Limitations of phosphatidylcholine/deoxycholate mixtures for the analysis of phospholipase A2 inhibition and activation: illustration with annexins.

The effects of human recombinant lipocortin I (annexin I) and bovine lung calpactin I (annexin II) on porcine pancreatic phospholipase A2 (PLA2) activity in phosphatidylcholine (PC)/deoxycholate (DOC) mixtures were investigated. Annexin-associated decreases in PLA2 activity were observed under some conditions, for example, at high DOC/PC molar ratios; however, activation was observed under other conditions. NaCl, which lowers the non-critical micellar concentration (NCMC) of deoxycholate, caused significant decreases in control PLA2 activity in the absence of annexins, and greater decreases in PLA2 activity in annexin-containing samples, resulting in an apparent increase in inhibition. The PC/DOC substrate mixtures themselves appeared unstable. Despite a large excess of detergent, precipitates were, at times, observed upon incubation of some PC/DOC mixtures at 37 degrees C. Such behavior is of interest in view of the numerous reports of PLA2 inhibition by annexins and annexin-derived peptides in the PC/DOC system. The influence of the annexins on activity in this system is consistent with effects on the phase behavior of the PC/DOC mixture and/or competition with the enzyme for available Ca2+. These results caution against use of the PC/DOC system for analysis of potential PLA2 inhibitors unless the phase behavior of the system is more fully delineated.     

Nitroarginine does not inhibit lysophosphatidylcholine (LPC)-induced vascular relaxation and accumulation of cyclic GMP.

Lysophosphatidylcholine (LPC) is a potent endothelium-dependent vascular smooth muscle relaxant. The possibility that its action is mediated through endothelium-derived nitric oxide (EDNO), although suggestive, has not been proven. Both lysophosphatidylcholine and endothelium-derived nitric oxide relax by activating guanylate cyclase to form cyclic GMP. Based on the finding that EDNO formation is inhibited by NNA (N-omega-nitro-L-arginine), we followed cyclic GMP changes in bovine intrapulmonary arteries with LPC after incubation with NNA. Inhibition of cyclic GMP by LPC following NNA exposure would be suggestive of the production of EDNO by LPC. However, while NNA significantly inhibited accumulation of cyclic GMP after exposure to the calcium ionophore A23187 which releases EDNO, NNA failed to inhibit LPC-induced accumulation of cyclic GMP. The results indicate that LPC relaxes vascular smooth muscle through a non NO-mediated pathway.     

Purification and regiospecificity of multiple enzyme activities of phospholipase A(1) from bonito muscle

Phospholipase A(1) (PLA(1)), which catalyzes the hydrolysis of the sn-1 ester bond of diacyl phospholipids, was purified from 100,000 x g supernatant of bonito muscle to homogeneity by ammonium-sulfate precipitation and four consecutive column chromatographies (DEAE anion-exchange, ether-Toyopeal, hydroxylapatite and Toyopeal HW 50S columns). The final preparation showed a single band above the 67-kDa molecular marker on SDS-PAGE, and the molecular mass was determined to be 71.5 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using bovine serum albumin as a standard for calibration. The N-terminal 8 amino residues were determined to be Ala-Pro-Ala-Glu-Lys-Val-Lys-Try. Regiospecificity of multiple enzyme activities of the PLA(1) was examined using positionally defined synthetic phosphatidylcholine (PC) and lysophosphatidylcholines (LPC). An acyl ester bond at the sn-1 position of PC was exclusively hydrolyzed by phospholipase activity, and 1-acyl LPC was cleaved to fatty acid and glycerophosphocholine by lysophospholipase (LPL) activity. However, the positional isomer, 2-acyl LPC was a poor substrate for LPL activity. PC/transacylation activity was also observed when excess 2-acyl LPC was supplied in the reaction mixture, and fatty acid at the sn-1 position of donor PC was transferred to the sn-1 position of acceptor LPC. These results demonstrate that the multiple enzyme activities of PLA(1), this is lysophospholipase, transacylase as well as phospholipase, have a strict regiospecificity at the sn-1 position of substrates.     

Metabolism of lysophosphatidyl ethanolamine and lysophosphatidyl choline by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages

A comparison has been made between the conversion of (32)P-labeled lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl choline (LPC) to their respective acylated and deacylated derivatives by homogenates of rabbit polymorphonuclear leukocytes and alveolar macrophages. Synthesis of PE by both homogenates and of PC by macrophage homogenates proceeded to about the same extent and is attributed to direct acylation of the lyso compounds. At higher LPC concentrations formation of PC by leukocytes is far greater than by macrophages. The mechanism of this enhanced synthesis of PC, which is brought out by higher substrate concentrations, is believed to be a transfer of the acyl group of one LPC molecule to another. Under optimal conditions macrophage homogenates deacylated LPE to a greater extent than LPC, while the reverse was true for leukocyte homogenates. Albumin inhibited deacylation of LPC and its conversion to PC by leukocytes, perhaps by binding the substrate (2 moles of LPC per mole of albumin). Other effects of albumin-stimulation of deacylation and acylation of LPE by macrophages, inhibition of deacylation and acylation of LPE by leukocytes-remain unexplained.     

Atriopeptin II relaxes and elevates cGMP in bovine pulmonary artery but not vein

Atriopeptin II, a 23-amino acid synthetic peptide fragment of atrial natriuretic factor, caused an endothelium-independent relaxation of isolated precontracted rings of bovine intrapulmonary artery that was accompanied by the concomitant accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) but not adenosine 3',5'-cyclic monophosphate. In contrast, rings of intrapulmonary vein were unaffected by atriopeptin II whether or not endothelium was present. Whereas methylene blue, an inhibitor of soluble guanylate cyclase, abolishes endothelium-dependent and independent arterial relaxation and cGMP accumulation in response to acetylcholine and glyceryl trinitrate, respectively, methylene blue failed to alter these responses to atriopeptin II. Similarly, the effects of atriopeptin II were unaltered by propranolol, indomethacin, or atropine. These results indicate that relaxation elicited by atriopeptin II may be selective for arterial smooth muscle receptors, does not require endothelial cells, and does not involve the soluble form of guanylate cyclase, although cGMP accumulation is stimulated.     

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for monitoring the digestion of phosphatidylcholine by pancreatic phospholipase A(2)

Different methods were established for monitoring the phospholipase A(2)(PLA(2)) activity but all of them are rather cumbersome and time consuming. In this paper we have investigated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the determination of the PLA(2) activity. Phosphatidylcholine (PC) was digested with pancreatic PLA(2) under different conditions, i.e., various Ca(2+), PC, and PLA(2) concentrations. The digestion products were analyzed by MALDI-TOF MS and the concentration of lysophosphatidylcholine (LPC)-generated upon PLA(2) digestion-was determined by the application of an internal standard (known concentration) and by a comparison of their signal-to-noise ratios. The results clearly demonstrate that the LPC concentration determined from the MALDI-TOF mass spectra correlates directly with the activity of the applied enzyme. Additionally, LPC concentration increased with an increase in Ca(2+), as well as in the PC concentration. A single MALDI-TOF mass spectrum provides immediate information on the digestion products as well as on the residual substrate without requirements for any previous derivatization. MALDI-TOF MS can be easily and simply applied for monitoring the PLA(2) activity and we assume that this method might also be useful for other types of phospholipases.     

Progesterone primes zona pellucida-induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.     

Krabbe disease: psychosine-mediated activation of phospholipase A2 in oligodendrocyte cell death

Globoid cell leukodystrophy (Krabbe disease) is an inherited neurological disorder caused by the pathogenomic accumulation of psychosine (galactosylsphingosine), a substrate for the deficient enzyme galactocerebroside beta-galactosidase. This study underscores the mechanism of action of psychosine in the regulation of oligodendrocyte cell death via the generation of lysophosphatidylcholine (LPC) and arachidonic acid (AA) by the activation of secretory phospholipase A2 (sPLA2). There was a significant increase in the level of LPC, indicating a phospholipase A2 (PLA2)-dependent pathobiology, in the brains of Krabbe disease patients and those of twitcher mice, an animal model of Krabbe disease. In vitro studies of the treatment of primary oligodendrocytes and the oligodendrocyte MO3.13 cell line with psychosine also showed the generation of LPC and the release of AA in a dose- and time-dependent manner, indicating psychosine-induced activation of PLA2. Studies with various pharmacological inhibitors of cytosolic phospholipase A2 and sPLA2 and psychosine-mediated induction of sPLA2 enzymatic activity in media supernatant suggest that psychosine-induced release of AA and generation of LPC is mainly contributed by sPLA2. An inhibitor of sPLA2, 7,7-dimethyl eicosadienoic acid, completely attenuated the psychosine-mediated accumulation of LPC levels, release of AA, and generation of reactive oxygen species, and blocked oligodendroyte cell death, as evident from cell survival, DNA fragmentation, and caspase 3 activity assays. This study documents for the first time that psychosine-induced cell death is mediated via the sPLA2 signaling pathway and that inhibitors of sPLA2 may hold a therapeutic potential for protection against oligodendrocyte cell death and resulting demyelination in Krabbe disease.     

Use of short-chain cyclopentano-phosphatidylcholines to probe the mode of activation of phospholipase A2 from bovine pancreas and bee venom

A great mystery in the mechanism of phospholipase A2 (PLA2) and many other lipolytic enzymes is the "interfacial activation" induced by micellar but not monomeric substrates. Equally mysterious is the lack of interfacial activation in bee venom PLA2, as opposed to PLA2s from pancreas and other sources. We have probed these problems using the conformationally restricted short-chain cyclopentano-analogues of diacylphosphatidylcholine (Cp-DCnPC, all-trans isomer). In the reaction catalyzed by bovine pancreatic PLA2, Cp-DC8PC behaved differently from DC8PC in that its monomers and micelles showed comparable activities (but lower than the activity of DC8PC). This result suggests that the activity of PLA2 can be regulated by substrate conformation and supports the "substrate conformation model" (Wells, M. A. (1974) Biochemistry 13, 2248-2257), but raises a question as to whether Cp-DC8PC mimics monomers or micelles of DC8PC. Conformational analysis by 1H NMR revealed that monomeric Cp-DC8PC was conformationally restricted near the carbonyl region, a property characteristic of micelles. Thus, monomeric CP-DC8PC can be considered as a conformational analogue of micelles, but the important structural feature lies in the CH2COO region instead of the glycerol backbone. CP-DC8PC was then used to test a previous proposal that the bee venom PLA2 hydrolyzes monomers but not micelles (which would predict little or no activity for Cp-DC8PC since its conformation is micelle-like whether below or above its critical micelle concentration). The results showed that Cp-DC8PC is a relatively good substrate for the bee venom PLA2 in comparison with the pancreatic PLA2. This and other evidence together suggest that the bee venom PLA2 is not sensitive to the conformation of monomeric and micellar substrates and hydrolyzes both monomers and micelles. The results in both PLA2s demonstrate the usefulness of cyclopentano-phospholipids in probing the mechanism of phospholipases and the roles of substrate conformation in the catalysis of PLA2.     

Involvement of tetrahydrobiopterin in local change of endothelium-dependent vasorelaxation in pulmonary hypertension

A deficiency of tetrahydrobiopterin (BH4), a NO-synthase co-factor, results in reactive oxygen species synthesis by NO-synthase. It leads to disturbances of endothelium-dependent vasorelaxation. We performed our study on the monocrotaline model of pulmonary hypertension. A decrease in endothelium-dependent relaxation was observed only in intrapulmonary arteries of monocrotaline-treated rats. A perfusion of BH4 (0.1 mol/liter) increased significantly endothelium-dependent dilation of hypertensive pulmonary arteries (p < 0.01). But BH4 did not influence the relaxation of systemic vessels and the dilation responses of pulmonary and systemic arteries of control rats. Measuring of superoxide by lucigenin-mediated chemiluminescence showed five-fold O2- production in intrapulmonary arteries of pulmonary hypertensive rats, that was activated by acetylcholine and inhibited by a nonselective NO-synthase blocker (L-NAME). However, activity of NO-synthase measured as [H3]arginine to [H3]citrulline conversion and assessed in pulmonary vessels and aortic tissue, did not differ in control and monocrotaline-treated groups. These data suggest, that there is a local deficiency of BH4--in pulmonary vessels, without significant changes of systemic circulation.     

Synthesis of structured phospholipids by immobilized phospholipase A2 catalyzed acidolysis

Acyl modification of the sn-2 position in phospholipids (PLs) was conducted by acidolysis reaction using immobilized phospholipase A(2) (PLA(2)) as the catalyst. In the first stage we screened different carriers for their ability to immobilize PLA(2). Several carriers were able to fix the enzyme and maintain catalytic activity; however, the final choice of carrier for the continued work was a non-ionic weakly polar macroreticular resin. Response surface methodology was applied to evaluate the influence of substrate ratio, reaction temperature, and water addition during acidolysis reaction between caprylic acid and soybean phosphatidylcholine (PC). Reaction temperature and water addition had significant effect on acidolysis reaction, however no effect was observed for substrate ratio (mol caprylic acid/mol PC) in range tested. In general an inverse relationship between incorporation of caprylic acid and PC recovery was observed. Highest incorporation obtained during acidolysis reactions was 36%. Such incorporation could be obtained under reaction temperature, 45 degrees C; substrate ratio, 9mol/mol caprylic acid/PC; water addition of 2%; 30wt.% immobilized enzyme; and reaction time, 48h. The yield under these conditions was however only 29%. Lysophosphatidylcholine (LPC) was the major by-product formed during the reaction. Incorporation of acyl donor into LPC was very low (<4%), which indicates that acyl migration is only a minor problem for PLA(2) catalyzed synthesis reaction. Conjugated linoleic acid and docosahexaenoic acid were also tested as acyl donors, and were able to be incorporated into PC with 30 and 20%, respectively.     

Secretory phospholipases A2 induce neurite outgrowth in PC12 cells through lysophosphatidylcholine generation and activation of G2A receptor

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A.     

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.     

Procyanidins in crataegus extract evoke endothelium-dependent vasorelaxation in rat aorta

The extract of Crataegus, a mixture of flavonoids and procyanidins extracted from hawthorn, Crataegus oxyacantha, L. and C. monogyna Jacq., relaxed vascular tone or increased production of cyclic GMP in the rat aorta, but flavonoid components of Crataegus extract, hyperoside, rutin and vitexin, did not affect the vascular tone. The aim of the present study was to characterize the endothelium-dependent relaxation elicited by procyanidins fractionated from Crataegus extract in isolated rat aorta. Procyanidins caused endothelium-dependent relaxation which was associated with the production of cyclic GMP. Both responses to these procyanidins were inhibited by methylene blue or N(G)-nitro-L-arginine, but not by indomethacin. Relaxation in response to procyanidins was not affected by atropine, diphenhydramine, [D-Pro2,D-Trp7,9]substance P, propranolol, nifedipine, verapamil and glibenclamide, but were markedly reduced by tetraethylammonium. These findings showed that procyanidins in Crataegus extract may be responsible for the endothelium-dependent nitric oxide-mediated relaxation in isolated rat aorta, possibly via activation of tetraethylammonium-sensitive K+ channels.     

The dihydropyridine calcium channel blocker benidipine prevents lysophosphatidylcholine-induced endothelial dysfunction in rat aorta

Background  

Lysophosphatidylcholine (LPC), an atherogenic component of oxidized low-density lipoprotein, has been shown to induce the attenuation of endothelium-dependent vascular relaxation. Although benidipine, a dihydropyridine-calcium channel blocker, is known to have endothelial protective effects, the effects of benidipine on LPC-induced endothelial dysfunction remain unknown. We examined the effects of benidipine on the impairment of endothelium-dependent relaxation induced by LPC.     

Platelet-activating factor promotes arachidonate incorporation into phosphatidylinositol and phosphatidylcholine in neutrophils

Platelet-activating factor (1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) promotes the incorporation of [1-14C]arachidonic acid most significantly into phosphatidylinositol (PI) and phosphatidylcholine (PC) during the early stages of guinea pig neutrophil-PAF interaction. The stimulation reached a maximum at 10(-7) M and started to decline at 10(-6) M. No changes in the mass of each phospholipid were detected in neutrophils challenged by PAF for 1 to 5 minutes. The stimulation by PAF on the formation of [14C]arachidonoyl-PC but not [14C]arachidonoyl-PI was dependent on the presence of external Ca2+. These results suggest that the increased acylation of PI and PC elicited by PAF is secondary to an increased deacylation of these phospholipids and the mechanisms by which PAF stimulates the deacylation of PI and PC may be different.     

Interaction mode of n-dodecylphosphorylcholine, a substrate analogue, with bovine pancreas phospholipase A2 as determined by X-ray crystal analysis.

Three-dimensional structure of a bovine pancreas phospholipase A2 (PLA2) crystal complexed with n-dodecylphosphorylcholine (n-C12PC), a substrate-type inhibitor, has been determined by the X-ray diffraction method. The present conventional R value is 0.275 at 2.3A resolution. The binding mode of n-C12PC to the PLA2 was clearly indicated, where the dodecyl chain was stably held by the hydrophobic contacts with the N-terminal region of PLA2 (Leu-2, Phe-5, and Ile-9), and the choline moiety was contacted with the hydrophobic space created by the side chains of Lys-53 and 56. The present result indicates that remarkable changes from the native PLA2 structure are caused at the N-terminal and middle (residues 60 to 70) regions by the binding of n-C12PC to the enzyme.     

GABA, progesterone and zona pellucida activation of PLA2 and regulation by MEK-ERK1/2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated whether GABA activates phospholipase A2 (PLA2) during acrosomal exocytosis, and if the MEK-ERK1/2 pathway modulates PLA2 activation initiated by GABA, progesterone or zona pellucida (ZP). In guinea pig spermatozoa prelabelled with [14C]arachidonic acid or [14C]choline chloride, GABA stimulated a decrease in phosphatidylcholine (PC), and release of arachidonic acid and lysoPC, during exocytosis. These lipid changes are indicative of PLA2 activation and appear essential for exocytosis since inclusion of aristolochic acid (a PLA2 inhibitor) abrogated them, along with exocytosis. GABA activation of PLA2 seems to be mediated, at least in part, by diacylglycerol (DAG) and protein kinase C since inclusion of the DAG kinase inhibitor R59022 enhanced PLA2 activity and exocytosis stimulated by GABA, whereas exposure to staurosporine decreased both. GABA-, progesterone- and ZP-induced release of arachidonic acid and exocytosis were prevented by U0126 and PD98059 (MEK inhibitors). Taken together, our results suggest that PLA2 plays a fundamental role in agonist-stimulated exocytosis and that MEK-ERK1/2 are involved in PLA2 regulation during this process.     

Thrombin receptor activating peptides induce Ca2+ mobilization,barrier dysfunction,prostaglandin synthesis,and platelet-derived growth factor mRNA expression in cultured endothelium

Endothelial cell activation by thrombin is a key event in wound healing, Inflammation, and hemostasis. To better define thrombin-endothelial cell interactions we synthesized several peptides of varying length corresponding to the initial 14 amino acid sequence of the cloned human platelet thrombin receptor after cleavage at an arginine⁴¹ site (R/SFLLRNPNDKYEPF). Thrombin receptor activating peptides (TRAPs) as short as 5 amino acids induced significant levels of PGl₂ synthesis and expression of PDGF mRNA in human endothelium and produced dose-dependent cellular contraction and permeability of confluent human umbilical vein and bovine pulmonary artery endothelial monolayers. To explore whether TRAPs utilized similar signal transducing pathways as α-thrombin to accomplish endothelial cell activation, phospholipase C production of the Ca²⁺ secretagogue IP₃ was measured and detected 10 seconds after either TRAP 7 or α-thrombin. Furthermore, TRAPs ranging from 5-14 residues induced significant dose-dependent incsreases in Fura-2 fluorescence indicative of Ca²⁺ ₁ mobilization. These results indicate that thrombin-mediated proteolytic cleavage of the human and bovine thrombin receptor initiates stimulus/coupling respones such phospholipase C activation, Ca²⁺ mobilization, and protein kinase C activation. The functional consequence of this cellular activation via the cleaved receptor is enhanced cellular contraction, barrier dysfunction, PGI₂ synthesis, and expression of PDGF mRNA. © 1993 Wiley-Liss, Inc.     

A strategy for solubilizing delipidated apolipoprotein with lysophosphatidylcholine and reconstitution with phosphatidylcholine

The apolipoproteins of insect lipophorin were dissociated in guanidinium chloride and isolated by gel permeation chromatography. Over 98% of the total lipid in lipophorin was associated with apolipophorin I (apoLp-I), thus suggesting this apolipoprotein to be the lipid binding component of the particle. ApoLp-I was delipidated with ethanol/ether and solubilized in buffer that contained radioactive lysophosphatidylcholine ([3H]LPC) above the critical micellar concentration. Sonic irradiation of radioactive phosphatidylcholine ([14C]PC) with [3H]LPC-solubilized apoLp-I at a molar ratio of 318 resulted in reconstituted lipophorin (RLp-I). [3H]LPC was bound to fatty acid free bovine serum albumin and was separated from RLp-I by density gradient ultracentrifugation and gel permeation chromatography. Negatively stained RLp-I particles were quasispherical with an average radium of 55 A, and their overall morphology and secondary structure were similar to those of native hemolymph lipophorin. The RLp-I particle had a rho = 1.137 g/mL, a Mr approximately 5.2 X 10(5), and a [14C]PC:apoLp-I molar ratio of 308. From the compositional analysis, molecular size, trypsinization, and lipolysis with phospholipase A2, we concluded that each RLp-I particle contained one molecule of apoLp-I and a monomolecular layer of [14C]PC. When injected into the hemolymph of adult moths in vivo, RLp-I was loaded with lipid, as judged by a decrease in its density both in the presence and in the absence of adipokinetic hormone. The similarities in morphology and immunology of RLp-I and native lipophorin, together with the ability of RLp-I to load lipid, suggest that reconstituted lipophorins may serve as models to probe lipophorin structure and function.