

Additional phospholipids were tested as possible substrates of conodipine-M. The reactions were carried out in 4 ml of 0.6 mM CaCl, 1 mM NaCl, pH 8.0, containing 0.6 mg of phospholipid, 20 µg of polymyxin B, and 0.07 µg of conodipine-M at 21 °C in the pH-stat. The enzyme readily hydrolyzes vesicles of 1,2-dioleoyl-sn-glycero-3-phosphomethanol, but vesicles of the D-stereoisomer were not detectably hydrolyzed. Thus, conodipine-M, like other PLAs, is stereospecific for phospholipids with the natural stereochemistry at the glycerol backbone. Under the same conditions the lysophospholipid 1-myristoyl-sn-glycero-3-phosphocholine is not a substrate. Conodipine-M hydrolyzes vesicles of the zwitterionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine, although the turnover number of 10 s is considerably less than 200 s measured with DMPM vesicles. Since fully vesicle-bound conodipine-M does not significantly discriminate between phospholipids with different polar head groups, the low turnover rate on zwitterionic vesicles may be due to poor binding of enzyme to the interface as is the case for many 14-kDa PLAs(45, 56, 61) , but this was not investigated further.

Conodipine-M has no triacylglycerol lipase activity. Adding 0.15 µg of conodipine-M to 7 mM tributyrin in 4 ml of 0.6 mM CaCl, 1 mM NaCl, 20 µg of polymyxin B, pH 8.0, at 21 °C in the pH-stat did not result in detectable hydrolysis, whereas adding 0.25 µg of pancreatic lipase gave rise to an immediate reaction. Conodipine-M also failed to hydrolyze 0.3 mM triolein in a reaction mixture containing 20 mM deoxycholate, 0.6 mM CaCl, 1 mM NaCl, pH 8.0, at 21 °C in the pH-stat. Under such conditions, hydrolysis was detected after adding pancreatic lipase.

Conodipine-M did not detectably hydrolyze [H]DPG present in DMPM vesicles, although [C]PAPC present in the same vesicles was hydrolyzed. Thus, conodipine-M has no diglyceride lipase activity.

Inhibitors of Conodipine-M

Figure 7: Inhibition of conodipine-M-catalyzed hydrolysis of DMPM vesicles by linoleoyl amide (filled circles) and MG14 (open circles) present in DMPM vesicles at the indicated mol fraction. Other conditions are given under ``Experimental Procedures.''

DISCUSSION

The results demonstrate that conodipine-M is a novel PLA; this is the first enzymatic activity extensively characterized from cone snail venom. We have named this enzyme conodipine-M and anticipate that this is only the first of a family of PLA enzymes present in these venoms. In another Conus venom, Conus caracteristicus, a related enzyme has been partially characterized. ()Thus, we propose that other PLA enzymes belonging to this family should also be referred to as conodipines (the major enzyme from C. caracteristicus would be referred to as conodipine-C; sequence variants of conodipine-M from C. magus would be referred to as conodipine-M1, conodipine-M2, etc.).

Conodipine-M was identified by an unusual route; the assay actually used to purify this PLA activity was not enzymatic activity but rather the inhibition of binding of a dihydropyridine drug to L-type calcium channels in rat brain membranes. We have demonstrated that purified conodipine-M potently inhibits [H]isradipine binding under standard assay conditions. There are two general possibilities. (i) The inhibition may be relatively nonspecific, i.e. PLA may be solubilizing the L-type calcium channel; if binding sites can no longer be filtered, this would appear as inhibition. (ii) An alternative explanation is that the PLA produces a metabolite (perhaps derived from arachidonic acid) which inhibits binding of isradipine to L-type calcium channels. These possibilities are presently being investigated. It is notable, however, that a 10-fold greater concentration of conodipine-M than is required to inhibit completely the binding of [H]isradipine to L-type calcium channels exhibits no detectable effect on the binding of I-labeled -conotoxin GVIA to N-type calcium channels, even though both assays employ filtration of membranes(55) .

Conodipine-M has two distinct polypeptide chains that are apparently linked to each other through one or more disulfide bonds; upon reduction of disulfides, conodipine-M is dissociated in two unequally sized subunits. A similar situation is found with several snake venom PLAs that have multiple subunits. In some cases, at least one of the subunits appears to be used for targeting the PLA to a particular membrane component (e.g. the -chain of bungarotoxin interacts with potassium channels)(64) . It is possible that one of the two subunits of conodipine-M is responsible for the catalytic activity, with the other subunit involved in targeting the enzyme to specific sites. The physiological function of PLA within C. magus venom remains a matter for conjecture, but it is of interest that this activity is apparently present in other Conus venoms, which specialize on different prey.

More than 50 PLAs from venoms have been sequenced to date, and three sequence groups are apparent(32) . Group I is composed of PLAs from elapid and Old World snakes. Members of group II include crotalids and viperids of the New World. Group I and group II sequences are highly homologous to each other. The major differences lie in the positioning of the one of the seven disulfides, and group II enzymes have a short COOH-terminal extension. Group III PLAs, which include the enzymes from bee and lizard venoms, are only about 20% homologous to group I and II enzyme(41) . A novel PLA, group IV, has recently been purified from the cytosol of a number of proinflammatory mammalian cells. It has a molecular weight of 85,000, and its amino acid sequence shows no homology to group I-III enzymes(21, 65, 66) .

The amino acid sequence of conodipine-M does not reveal any obvious homology with group I-IV PLAs. However, some similarity may exist in sequences including and near amino acid residues that are known to function in the active site. In group I-III PLAs a histidine residue, which functions as a general base in assisting the attack of a water molecule on the substrate ester, is followed by an aspartate that is one of the ligands for the active-site calcium cofactor. This pair of residues is flanked by 3 cysteines ( Table 2and Table 4). The -chain of conodipine-M has a histidine-aspartate pair flanked by 2 cysteines instead of three, but it is not known whether these residues function in a way analogous to those in group I-III PLAs. Group I-III PLAs have an aspartate that is hydrogen-bonded to the catalytic histidine residue and is flanked by 3 cysteines ( Table 2and Table 4). The -chain of conodipine-M has an aspartate residue with only 1 nearby cysteine ( Table 2and Table 4). The sequence Tyr/Trp-Cys-Gly-Xaa-Gly is found in all group I-III PLAs. The carbonyl oxygens of the 2 glycines of this sequence are ligands to the calcium cofactor. Conodipine-M lacks such a sequence.

Like the other enzymes, conodipine-M requires calcium for its PLA activity, although the concentration of calcium that gives 50% of maximal activity (about 20 µM) is considerably lower than typical values for group I-III enzymes (a few hundred µM). It is thus significant that conodipine-M lacks the otherwise conserved calcium binding residues. All sequence factors considered, conodipine-M clearly represents a very structurally diverse enzyme, and we therefore propose to classify it as a group V PLA.

Given the low homology to other PLAs, it is remarkable that conodipine-M displays many other features of venom PLAs as well. The phospholipid analog MG14 is a transition state analog inhibitor of group I-III enzymes, and the fact that it is also a potent inhibitor of conodipine-M suggests that the catalytic mechanism of the Conus enzyme may be similar to the other venom enzymes. Also, the pH-rate profile for conodipine-M is similar to those for group I-III enzymes. For these latter enzymes the pH dependence reflects, in part, that the active-site histidine is in the nonprotonated form to function as a general base in the catalytic reaction(67) . Conodipine-M, like other venom PLAs, does not significantly discriminate between phospholipids with different polar head groups or unsaturated versus saturated acyl chains. Further structural studies of conodipine-M will be required to understand fully its catalytic mechanism and to compare it with other known PLAs. It is not known which conodipine-M subunit contains the phospholipase activity, but as discussed above, the -chain may have similar catalytic sequence motifs. The amino acids that we have postulated as key catalytic residues need to be confirmed experimentally. Our speculation clearly predicts, however, that catalytic activity will be carried out primarily by the -chain.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants K20 MH00929 (to J. M. M.), PO1 GM 48677 (to B. M. O.), and HL36235 (to M. H. G.) and by Research Career Development Award GM562 (to M. H. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: University of Utah, 201 S. Biology, Salt Lake City, UT 84112. Tel.: 801-581-7951; Fax: 801-585-5010.

()

The abbreviations used are: PLA, phospholipase A; DMPM, 1,2-dimyristoyl-sn-glycero-3-phosphomethanol; [H]DPG, 1-palmitoyl-2-[9,10-[H]]palmitoyl-sn-glycerol; [H]DPPA, 1-palmitoyl-2-[9,10-[H]] palmitoyl-sn-glycero-3-phosphate; [H]DPPC, 1-palmitoyl-2-[9,10-[H]] palmitoyl-sn-glycero-3-phosphocholine; [H]DPPE, 1-palmitoyl-2-[9,10-[H]]palmitoyl-sn-glycero-3-phosphoethanolamine; [C]PAPC, 1-palmitoyl-2-[1-C]arachidonyl-sn-glycero-3-phosphocholine; [C]SAPI, 1stearoyl-2-[1-C]arachidonyl-sn-glycero-3-phosphoinositol; [H]SAPS, 1-stearoyl-2-[5,6,8,9,11,12,14,15-[H]]arachidonyl-sn-glycero-3-phosphoserine; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

()

S. R. Naisbitt and J. M. McIntosh, unpublished results.

ACKNOWLEDGEMENTS

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