Lys49 Phospholipase Research Articles

Lys49 phospholipase A 2 myotoxins lyse cell cultures by two distinct mechanisms

Three Lys49 phospholipase A 2 (PLA 2) myotoxins from Agkistrodon contortrix laticinctus (ACLMT), Bothrops jararacussu (bothropstoxin-I) and Bothrops asper (myotoxin II) snake venoms are enzymatically inactive on artificial substrates, yet addition of these toxins to cell cultures causes the release of fatty acids derived from the hydrolysis of membrane phospholipids. Bothropstoxin-I treated with p-bromophenacyl bromide is no longer enzymatically active on cell cultures, suggesting the toxin, not tissue PLA 2, may hydrolyze the phospholipids. The NB41A3 cell line is sensitive to lysis by ACLMT by two separate mechanisms. The first mechanism is predominant at lower concentrations of ACLMT (0.1–0.5 μM) and over long incubation periods (24 h) with toxin. This mechanism is antagonized by methylprednisolone (MePDN). The second is predominant at higher concentrations of toxin (1–5 μM) incubated over a short period (1 h) and is not antagonized by MePDN. There is no correlation between enzymatic activity and toxicity at the higher concentrations (5 μM; 1 h) when the enzymatic activity of ACLMT is compared with a noncytolytic PLA 2 from Naja naja atra venom (1 μM). However, over a 24 h period, triglyceride formation relative to fatty acids remaining free is about 10-fold greater for ACLMT (ratio about 40:1) than for the PLA 2 from Naja naja atra venom (ratio about 4:1), suggesting the two enzymes act on substrates associated with different cellular compartments under this condition. Therefore, two mechanisms of Lys49 PLA 2-induced myonecrosis exist and these are dependent on toxin concentration. The MePDN-sensitive mechanism associated with triglyceride accumulation correlates with myotoxicity.

Immunochemical Characterization and Role in Toxic Activities of Region 115–129 of Myotoxin II, a Lys49 Phospholipase A2fromBothrops asperSnake Venom

The region 115–129 of myotoxin II, a catalytically inactive Lys49 phospholipase A2, was previously shown to constitute a heparin-binding site and to be involved in its cytolytic actionin vitro.An immunochemical approach was utilized to further explore the role of this region in the toxic activities of myotoxin II. By using a carrier-linked 13-mer synthetic peptide as immunogen, rabbit polyclonal antibodies against region 115–129 were obtained. These antibodies were able to bind to the native protein and to inhibit its myotoxic and cytolytic effects in preincubation-type neutralization experiments. Antibodies to peptide 115–129 formed precipitating macromolecular complexes in gel immunodiffusion, demonstrating the oligomeric state of myotoxin II not only in its crystalline structure (dimeric), but also in solution. Analyses of the antibody response to carrier-linked peptide 115–129 and native myotoxin II suggest that region 115–129, although potentially immunogenic, is not an immunodominant B-cell epitope of this protein, failing to elicit significant antibody responses in animals immunized with the native toxin. Antibodies to peptide 115–129 cross-reacted with 15 purified class II myotoxic phospholipases A2found in snake venoms of the generaBothrops, Agkistrodon, Trimeresurus,andVipera,but not with the recombinant human class II phospholipase A2, for which no toxic actions have been described. Myotoxic phospholipases of the class I (notexin) and class III (bee venom) groups were not recognized by antibodies to p115–129. These results demonstrate that the overall antigenic structure of region 115–129 is conserved among class II myotoxic phospholipases A2, despite differences in their corresponding amino acid sequences. Based on the accumulated experimental evidence, a model of the myotoxic region of myotoxin II, and possibly of related class II Lys49 phospholipase A2myotoxins, is proposed.

Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom--synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region.

Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.

Sequence analysis of Lys49 phospholipase A 2 myotoxins: A highly conserved class of proteins

A cDNA clone coding for a putative Lys49 phospholipase A 2 myotoxin (ACL myotoxin) from Agkistrodon contortrix laticinctus was isolated from a venom gland library and sequenced. The sequence of the first 40 amino acid residues of the predicted protein matches exactly with the N-terminal sequence of the purified myotoxin. Sequence comparison of the predicted sequence of ACL myotoxin and other Lys49 and Asp49 phospholipase A 2 enzymes shows that the Lys49 phospholipase toxins form a highly conserved protein family. In addition to the change at position 49, Lys49 myotoxins have several invariant residues not found in the Asp49 group, like Lys7, Glu 12, Thrl3, Lysl6, Lys78, Lys80, Lys115, and Lys116. There are also some conserved residues in the Asp49 group that are not conserved in the Lys49 group: Tyr28, Gly32, Gly33, Gly53. Lys49 myotoxins also have a Lys-rich region in the C-terminus, which is not present in the Asp49 group. These differences clearly indicate that Lys49 myotoxins comprise a conserved and distinct class of phospholipase A 2 enzymes.

Similarities and differences in mechanisms of cardiotoxins, melittin and other myotoxins

Myonecrosis induced in vivo by cardiotoxin, melittin, and Asp49 and Lys49 phospholipase A 2 (PLA 2) myotoxins involves rapid lysis of the sarcolemma, myofibril clumping, and hypercontraction of sarcomeres. In contrast, skeletal muscle necrosis induced by crotamine and myotoxin a is much slower, consisting of mitochondrial and sarcoplasmic reticulum swelling, myofibril degeneration, and lack of sarcolemma or transverse tubule damage. The mechanisms contributing to the myonecrosis induced by these peptides were evaluated. Two cardiotoxins and two Lys49 PLA 2 myotoxins lysed primary cultures of human skeletal muscle within 24 hr at a concentration of 0.25 μM, while melittin, crotamine, and myotoxin a, and an Asp49 PLA 2 myotoxin were non-cytolytic at concentrations up to 5.0 μM, suggesting that cytolysis is not a good measure of myotoxicity. Crotamine and the Lys49 PLA 2 myotoxin altered Ca 2+ ion flux in human heavy sarcoplasmic reticulum by opening the ryanodine receptor. Whole-cell patch-clamp studies demonstrated that administrating crotamine intracellularly increased Na + currents. Free fatty acids, liberated by activation of tissue phospholipase C or by the PLA 2 activity of the myotoxins, were monitored for crotamine, myotoxin a and a Lys49 PLA 2 myotoxin in cell cultures in which the lipids had been radiolabeled. Only the Lys49 myotoxin produced significant amounts of fatty acid in cell cultures, supporting a potential role for fatty acid production only in the mechanism of sarcolemma-destroying myotoxins. These findings, coupled with those in the literature, support a hypothesis in which the myotoxins and/or products of lipase activity (e.g. fatty acids) are acting at a site existing on both the Na + channel and a protein involved in Ca 2+ release and probably serving a modulatory function for ion regulation. Based on the similarities in mechanisms between the toxins and fatty acids, the most likely site would be a fatty acid binding site on the protein (either similar to that on fatty acid binding proteins, or an acylated cysteine residue) or in the membrane.

Specificities of Asp49, Asp35 and Lys49 phospholipase A 2 enzymes on skeletal muscle lipid substrates

Specificities of Asp49, Asp35 and Lys49 phospholipase A 2 enzymes on skeletal muscle lipid substrates

No role for enzymatic activity or dantrolene-sensitive Ca 2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A 2 myotoxin

The role of low levels of phospholipase A 2 (PLA 2) activity and intracellular Ca 2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA 2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA 2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA 2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA 2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr 2+ (4.0 mM). However, the replacement of 1.8 mM Ca 2+ by 4 mM Sr 2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr 2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca 2+ release channel (ryanodine receptor) and lowered the threshold of Ca 2+-induced Ca 2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca 2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA 2 activity, or dantrolene-sensitive intracellular Ca 2+ stores, in the action of BthTX. The mechanism whereby Sr 2+ stimulates the pharmacological activity of BthTX remains to be clarified.

Membrane leakage induced by synergetic action of Lys-49 and Asp-49 Agkistrodon piscivorus piscivorus phospholipases A 2: Implications in their pharmacological activities

To understand the mechanism by which Lys-49 phospholipase A 2 from the venom of Agkistrodon piscivorus piscivorus exerts its myotoxic activities, we studied the interactions of this non-catalytic phospholipase A 2 with various phospholipid monolayers and liposomes. We measured the interactions of A. p. piscivorus Lys-49 phospholipase A 2 with phospholipid monolayers in terms of the change in surface pressure of monolayer and with liposomes in terms of the leakage of 6-carboxyfluorescein trapped in liposomes. A. p. piscivorus Lys-49 phospholipase A 2 showed the ability to rapidly and extensively induce the leakage of 6-carboxyfluorescein from anionic liposomes. The extent of leakage was sensitive to the ionic strength of medium and the surface charge density of liposomes, indicating the electrostatic nature of the interactions. Also, this protein was able to penetrate into anionic phospholipid monolayers with surface pressure of up to 38 dyn/cm, indicating the involvement of hydrophobic interactions in its binding to anionic liposomes. Another phospholipase A 2 from the same venom, Asp-49 phospholipase A 2, was shown to assist the lytic activity of Lys-49 phospholipase A 2 toward neutral liposomes by hydrolytically generating anionic patches of reaction products on the liposome surface. These results indicate that Lys-49 phospholipase A 2 binds to and disrupts anionic membranes via both electrostatic and hydrophobic interactions and that two phospholipases A 2 from the same venom work synergetically to enhance their pharmacological activities.

Crystallization and preliminary diffraction data of bothropstoxin I isolated from the venom of Bothrops jararacussu

The myotoxic Lys-49 phospholipase bothropstoxin I was crystallized, and X-ray diffraction data were collected to 3.5 Å resolution. Preliminary analysis reveals the presence of four molecules in the asymmetric unit.

Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling.

Heparin binds to phospholipase A2 myotoxins from Bothrops asper snake venom, inhibiting their toxic activities. This interaction was investigated using purified myotoxin II, a Lys-49 phospholipase A2 of this venom, and a series of heparin variants, fragments, and other glycosaminoglycans. The binding was correlated to toxin neutralization, using endothelial cells as a target. Myotoxin II binds radiolabeled heparin in solution unselectively, and forms macromolecular complexes with an optimum at a heparin:toxin molar ratio of 1:5. Both O-sulfates and N-sulfates play a role in heparin binding, in the order of importance 2-O-sulfates > 6-Osulfates > N-sulfates. The shortest heparin oligosaccharides interacting with myotoxin II are hexasaccharides. The binding of a neutralizing monoclonal antibody (MAb-3) to myotoxin II was not inhibited by heparin, indicating that the two molecules interact with different sites on the toxin. A synthetic peptide (residues 115-129 in the numbering system of Renetseder et al. (Renetseder, R., Brunie, S., Dijkstra, B. W., Drenth, J., and Sigler, P. B. (1985) J. Biol. Chem. 260, 11627-11634) of myotoxin II displays both heparin-binding and cytolytic activities. It is concluded that heparin neutralizes myotoxin II by binding to a strongly cationic site in the region of residues 115-129, a possible contribution of lysines 36 and 38 suggested by molecular modeling studies. As this cationic region appears to be responsible for the cytolytic activity of the toxin, the present report constitutes the first identification of a cytotoxic region on a phospholipase A2 myotoxin.

The dynamics of local tissue damage induced by Bothrops asper snake venom and myotoxin II on the mouse cremaster muscle: An intravital and electron microscopic study

The acute tissue damaging effects of Bothrops asper snake venom and a myotoxic Lys-49 phospholipase A 2 (myotoxin II) on the mouse cremaster muscle were studied by intravital and electron microscopy. Both venom and myotoxin induced local contractions of the muscle fibres within 10–60 sec after exposure, which disappeared after 1–2 min. This observation is consistent with the hypothesis that Bothrops myotoxins act initially at the sarcolemma by affecting its permeability and allowing an influx of calcium. The venom also induced an early but transient vasoconstriction of arterioles. The development of edema was monitored using i.v. FITC-dextran as a marker. Plasma leakage started after about 2 min of exposure to venom or myotoxin, was extensive by 4–5 min, and originated from small venules and their adjoining capillary segments. The venom induced formation of thrombi and emboli in venules, but not in arterioles. Haemorrhage appeared after 4–6 min of exposure, the bleedings always originating from capillaries and small venules. The microbleedings were explosive, appearing as rapid bursts of erythrocytes into the extravascular space, and suggesting a per rhexis type of haemorrhage. This was confirmed by electron microscopy evaluation of the same microvessels observed intravitally, which showed erythrocyte extravasation through gaps in damaged endothelial cells. Other phenomena in the microcirculation included blood-flow disturbances, crenation and sphering of erythrocytes, and stasis with dense packing of cells in capillary networks. Muscle necrosis, caused by either venom or myotoxin, started 3–4 min after application. The first sign of damage in the fibres was the development of a narrow, transverse band with local loss of striation. This was followed by slow retraction of myofibrils until there was a complete transverse rupture of the fibre. This process was often repeated along the same fibre, leaving a row of fragments separated by spaces apparently devoid of myofibrillar material. The results confirm the rapid tissue damaging effects of B. asper venom, implying that potentially useful blocking agents must be administered early and have the ability to diffuse rapidly into the tissues.

Bothropstoxin-I: Amino acid sequence and function

The complete amino acid sequence of bothropstoxin-I (BthTX-I), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-I is a Lys49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 121 amino acid residues (M(r) = 13,720), containing one methionine and 14 half-cystines. Although deprived of any detectable PLA2 activity, BthTX-I reveals a high degree of sequence homology with Asp49-PLA2s and with other Lys49-myotoxins. Critical mutations--such as Leu5 for Phe5; Gln11 for X11; Asn28 for Tyr28; Leu32 for Gly32; Lys49 for Asp49; and Asp71 for Asn71--which are apparently involved with the decreasing or elimination of PLA2 activity, have been detected. The same mutations occurred in myotoxin II from Bothrops asper venom, but five extra changes--namely, Pro90 for Ser90; Gly111 for Asn111; His120 for Tyr120; Phe124 for Leu124; and Pro132 for Ala132--have been found relative to myotoxin II.

The amino acid sequence and properties of an edema-inducing Lys-49 phospholipase A 2 homolog from the venom of Trimeresurus mucrosquamatus

Three phospholipase A 2 enzymes or homologs were purified the venom of Trimeresurus mucrosquamatus (Taiwan habu). The most abundant one was found to be a phospholipase homolog without enzyme activity, and its complete amino acid sequence was determined using oligopeptide fragments derived from digestion by endopeptidases Glu C. Asp N, Lys C and α-chymotrypsin, and by means of gas-phase sequencing. The sequence revealed that the protein belonged to the Lys-49 family of snake venom phospholipase A 2. This protein's function was characterized as edema-inducing. The Lys-49 protein has the potential to bind membrane phospholipid and Ca 2+ ( K d = 1.6·10 −4 M) as shown by ultraviolet difference spectra; however, the catalytic site appeared to be inactive and the edematous response was independent of the protein's hydrolytic activity. Mast cells and platelets were shown to be subject to activation by the Lys-49 protein.

Myotoxin II from Bothrops asper (terciopelo) venom is a lysine-49 phospholipase A 2

A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A 2, except that it shows only 0.1% of the phospholipase activity against l-α-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A 2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca 2+-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms ( Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A 2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.

The role of Asp-49 and other conserved amino acids in phospholipases A2 and their importance for enzymatic activity.

The role of aspartic acid-49 (Asp-49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions, in particular for the lysine mutant, but the affinity for substrate analogues is hardly affected. Extensive purification of naturally occurring Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself has no enzymatic activity. Our results indicate that Asp-49 is essential for the catalytic action of phospholipase A2. The importance of Asp-49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipase A2 homologues.

Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A 2 from Agkistrodon piscivorus piscivorus snake venom

The basic Lys-49 phospholipase A 2 (PLA 2) from Agkistrodon pisciuorus piscivorus venom is homologous to the basic Asp-49 PLA 2 from the same venom as well as other snake venom PLA 2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent V max of the Lys-49 PLA 2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA 2, compared to the Asp-49 PLA 2 had < 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA 2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA 2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA 2. Evaluation of the present results, along with earlier findings using Asp-49 PLA 2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: (1) The A. p. piscivorus Asp-49 PLA 2 enzyme resembles the Asp-49 enzymes fron N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA 2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. (2) In contrast to some previous suggestions, basic PLA 2 enzymes are not necessarily more toxic than neutral or acidic enzymes. (3) Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA 2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA 2 enzymes exert effects independent of phospholipid hydrolysis.

Crystallization and preliminary diffraction studies of the Lys-49 phospholipase A2 from Agkistrodon piscivorus piscivorus.

Previous chemical and structural studies have proposed a major role for Asp-49 in the calcium-mediated activation of phospholipases A2. Recently, a new class of phospholipases A2 has been characterized with a lysine in the place of aspartate at position 49 (Maraganore, J. M., Merutka, G., Cho, W., Welches, W., Kézdy, F. J., and Heinrikson, R. L. (1984) J. Biol. Chem. 259, 13839-13843; Maraganore, J. M., and Heinrikson, R. L. (1986) J. Biol. Chem. 261, 4797-4804). Although both the Lys-49 and Asp-49 phospholipases require calcium for enzymatic activity, the Lys-49 enzymes appear to be unique in their ability to bind phospholipids prior to undergoing calcium-mediated activation. We have successfully crystallized the Lys-49 phospholipase A2 from the venom of the American cottonmouth water moccasin (Agkistrodon piscivorus piscivorus). The crystals are tetragonal, the space group being P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 71.05 A, and c = 57.76 A. There is only one molecule in the asymmetric unit and the crystals provide good quality diffraction data to 2.2 A.

The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2.

A new class of phospholipases A2 that have a lysine at position 49 differ from the more conventional Asp-49 enzymes with respect to the sequential binding of the essential cofactor, calcium, and the substrate, phospholipid, in the formation of the catalytic complex (Maraganore, J.M., Merutka, G., Cho, W., Welches, W., Kézdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843). We report here the complete amino acid sequence of the Lys-49 enzyme from Agkistrodon piscivorus piscivorus. The sequence was determined by automated Edman degradation of the intact, S-carboxymethylcysteinyl protein and of peptides derived therefrom by cleavage with cyanogen bromide, chymotrypsin, trypsin, and endoproteinase Lys-C. Despite several changes at amino acid residues previously considered to be invariant, the Lys-49 enzymes are homologous to the Asp-49 phospholipases. Homology is especially apparent in the following: 1) the pattern of 14 half-cystine residues, 2) conservation of hydrophobic residues which have been shown to encircle the active site, and 3) conservation of Asp-99 and His-48 which have been implicated in the catalytic reaction itself. These observations together with kinetic and binding data imply that the Lys-49 phospholipases have a catalytic mechanism and a three-dimensional architecture similar to those of the Asp-49 enzymes. Modeling of the Lys-49 enzyme based upon the structure of bovine pancreatic phospholipase reveals that the epsilon-amino group of Lys-49 can fit easily in the calcium-binding site and, moreover, that this orientation of a cationic side chain at position 49 could account for the characteristic and novel feature of the Lys-49 phospholipases, i.e. that they are able to form complexes with phospholipid in the absence of calcium.

A new class of phospholipases A2 with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding.

We report here the discovery of a new class of phospholipases A2 in which Asp-49, a residue considered to be an obligate component of the catalytic apparatus, is replaced by a lysine. Asp-49 is invariant among the more than 30 venom and pancreatic phospholipases A2 sequenced to date, and its beta-carboxylate group has been shown to be a ligand for calcium in a binding site which also involves contributions from the peptide carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, the so-called calcium-binding loop. The change of Asp-49 to a lysine, and other substitutions in regions heretofore thought to be invariant, including the calcium-binding loop, suggested that the new phospholipases might differ functionally with respect to calcium and/or substrate binding. Indeed, although the Lys-49 phospholipases A2 show a dependence on calcium similar to that of the Asp-49 enzymes, they may be distinguished by the fact that, in the absence of phospholipid, they do not bind calcium to any measurable extent under conditions where Asp-49 enzymes bind a stoichiometric amount of calcium. Furthermore, in the absence of calcium, they show binding to single bilayer phospholipid vesicles under conditions where Asp-49 phospholipases do not bind at all. These results suggest a reversed order of addition of calcium and substrate in the formation of the ternary catalytic complex in the Lys-49 phospholipases A2. Although the mechanistic implications of these structural and functional alterations are not defined at present, it is clear that Asp-49 is not essential for phospholipase A2 catalysis and that it does not participate in the enzyme-calcium-phospholipid catalytic complex.

Endogenous prostaglandins and osmotic water flow in the toad bladder.

Endogenous prostaglandins and osmotic water flow in the toad bladder.