Benzylideneacetone, an eicosanoid biosynthesis inhibitor enhances baculovirus pathogenicity in the diamondback moth, Plutella xylostella

Benzylideneacetone (BZA) is a monoterpenoid compound produced by an entomopathogenic bacterium, Xenorhabdus nematophila. BZA inhibits phospholipase A₂ to suppress biosynthesis of eicosanoids that mediate immune responses in insects. In response to per os infection of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), the diamondback moth, Plutella xylostella, developed red spots on the midgut epithelium. The midgut exhibiting red spot formation suffered abnormal cell integrity, such as genomic DNA fragmentation and condensed spots in the nucleoplasm. The number of red spots increased with viral dose and incubation time after the viral treatment. BZA inhibited the formation of the midgut red spots in a dose-dependent manner. However, the inhibitory effect of BZA on the red spot formation was reversed by addition of arachidonic acid, suggesting that the red spot response may be mediated by eicosanoids. BZA treatment resulted in significant enhancement of AcMNPV occlusion body (OB) pathogenicity to P. xylostella.     

Dexamethasone,a Specific PLA2 Inhibitor,Allows Nonpathogenic Pseudomonas fluorescens to Induce Virulence Against Spodoptera exigua

Phospholipase A₂ (PLA₂) catalyzes phospholipids at sn-2 position to release arachidonic acid (20:4n-6). The arachidonic acid is further oxidized to form different eicosanoids, which play biological mediators to express cellular or humoral immune reactions in response to pathogen infection. Xeno-rahbdus and Photorhabdus, the symbiotic bacteria of entomopathogenic nematodes, have been known to inhibit PLA₂ to express their pathogenicity. This research aimed to test a hypothesis that other entomopathogenic bacteria also inhibit PLA₂ to express their pathogenicity in Spodopera exigua. Two bacterial species of Enterococcus faecalis and Pseudomonas fluorescens presumably different in ento-mopathogenicity were analyzed in their PLA₂ inhibitory activities. A pathogenic E. faecalis induced significantly immunodepression of S. exigua by inhibiting PLA₂ activity because the bacteria-infected S. exigua recovered immune reactions after the addition of arachidonic acid. However, the nonpathogenic P. fluorescens did not induce immunodepression because the addition of arachidonic acid to P. fluorescens-infected S. exigua did not further increase immune capacities while dexamethasone, a PLA₂ inhibitor, could decrease the immune activities. Injection of E. faecalis along with 10 μg of dexamethasone significantly increased pathogenicity in comparison with the bacteria alone. Moreover, the addition of dexamethasone transformed nonpathogenic P. fluorescens into pathogenic bacterium. This study suggests an evidence that PLA₂ is an inhibitory target even for entomopathogenic bacteria not related with entomopathogenic nematodes, and that the inhibition of PLA₂ determines the bacterial virulence in S. exigua.     

Benzylideneacetone suppresses both cellular and humoral immune responses of Spodoptera exigua and enhances fungal pathogenicity

An entomopathogenic fungus, Beauveria bassiana, had significant insecticidal activity against the beet armyworm, Spodoptera exigua. However, it took almost one week to cause significant mortality. This study used a mixture treatment with an immunosuppressant to enhance the fungal pathogenicity. A bacterial metabolite, benzylideneacetone (BZA), had a significant synergistic effect on the fungal pathogenicity against S. exigua, although it had little insecticidal activity by itself. The mixture treatment shortened median lethal time of B. bassiana by approximately 2 days. The synergistic activity of BZA on the pathogenicity of B. bassiana was induced by its immunosuppressive effects on both cellular and humoral antifungal responses of S. exigua. In response to B. bassiana, S. exigua larvae can form hemocytic nodules. Nodules were significantly suppressed by BZA treatment. Moreover, BZA inhibited expression of some antimicrobial peptide genes of S. exigua in response to fungal challenge. The immunosuppressive condition induced by BZA allowed B. bassiana to easily colonize and multiply in the hemocoel of treated larvae, which resulted in significant enhancement of the pathogenicity of B. bassiana.     

Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Phospholipase A₂ (PLA₂) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA₂ and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA₂ and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA₂. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA₂ and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA₂ activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca²⁺ binding and antigenicity of Lys-6-modified PLA₂ were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA₂ to 32% of that of PLA₂. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA₂ activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA₂ is almost the same as that of native PLA₂. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA₂, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA₂.     

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A₂ (PLA₂s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA₂ inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA₂s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated αBaltMIP, showed it to be a glycoprotein with Mr of ∼24,000 for the monomeric subunit. CD spectra of the PLA₂/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of αBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization.     

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins’ mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD₅₀ concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.     

Modification of Lys-6 and Lys-65 Affects the Structural Stability of Taiwan Cobra Phospholipase A2

To assess whether chemical modification of phospholipase A₂ (PLA₂) enzymes may affect their fine structure and consequently alter their enzymatic activity, the present study was carried out. Both Lys-6 and Lys-65 in the Taiwan cobra (Naja naja atra) PLA₂ were selectively modified with trinitrobenzene sulfonate and pyridoxal-5′-phosphate (PLP), respectively. Incorporation of either trinitrophenylated (TNP) or PLP groups on Lys-6 and Lys-65 caused a drop in PLA₂ activity, but the Ca²⁺-binding ability and global conformation of modified derivatives were not significantly different from that of native enzyme. A distinct enhancement of stability was observed with native PLA₂ when thermal unfolding was conducted in the presence of 20 mM Ca²⁺. Conformational transition induced by guanidine hydrochloride was also attenuated by the addition of Ca²⁺. Conversely, a marked decrease in the structural stability was noted with modified derivatives, and the enhancing effect of Ca²⁺ pronouncedly decreased. Together with the finding that the incorporated TNP and PLP groups did not equally affect enzymatic activity and structural stability of PLA₂, our data suggest that an alteration in the fine structure owing to the incorporated groups should contribute to the observed decrease in PLA₂ activity.     

Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Phospholipase A₂ (PLA₂) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA₂ and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA₂ and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA₂. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA₂ and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA₂ activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca²⁺ binding and antigenicity of Lys-6-modified PLA₂ were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA₂ to 32% of that of PLA₂. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA₂ activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA₂ is almost the same as that of native PLA₂. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA₂, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA₂.     

Preliminary Characterization of Phospholipase A2 in Lagenidium giganteum

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A₂ in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A₂ (PLA₂) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA₂ activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA₂ activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-¹⁴C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ all enhanced PLA² activity, while Co²⁺, Fe²⁺, and Zn²⁺ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca²⁺ and Mn²⁺ than Mg²⁺, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA₂ activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA₂ hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA₂ activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA₂ activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.     

Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.

Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in β-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC₅₀ value was 0.0103 μg.mL^?1. The two mutations in β-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.     

Changes in PLA(2) activity after interacting with anti-inflammatory drugs and model membranes: evidence for the involvement of tryptophan residues

Phospholipase A₂ (PLA₂) lipolytic activity can be regarded as a limiting factor for the development of inflammatory processes by restricting the production of pro-inflammatory mediators, hence representing a valuable therapeutic target for drugs that are able to modulate the activity of this enzyme. In the current work, the hydrolysis of phospholipids by PLA₂ was monitored with acrylodan-labelled intestinal fatty acid binding protein (ADIFAB) and this fluorescence based technique was also used to access the enzymatic inhibitory effect of non-steroidal anti-inflammatory drugs (NSAIDs). The intrinsic fluorescence of PLA₂ tryptophan residues was further used to gain complementary information regarding the accessibility of these residues on the PLA₂ structure upon interaction with the NSAIDs tested; and to calculate the NSAIDs-PLA₂ binding constants. Finally, circular dichroism (CD) measurements were performed to evaluate changes in PLA₂ conformation resultant from the inhibitory effect of the drugs tested. Overall, results gathered in this study point to the conclusion that the studied NSAIDs inhibit PLA₂ activity due to a disturbance of the enzyme binding efficiency to membrane interface possibly by a shielding effect of the Trp residues required for the membrane interfacial binding step that precedes lipolysis process.     

cDNA cloning and bacterial expression of phospholipase A2 inhibitor PLIα from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus1

The cDNA encoding of a phospholipase A₂ inhibitor (PLIα) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIα. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIα. The PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIα expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIα fusion protein underwent folding to form a trimeric structure like the intact PLIα, and showed inhibitory activity against the group II acidic PLA₂ from A. blomhoffii siniticus venom; although its binding constant (1/K_i) value was 30-fold lower than that of the natural PLIα. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIα against the acidic PLA₂. Furthermore, the carbohydrate chains of the natural PLIα were found to play an important role in the inhibitory activity against the basic PLA₂.     

Effects of stilbene optical brighteners on the insecticidal activity of Bacillus thuringiensis and a single nucleopolyhedrovirus on Helicoverpa armigera

The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC₅₀ values) but their effect on Bacillus thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and occlusion bodies (OBs) of a Spanish isolate of H. armigera single nucleocapsid nucleopolyhedrovirus (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD-1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its insecticidal activity. Mixtures of HearNPV-SP1 OBs with Tinopal UNPA-GX or Leucophor UO resulted in 31.4- and 11.4-fold increases in pathogenicity, respectively, at 1%, and 11.4- and 6.3-fold increases in pathogenicity, respectively, at 0.1%, compared to the OBs alone. However, none of these brighteners increased Bt HD-1 activity. These results appear consistent with the hypothesis that the enhancement of HearNPV-SP1 pathogenicity and the null or antagonistic effects observed in Bt HD-1 against H. armigera were due to optical brightener-mediated degradation of the peritrophic membrane, but additional systematic studies involving a broad range of brighteners and electron microscope observations are required to confirm this premise.     

Phospholipase A2 has a role in proliferation but not in differentiation of HL-60 cells

The role of phospholipase A₂ (PLA₂) and its metabolite arachidonic acid (AA) in the proliferation and differentiation of HL-60 cells was investigated. Addition of either 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) or retinoic acid (RA) to HL-60 cells for 2 h inhibited PMA-stimulated PLA₂ activity measured by [³H]AA release. The inhibitor of PLA₂ activity, p-bromophenacyl bromide (BPB), significantly inhibited the proliferation of HL-60 cells and of fibroblast L929 and Swiss 3T3 cells in a dose-dependent manner. The effect of BPB on proliferation is probably through its inhibitory effect on PLA₂ activity, since the same doses of BPB which inhibited proliferation also inhibited PLA₂ activity determined by [³H]AA release. The importance of PLA₂ activity for cell growth was further supported by the effect of two other PLA₂ inhibitors, AACOCF₃ and scalaradial, which inhibited HL-60 proliferation in a dose-dependent manner. BPB, AACOCF₃ and scalaradial significantly increased the doubling time to 32.4 h, 34.0 h and 31.8 h, respectively, compared with 24.6 h in the control. The inhibitory effect of BPB on HL-60 proliferation was reversed by addition of exogenous free AA to HL-60 cells, indicating the importance of this metabolite for the proliferation process. This reversible effect is specific for AA since it was not achieved by other fatty acids like linolenic acid (LA) or oleic acid (OA). Addition of free AA to HL-60 cells did not induce differentiation, as expected. Although BPB, AACOCF₃, or scalaradial inhibited proliferation, they did not induce differentiation nor affect the differentiation induced by 1,25(OH)₂D₃ or RA. These results implicate that PLA₂ activity has no regulatory role in differentiation of HL-60 cells. The differential effect of PLA₂ inhibitors on proliferation and differentiation of HL-60 cells suggests that these two processes function under different regulatory mechanisms.     

Effects of temperature on baseline susceptibility and stability of insecticide resistance against Plutella xylostella (Lepidoptera: Plutellidae) in the absence of selection pressure

Plutella xylostella L. (Lepidoptera: Plutellidae) is an important pest causing significant losses to vegetables worldwide. Insecticides resistance in P. xylostella is a serious issue for scientists since last 30 years. However, deltamethrin and Bt Cry1Ac are commonly used insecticides against P. xylostella but studies involving development of resistance in P. xylostella against these two insecticides at different temperatures are lacking. The current study was aimed to find out the toxicity of deltamethrin and Bt Cry1Ac, and resistance development in P. xylostella. Results showed that the positive correlation between the temperature and toxicities of deltamethrin and Bt Cry1Ac. The results indicated −0.051, −0.049, −0.047, and −0.046 folds of deltamethrin resistance at 15 °C, 20 °C, 25 °C, and 30 °C temperatures, respectively from 1^st to 12^th generations. The toxicity of Bt Cry1Ac after 24 h was 2.2 and 4.8 folds on 1^st generation at 20 °C and 25 °C temperatures, respectively compared to the toxicity recorded at 15 °C (non-overlapping of 95% confidence limits). Based on the results of this study, it is concluded that the temperature has a positive correlation with the toxicity of deltamethrin and Bt Cry1Ac against the larvae of P. xylostella. This study suggests that deltamethrin and Bt Cry1Ac can be included in the management program of P. xylostella on many vegetable crops. The baseline susceptibility data might be helpful to understand the resistance mechanisms in P. xylostella.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

The Structural Variations of ε-Amino Groups in Phospholipase A2 Enzymes from Naja naja atra and Bungarus Multicinctus Venoms

Comparative studies on Naja naja atra phospholipase A₂ (NNA-PLA₂), Bungarus multicinctus phospholipase A₂ (BM-PLA₂), and their Lys-modified derivatives were made to assess the differences in the fine structures around the conserved Lys residues of PLA₂ enzymes. It was found that the accessibility of Lys residues of PLA₂ enzymes toward modified reagent, trinitrobenzene sulfonate, were not the same. Moreover, the extent of decrease in pI values of PLA₂ enzymes that resulted from trinitrophenylation of lysine residues was different between NNA-PLA₂ and BM-PLA₂. The Lys-6 of BM-PLA₂ mostly contributed to the positively charged character of the enzyme molecule, whereas the contribution of Lys-6 of NNA-PLA₂ to its molecular charge was not notably different from other Lys residues. A linear relationship was observed by plotting the mobilities of PLA₂ enzymes and their TNP derivatives against their pI values. However, native and Lys-modified NNA-PLA₂ were not aligned with those of BM-PLA₂ in the same line. Apparently the gross conformation of PLA₂ enzymes was not notably perturbed by the modification of Lys residues, but the fine structure of NNA-PLA₂ was not the same as that of BM-PLA₂. These results indicate that the positioning of side chains of the conserved Lys residues in the two PLA₂ enzymes is essentially different, and suggest that the variations in the fine structures of homologous proteins could be effectively explored by chemical modification studies and electrophoretic analysis.     

PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

Phospholipase A₂ (PLA₂) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA₂-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca²⁺-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 μg/ml significantly stimulated the PLA₂ activity and the release of free arachidonic acid (AA) from 1-acyl 2-¹⁴C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA₂ hyperactivity is Ca²⁺-dependent. There was no considerable alteration in the PLA₂ activity by stimulation of the activity by tyrosine kinase (with vanadate, H₂0₂), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF₄ thus in Tetrahymena PLA₂ activity seems to be independent of these—in Tetrahymena (also functioning)—signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA₂ activity is in connection with the phospholipid metabolism of Tetrahymena.