Protein recognition of hetero-/homoleptic ruthenium(II) tris(bipyridine)s for α-chymotrypsin and cytochrome c

We examined the relationship between the structures of hetero-/homoleptic ruthenium(II) tris(bipyridine) metal complexes (Ru(II)(bpy)(3)) and their binding properties for α-chymotrypsin (ChT) and cytochrome c (cyt c). Heteroleptic compound 1a binds to both ChT and cyt c in 1:1 ratio, whereas homoleptic 2 forms 1:2 protein complex with ChT but 1:1 complex with cyt c. These results suggest that the structure of the recognition cavity in Ru(II)(bpy)(3) can be designed for shape complementarity to the targeted proteins. In addition, Ru(II)(bpy)(3) complexes were found to be potent inhibitors of cyt c reduction and to permeate A549 cells.     

Phosphorogenic and spontaneous formation of tris(bipyridine)ruthenium in peptide scaffolds

Redox‐active ruthenium complexes have been widely used in various fields; however, the harsh conditions required for their synthesis are not always conducive to their subsequent use in biological applications. In this study, we demonstrate the spontaneous formation of a derivative of tris(bipyridine)ruthenium at 37°C through the coordination of three bipyridyl ligands incorporated into a peptide to a ruthenium ion. Specifically, we synthesized six bipyridyl‐functionalized peptides with randomly chosen sequences. The six peptides bound to ruthenium ions and exhibited similar spectroscopic and electrochemical features to tris(bipyridine)ruthenium, indicating the formation of ruthenium complexes as we anticipated. The photo‐excited triplet state of the ruthenium complex formed in the peptides exhibited an approximately 1.6‐fold longer lifetime than that of tris(bipyridine)ruthenium. We also found that the photo‐excited state of the ruthenium complexes was able to transfer an electron to methyl viologen, indicating that the ruthenium complexes formed in the peptides had the same ability to transfer charge as tris(bipyridine)ruthenium. We believe that this strategy of producing ruthenium complexes in peptides under mild conditions will pave the way for developing new metallopeptides and metalloproteins containing functional metal‐complexes.     

Synthesis and DNA binding/cleavage of mononuclear copper(II) phenanthroline/bipyridine proline complexes

The complexes [Cu(II)(phen)(L-Pro)(H2O)]+ ClO4(-) (1; phen = 1,10-phenanthroline) and [Cu(II)(bipy)(L-Pro)(H2O)]+ ClO4(-) (2; bipy = 2,2'-bipyridine) were synthesized and characterized by IR, magnetic susceptibility, UV/VIS, EPR, ESI-MS, elemental analysis, and theoretical calculations. The metal center was found in a square-pyramidal geometry. UV/VIS, thermal-denaturation, and fluorescence-spectroscopic studies were conducted to assess the interaction of the complexes with CT-DNA. An intercalative mode of binding was found, with intrinsic binding constants (Kb) of 3.86x10(3) and 4.6x10(3) M(-1) and Stern-Volmer quenching constants (K) of 0.15 and 0.11 for 1 and 2, respectively. Interestingly, none of the Cu(II) complexes was able to cleave pUC-19 DNA, which is attributed to the absence of a Pro amide H-atom and inhibition of the formation of an OH radical from the axially coordinated H2O molecule.     

Heparin reduces the alpha-helical content of cobra basic phospholipase A(2) and promotes its complex formation

The interaction of phospholipase A(2) (PLA(2)) with glycosaminoglycans (GAGs) has recently attracted attention in view of its implication on inflammation and cell proliferation. By using Fourier Transformed Infrared (FTIR) spectroscopic measurements, we demonstrate here that binding of cobra basic phospholipase A(2) from Naja nigricollis (N-PLA(2)) to heparin may induce a significant conformational change observed in the amide I region of the enzyme's alpha-helical and beta-sheet structure. It is observed that notable conformational change of N-PLA(2) due to heparin binding occurs only when heparin's chain length is at least an octasaccharide as evidenced by circular dichroism and optical density measurements. This correlation may be an important factor in the aggregation of N-PLA(2) and N-PLA(2)-heparin complexes. Heparin induced change in conformation of PLA(2) is suggested to be a notable link in understanding the diversity in PLA(2) activity when rendered to the extracellular matrix of cell membranes that is full of GAG molecules.     

Cooperative binding of monodisperse anionic amphiphiles to the i-face: phospholipase A2-paradigm for interfacial binding

Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A(2) (PLA2) to form the premicellar complexes (E(i)(#)) are characterized to discern the short-range specific interactions. Typically, E(i)(#) complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (E(i)(#), i = 1, 2, 3). In the presence of calcium, the dissociation constant K(1) for the E(1)(#) complex of PLA2 with decyl sulfate (CMC = 4500 microM) is 70 microM with a Hill coefficient n(1) = 2.1 +/- 0.2; K(2) for E(2)(#) is 750 microM with n(2) = 8 +/- 1, and K(3) for E(3)(#) is 4000 microM with an n(3) value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of E(i)(#); (c) K(i) and n(i) do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the E(i)(#) complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in E(i)(#). The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of E(i)(#). We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E and E(i)(#) aggregates is discussed.     

Exposure of bovine cytochrome c oxidase to high triton X-100 or to alkaline conditions causes a dramatic change in the rate of reduction of compound F.

The final step in the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a(3) in compound F, was investigated using a binuclear polypyridine ruthenium complex ([Ru(bipyridine)(2)](2)(1,4-bis[2-(4'-methyl-2, 2'-bipyrid-4-yl)ethenyl]benzene)(PF(6))(4)) as a photoactive reducing agent. In the untreated dimeric enzyme, the rate constant for reduction of compound F decreased from 700 s(-1) to 200 s(-1) as the pH was increased from 7.5 to 9.5. Incubation of dimeric enzyme at pH 10 led to an increase in the rate constant to 1650 s(-1), which was independent of pH between pH 7.4 and 10. This treatment resulted in a decrease in the sedimentation coefficient consistent with the irreversible conversion of the enzyme to a monomeric form. Similar results were obtained when the enzyme was incubated with Triton X-100 at pH 8.0. These treatments, which have traditionally been used to convert dimeric enzyme to monomeric form, have no effect on the steady-state activity. The data indicate that either the conversion of the bovine oxidase to a monomeric form or some structural change coincident with this conversion strongly influences the rate constant of this step in the catalytic cycle, perhaps by influencing the proton access to the heme-copper binuclear center.     

Biological activity and binding properties of [Ru(II)(dcbpy)<Subscript>2</Subscript>Cl<Subscript>2</Subscript>] complex to bovine serum albumin,phospholipase A<Subscript>2</Subscript> and glutathione

Ruthenium compounds are highly regarded as metallo-drug candidates. Many studies have focused their attention on the interaction between ruthenium complexes with their possible biological targets. The interaction of ruthenium complexes with transport proteins, enzymes and peptides is of great importance for understanding their biodistribution and mechanism of action, therefore, the development of an anti-cancer therapy involving ruthenium complexes has recently shifted from DNA targeting towards protein targeting. With the aim of gaining insight into possible interactions between ruthenium complexes with biologically relevant proteins, we have studied the interaction of cis-dichlorobis(2,2′-bipyridyl-4,4′-dicarboxylic acid)ruthenium(II) complex [Ru(II)(dcbpy)₂Cl₂], which previously showed good potency in photo-dynamic chemotherapy, with bovine serum albumin (BSA), phospholipase A₂ (PLA₂) and glutathione (GSH). Binding constants and possible number of binding sites to mentioned proteins and peptide are investigated by ultraviolet–visible spectroscopy and Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI TOF MS). The complex binding affinities were in the following order: PLA₂ > BSA > GSH. Moreover, genotoxic profile of the complex, tested on peripheral blood lymphocytes as a model system, was also promising.     

Resonance Raman and lifetime studies on regioselectively deuteriated ruthenium(II) polypyridyl complexes.

Two series of ruthenium(II) polypyridyl complexes [Ru(bipy)(2)(phpytr)](+) and [Ru(bipy)(2)(phpztr)](+) (where Hphpytr = 2-(5-phenyl-1H-[1,2,4]triazol-3-yl)-pyridine and Hphpztr = 2-(5-phenyl-1H-[1,2,4]triazol-3-yl)-pyrazine) are examined by electrochemistry, UV/Vis, emission, resonance Raman, transient resonance Raman and transient absorption spectroscopy, in order to obtain a more comprehensive understanding of their excited state electronic properties. The interpretation of the results obtained is facilitated by the availability of several isotopologues of each of the complexes examined. For the pyridine-1,2,4-triazolato based complex the lowest emissive excited state is exclusively bipy based, however, for the pyrazine based complexes excited state localisation on particular ligands shows considerable solvent and pH dependency.     

A method for the preparation of Tetrahymena thermophila phospholipase A1 suitable for large-scale production

A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation. Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis. In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416-fold increase in specific activity. This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources. This practical method for the purification of a microbial PLA1 opens the way to large-scale production of these types of enzyme, which are not as yet commercially available.     

Copper(II)-fluoroquinolone complexes with anti-Trypanosoma cruzi activity and DNA binding ability

Copper(II) complexes of fluoroquinolone antibacterial agents levofloxacin (LEV) and sparfloxacin (SPAR), containing or not a nitrogen donor heterocyclic ligand, 2,2'-bipyridine (bipy) or 1,10-phenathroline (phen), were prepared and characterized. The complexes are of the type [CuCl(2)(H(2)O)(L)], [CuCl(bipy)(L)]Cl and [CuCl(2)(phen)(L)], where L?=?LEV or SPAR. The data suggest that LEV and SPAR act as zwitterionic bidentade ligands coordinated to Cu(II) through the carboxylate and ketone oxygen atoms. The electron paramagnetic resonance spectra of the [CuCl(bipy)(L)]Cl and [CuCl(2)(phen)(L)] complexes (L?=?LEV and SPAR) in aqueous and DMSO solutions indicate mixture of mononuclear and binuclear forms. The Cu(II) complexes, together with the corresponding ligands, were evaluated for their trypanocidal activity in vitro against Trypanosoma cruzi, the causative agent of Chagas disease. The assays performed against bloodstream trypomastigotes showed that all complexes were more active than their corresponding ligands. Complexes [CuCl(2)(phen)(LEV)] and [CuCl(2)(phen)(SPAR)] were revealed, among all studied compounds, to be the most active with IC(50)?=?1.6 and 4.7?μM, respectively, both presenting a superior effect than benznidazole. The interactions of fluoroquinolones and their Cu(II) complexes with calf-thymus DNA were investigated. These compounds showed binding properties towards DNA, with moderated binding constants values, suggesting that this structure may represent a parasite target.     

Cytoxicity and Apoptotic Mechanism of Ruthenium(II) Amino Acid Complexes in Sarcoma-180 Tumor Cells

Over the past several decades, much attention has been focused on ruthenium complexes in antitumor therapy. Ruthenium is a transition metal that possesses several advantages for rational antitumor drug design and biological applications. In the present study, five ruthenium complexes containing amino acids were studied in vitro to determine their biological activity against sarcoma-180 tumor cells. The cytotoxicity of the complexes was evaluated by an MTT assay, and their mechanism of action was investigated. The results demonstrated that the five complexes inhibited the growth of the S180 tumor cell line, with IC₅₀ values ranging from 22.53 µM to 50.18 µM, and showed low cytotoxicity against normal L929 fibroblast cells. Flow cytometric analysis revealed that the [Ru(gly)(bipy)(dppb)]PF₆ complex (2) inhibited the growth of the tumor cells by inducing apoptosis, as evidenced by an increased number of Annexin V-positive cells and G0/G1 phase cell cycle arrest. Further investigation showed that complex 2 caused a loss of mitochondrial membrane potential; activated caspases 3, caspase-8, and caspase-9 and caused a change in the mRNA expression levels of caspase 3, caspase-9 as well as the bax genes. The levels of the pro-apoptotic Bcl-2 family protein Bak were increased. Thus, we demonstrated that ruthenium amino acid complexes are promising drugs against S180 tumor cells, and we recommend further investigations of their role as chemotherapeutic agents for sarcomas.     

Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions

The effects of gold(I) complexes (auranofin, triethylphosphine gold and aurothiomalate), gold(III) complexes ([Au(2,2'-diethylendiamine)Cl]Cl(2), [(Au(2-(1,1-dimethylbenzyl)-pyridine) (CH(3)COO)(2)], [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine)(OH)](PF(6)), [Au(bipy(dmb)-H)(2,6-xylidine)](PF(6))), metal ions (zinc and cadmium acetate) and metal complexes (cisplatin, zinc pyrithione and tributyltin) on mitochondrial thioredoxin reductase and mitochondrial functions have been examined. Both gold(I) and gold(III) complexes are extremely efficient inhibitors of thioredoxin reductase showing IC(50) ranging from 0.020 to 1.42 microM while metal ions and complexes not containing gold are less effective, exhibiting IC(50) going from 11.8 to 76.0 microM. At variance with thioredoxin reductase, auranofin is completely ineffective in inhibiting glutathione peroxidase and glutathione reductase, while gold(III) compounds show some effect on glutathione peroxidase. The mitochondrial respiratory chain is scarcely affected by gold compounds while the other metal complexes and metal ions, in particular zinc ion and zinc pyrithione, show a more marked inhibitory effect that is reflected on a rapid induction of membrane potential decrease that precedes swelling. Therefore, differently from gold compounds, the various metal ions and metal complexes exert their effect on different targets indicating a lower specificity. It is concluded that gold compounds are highly specific inhibitors of mitochondrial thioredoxin reductase and this action influences other functions such as membrane permeability properties. Metal ions and metal complexes markedly inhibit the activity of thioredoxin reductase although to an extent lower than that of gold compounds. They also inhibit mitochondrial respiration, decrease membrane potential and, finally, induce swelling.     

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.     

Modeling of acanthoxin A1, a PLA2 enzyme from the venom of the common death adder (Acanthophis antarcticus)

The phospholipase A2 enzyme, acanthoxin, found in the venom of the common death adder (Acanthophis antarcticus) as with other snake PLA2 enzymes displays neurotoxic activity. It is unclear whether this neurotoxic activity particular to some snake PLA2 enzymes is a result of structural differences solely within the catalytic sites or at a distant location upon the molecules. We have predicted the three-dimensional structure of one of the two predominant isoforms of acanthoxin (A1) using comparative protein modeling techniques. Given the high degree of homology and the availability of a high quality crystallographic structure, notexin was used as a molecular template to construct an all atom model of acanthoxin. The model was made using the program MODELLER3 and then refined with X-PLOR. Comparison between the predicted structure of acanthoxin and several X-ray structures of toxic and nontoxic PLA2 enzymes has led to a testable two-step proposal of neurotoxic PLA2 activity; involving the favorable binding to acceptor molecules followed by enzymatic intrusion upon the target membrane. The electrostatic potentials across the molecular surfaces of toxic and nontoxic PLA2 enzymes were calculated (GRASP) and it was found that the toxic PLA2 enzymes possessed a charge distribution on the noncatalytic surface not identified in the nontoxic PLA2 enzymes. Thus we have identified residues potentially involved in the interaction of the PLA2 enzymes with their acceptor molecules. Furthermore, the proposed acceptor molecule recognition site is distant from the catalytic site which upon binding of the PLA2 to the acceptor molecule may enhance the enzymatic ability of the toxic PLA2 enzymes on particular cell types.     

Crystal structures of the complexes of a group IIA phospholipase A2 with two natural anti-inflammatory agents, anisic acid, and atropine reveal a similar mode of binding

Secretory low molecular weight phospholipase A(2)s (PLA(2)s) are believed to be involved in the release of arachidonic acid, a precursor for the biosynthesis of pro-inflammatory eicosanoids. Therefore, the specific inhibitors of these enzymes may act as potent anti-inflammatory agents. Similarly, the compounds with known anti-inflammatory properties should act as specific inhibitors. Two plant compounds, (a) anisic acid (4-methoxy benzoic acid) and (b) atropine (8-methyl-8-azabicyclo oct-3-hydroxy-2-phenylpropanoate), have been used in various inflammatory disorders. Both compounds (a) and (b) have been found to inhibit PLA(2) activity having binding constants of 4.5 x 10(-5) M and 2.1 x 10(-8) M, respectively. A group IIA PLA(2) was isolated and purified from the venom of Daboia russelli pulchella (DRP) and its complexes were made with anisic acid and atropine. The crystal structures of the two complexes (i) and (ii) of PLA(2) with compounds (a) and (b) have been determined at 1.3 and 1.2 A resolutions, respectively. The high-quality observed electron densities for the two compounds allowed the accurate determinations of their atomic positions. The structures revealed that these compounds bound to the enzyme at the substrate - binding cleft and their positions were stabilized by networks of hydrogen bonds and hydrophobic interactions. The most characteristic interactions involving Asp 49 and His 48 were clearly observed in both complexes, although the residues that formed hydrophobic interactions with these compounds were not identical because their positions did not exactly superimpose in the large substrate-binding hydrophobic channel. Owing to a relatively small size, the structure of anisic acid did not alter upon binding to PLA(2), while that of atropine changed significantly when compared with its native crystal structure. The conformation of the protein also did not show notable changes upon the bindings of these ligands. The mode of binding of anisic acid to the present group II PLA(2) is almost identical to its binding with bovine pancreatic PLA(2) of group I. On the other hand, the binding of atropine to PLA(2) is similar to that of another plant alkaloid aristolochic acid.     

Inhibition of topoisomerase II catalytic activity by two ruthenium compounds: a ligand-dependent mode of action

The ability of two structurally different ruthenium complexes to interfere with the catalytic activity of topoisomerase II was studied to elucidate their molecular mechanism of action and relative antineoplastic activity. The first complex, [RuCl2(C6H6)(dmso)], could completely inhibit DNA relaxation activity of topoisomerase II and form a drug-induced cleavage complex. This strongly suggests that the drug interferes with topoisomerase II activity by cleavage complex formation. The bi-directional binding of [RuCl2(C6H6)(dmso)] to DNA and topoisomerase II was verified by immunoprecipitation experiments which confirmed the presence of DNA and ruthenium in the cleavage complex. The second complex, Ruthenium Salicylaldoxime, could not inhibit topoisomerase II relaxation activity appreciably and also could not induce cleavage complex formation, though its DNA-binding characteristics and antiproliferation activity were almost comparable to those of [RuCl2(C6H6)(dmso)]. The results suggest that the difference in ligands and their orientation around a metal atom may be responsible for topoisomerase II poisoning by the first complex and not by the second. A probable mechanism is proposed for [RuCl2(C6H6)(dmso)], where the ruthenium atom interacts with DNA and ligands of the metal atom form cross-links with topoisomerase II. This may facilitate the formation of a drug-induced cleavage complex.     

A spectroscopic investigation of the self-association and DNA binding properties of a series of ternary ruthenium(II) complexes

Six ruthenium(II) complexes of the general form cis-alpha-[Ru(N4-tetradentate)(N2-bidentate)]Cl2 have been synthesized from the two related tetradentate ligands 1,6-di(2'-pyridyl)-2,5-dimethyl-2,5-diazahexane (picenMe2) and 1,6-di(2'-pyridyl)-2,5-dibenzyl-2,5-diazahexane (picenBz2) and the bidentate ligands 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) and dipyrido[3,2-f:2'3'-h]quinoxaline (dpq). Synthetic intermediate species of the general form cis-alpha-[Ru(II)(N4-tetradentate)(DMSO)Cl][PF6] were isolated. The N4-tetradentate ligand picenMe2 formed only the cis-alpha stereoisomer, while picenBz2 formed both the cis-alpha and cis-beta stereoisomers. These latter stereoisomers were resolved by fractional crystallisation. Dimer self-association constants, K(D), were estimated from the concentration dependence of the 1H NMR shifts for some of these complexes in aqueous solutions at 25 degrees C. The values of K(D) ranged from 0.6 to 7.9 M(-1) and a relationship was observed between the aromatic surface area of the bidentate component and the degree to which self-association occurred, whereby a greater level of self-association correlates with a larger surface area for the bidentate ligand. Some of these complexes demonstrate an ability to bind to DNA that is consistent with intercalation of the bidentate molecular component between the base pairs of the DNA molecule. Using calf-thymus DNA, the equilibrium binding constants, K(B), were determined for some of the complexes using intrinsic methods and these ranged from 3.32 to 5.11 M(-1), the intercalating abilities of the different bidentate ligands being in the order dp q > phen > bipy. This relationship between aromatic surface area of the bidentate ligand and the degree of DNA binding activity is the same as that observed in the self-association study.     

Artificial peptides with unnatural components designed for materializing protein function

In order to design functional peptides, we employed two strategies. The first one is to incorporate rigid unnatural amino acids into peptides to make the peptide backbone rigid. Functions were expected to appear through the conformational control by the strategy. A series of cyclic peptides constituted of alternating natural amino acids and 3-aminobenzoic acid, used as an unnatural amino acid, were synthesized. These cyclic peptides were found to function as strong binders for phosphomonoester, catalysts for ester hydrolysis, and/or ion channels. The second strategy is to conjugate peptides with unnatural and inherently functional molecules. Following this strategy, oligo(L-leucine)- or oligo(L-phenylalanine)-modified ruthenium tris(bipyridine) complexes were synthesized. Distance dependence of the photoinduced electron transfer from the ruthenium complexes and the function as sensors for phosphate anion (H(2)PO(-)(4)) are discussed.     

Migration of vascular smooth muscle cells by phospholipase A2 via specific binding sites.

Pancreatic-type group I phospholipase A2 (PLA2-I), EC 3.1.1.4, long thought to act as a digestive enzyme, has a specific binding site in several types of tissues and cells and these sites promote PLA2-I-stimulated DNA synthesis. In this study we report a PLA2-I action on the migration of rat embryonic thoracic aorta smooth muscle cells (A7r5). A7r5 cells had a single class of PLA2-I binding site with an equilibrium binding constant (Kd) value of 1.7 nM and a maximum binding capacity (Bmax) of 40,000 sites/cell. The migration activity of PLA2-I for A7r5 cells was examined using modified Boyden chambers. PLA2-I stimulated the migration dose-dependently, and the ED50 value was about 1 nM, which was almost the same as the Kd value for PLA2-I binding. Checkerboard analysis showed that the response of A7r5 cells to PLA2-I was chemokinetic, but not chemotactic. These findings reveal a new aspect of PLA2-I in the modulation of vascular function.     

1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid

1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of lysophospholipid.