Pancreatic Phospholipase Research Articles

Desolvation map of the i-face of phospholipase A2

The changes in the microenvironment of the Trp-3 on the i-face of pig pancreatic IB phospholipase A 2 (PLA2) provide a measure of the tight contact (Ramirez and Jain, Protein Sci. 9, 229–239, 1991) with the substrate interface during the processive interfacial turnover. Spectral changes from the single Trp-substituent at position 1, 2, 6, 10, 19, 20, 31, 53, 56 or 87 on the surface of W3F PLA2 are used to probe the Trp-environment. Based on our current understanding only the residue 87 is away from i-face, therefore all other mutants are well suited to report modest differences along the i-face. All Trp-mutants bind tightly to anionic vesicles. Only those with Trp at 1, 2 or 3 near the rim of the active site on the i-face cause significant perturbation of the catalytic functions. Most other Trp-mutants showed < 3-fold change in the interfacial processive turnover rate and the competitive inhibition by MJ33. Binding of calcium to the enzyme in the aqueous phase had modest effect on the Trp-emission intensity. However, on the binding of the enzyme to the interface the fluorescence change is large, and the rate of oxidation of the Trp-substituent with N-bromosuccinimide depends on the location of the Trp-substituent. These results show that the solvation environment of the Trp-substituents on the i-face is shielded in the enzyme bound to the interface. Additional changes are noticeable if the active site of the bound enzyme is also occupied, however, the catalytically inert zymogen of PLA2 (proPLA2) does not show such changes. Significance of these results in relation to the changes in the solvent accessibility and desolvation of the i-face of PLA2 at the interface is discussed.

Third calcium ion found in an inhibitor-bound phospholipase A2

The lipolytic enzyme phospholipase A2 plays a crucial role in lipid metabolism and catalyzes hydrolysis of the fatty-acid ester bond at the sn-2 position of phospholipids. Here, the crystal structure (1.7 A resolution) of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 complexed with an organic molecule, p-methoxybenzoic acid (anisic acid), is reported. Residues 60-70 (the surface-loop residues) are ordered and adopt conformations which are different from those normally found in structures in which a second calcium ion is present. It is interesting to note that for the first time a third calcium ion has been identified. In addition, four Tris (2-amino-2-hydroxymethyl-1,3-propanediol) molecules were located. It is believed that one of the Tris molecules plays a role in clamping the third calcium ion and that another is involved in controlling the dynamics of the surface loop through hydrogen bonds.

POSITIONAL DISTRIBUTION OF FATTY ACIDS IN EGG YOLK LIPIDS

ABSTRACT Numerous methods for the assessment of the positional distribution of fatty acids (FAs) in triacylglycerols (TGs) and phospholipids (PLs) have been described in the literature. Most of them employ enzymatic reactions or chiral separation by high‐performance liquid chromatography. Compared with most other food lipids, egg lipid has a rather unique composition, containing high amounts of TG as well as PL organized in lipoproteins. In this study, we demonstrate that the classical approach employing pancreatic lipase and phospholipase A2 is suitable for application to egg lipids. By using this method, the total distribution of FAs among the single positions of the main lipid classes in egg lipids can be measured in a rather straightforward way. Problems regarding accuracy, especially for the determination of minor and polyunsaturated fatty acids, are discussed. The presented method gave satisfactory results even for polyunsaturates such as docosahexaenoic acid down to amounts of 1% in a specific position.

Hexadecylphosphocholine (miltefosine) has broad-spectrum fungicidal activity and is efficacious in a mouse model of cryptococcosis.

The alkyl phosphocholine drug miltefosine is structurally similar to natural substrates of the fungal virulence determinant phospholipase B1 (PLB1), which is a potential drug target. We determined the MICs of miltefosine against key fungal pathogens, correlated antifungal activity with inhibition of the PLB1 activities (PLB, lysophospholipase [LPL], and lysophospholipase-transacylase [LPTA]), and investigated its efficacy in a mouse model of disseminated cryptococcosis. Miltefosine inhibited secreted cryptococcal LPTA activity by 35% at the subhemolytic concentration of 25 microM (10.2 microg/ml) and was inactive against mammalian pancreatic phospholipase A2 (PLA2). At 250 microM, cytosolic PLB, LPL, and LPTA activities were inhibited by 25%, 51%, and 77%, respectively. The MICs at which 90% of isolates were inhibited (MIC90s) against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Cryptococcus gattii, Aspergillus fumigatus, Fusarium solani, Scedosporium prolificans, and Scedosporium apiospermum were 2 to 4 microg/ml. The MICs of miltefosine against Candida tropicalis (n = 8) were 2 to 4 microg/ml, those against Aspergillus terreus and Candida parapsilosis were 8 microg/ml (MIC90), and those against Aspergillus flavus (n = 8) were 2 to 16 microg/ml. Miltefosine was fungicidal for C. neoformans, with rates of killing of 2 log units within 4 h at 7.0 microM (2.8 microg/ml). Miltefosine given orally to mice on days 1 to 5 after intravenous infection with C. neoformans delayed the development of illness and mortality and significantly reduced the brain cryptococcal burden. We conclude that miltefosine has broad-spectrum antifungal activity and is active in vivo in a mouse model of disseminated cryptococcosis. The relatively small inhibitory effect on PLB1 enzyme activities at concentrations exceeding the MIC by 2 to 20 times suggests that PLB1 inhibition is not the only mechanism of the antifungal effect.

Absorption and lipoprotein transport of sphingomyelin

Dietary sphingomyelin (SM) is hydrolyzed by intestinal alkaline sphingomyelinase and neutral ceramidase to sphingosine, which is absorbed and converted to palmitic acid and acylated into chylomicron triglycerides (TGs). SM digestion is slow and is affected by luminal factors such as bile salt, cholesterol, and other lipids. In the gut, SM and its metabolites may influence TG hydrolysis, cholesterol absorption, lipoprotein formation, and mucosal growth. SM accounts for approximately 20% of the phospholipids in human plasma lipoproteins, of which two-thirds are in LDL and VLDL. It is secreted in chylomicrons and VLDL and transferred into HDL via the ABCA1 transporter. Plasma SM increases after periods of large lipid loads, during suckling, and in type II hypercholesterolemia, cholesterol-fed animals, and apolipoprotein E-deficient mice. SM is thus an important amphiphilic component when plasma lipoprotein pools expand in response to large lipid loads or metabolic abnormalities. It inhibits lipoprotein lipase and LCAT as well as the interaction of lipoproteins with receptors and counteracts LDL oxidation. The turnover of plasma SM is greater than can be accounted for by the turnover of LDL and HDL particles. Some SM must be degraded via receptor-mediated catabolism of chylomicron and VLDL remnants and by scavenger receptor class B type I receptor-mediated transfer into cells.

Reorganization of lipid nanocapsules at air–water interface: 3. Action of hydrolytic enzymes HLL and pancreatic PLA 2

The action of the hydrolytic enzymes humicola lanuginosa lipase (HLL) and pancreatic phospholipase A 2 (PLA 2) on monolayers formed from lipid nanocapsules (LNC) and model monolayers containing their components, Labrafac ®, Solutol ® and Lipoid ®, is studied by simultaneous measuring the changes in the film area and the surface potential in the “zero order” trough at constant surface pressure ( π). The kinetic models describing the hydrolysis by HLL of the Labrafac, Solutol and their mixtures have been proposed. By using the developed theoretical approach together with the experimental results the surface concentrations of the substrates, hydrolysis products and values of the global kinetic constants were obtained. The comparison between the global kinetic constants in the case of HLL hydrolysis of pure Labrafac, Solutol monolayers and those of the model mixed Labrafac/Solutol monolayers, shows that the rates of hydrolysis are of the same order of magnitude, i.e. an additively of the HLL enzyme action is observed. The composition of the mixed Labrafac/Solutol monolayer, formed after the interfacial LNC destabilization, was estimated.

Amino-acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus).

The amino acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan, has been determined. The beta 1 isosubunit, which is neither toxic nor enzymatically active on its own, consists of a single polypeptide chain of 118 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined by automated Edman degradation. The taipoxin beta 1 isosubunit is closely homologous to the taipoxin alpha and gamma subunits and to enzymatically active pancreatic and elapid snake venom phospholipases A2.

Indirect Pancreatic Function Tests in Children

Indirect Pancreatic Function Tests in Children

Sol i 1, the phospholipase allergen of imported fire ant venom

Sol i 1, the venom phospholipase of imported fire ant venom is an important allergen and exhibits some cross-reactivity with IgE antibodies from patients sensitized to other Hymenoptera venoms. To determine the primary structure of Sol i 1 and evaluate the roles of protein and carbohydrate epitopes in its cross-reactivity. Sol i 1 was purified from venom, proteolytic peptides prepared and amino acid sequences obtained. The cDNA for Sol i 1 was cloned, sequenced, and compared with sequences of other wasp venom phospholipases. The role of carbohydrate epitopes in the cross-reactivity with other Hymenoptera venoms was studied by RAST inhibition. The sequence identified Sol i 1 as a lipase of the GX class, lipoprotein lipase superfamily, pancreatic lipase homologous family and RP2 subgroup phospholipases as are the vespid venom phospholipases. The 148 residues identified by amino acid sequencing represent about 48% of the translated cDNA sequence. Sol i 1 was 31-32% identical to yellow jacket phospholipases. The identical regions of sequence were clustered in the domain which forms the serine hydrolase active site. Mannosylated N-glycans could completely inhibit binding of IgE from honeybee venom sensitized patients to Sol i 1. Inhibition by glycan of IgE binding from yellow jacket venom sensitized patients was low or absent for three of eight sera and substantial, but not complete for five sera. Sol i 1 is related to wasp venom phospholipases. Cross-reactivity with honeybee venom is caused by carbohydrate, whereas cross-reactivity with yellow jacket venom involves reactivity with both carbohydrate determinants of hyaluronidase and high molecular weight proteins and phospholipase protein determinants.

Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts

Previous studies suggest that antagonists of cyclooxygenases 1 and 2 (COX-1, -2) inhibit angiogenesis in tumor xenografts, but the molecular mechanisms involved remain unclear. Here we characterized the effects of non-selective (indomethacin) and selective (NS398, celecoxib) cyclooxygenase inhibitors on parameters of angiogenesis in human pancreatic adenocarcinoma cells. COX-1 expression was constitutive in 9/9 pancreatic cancer cell lines, whereas COX-2 and cytosolic phospholipase A2 (cPLA2 ) expression were observed in 4/9 cell lines (BxPC3, Capan2, Cfpac1, and L3.6pl). Production of the COX product, prostaglandin E2, correlated with expression of cPLA2 and COX-2 and was blocked by non-steroidal anti-inflammatory drugs (NSAIDs, indomethacin or NS398). In contrast to the findings of others, neither indomethacin nor NS398 affected tumor cell proliferation or secretion of angiogenic factors (VEGF, bFGF, IL-8) at concentrations that produced maximal inhibition of PGE2 production, and higher concentrations increased angiogenic factor production. We also studied the effects of celecoxib in orthotopic L3.6pl xenografts. Immunofluorescence analyses revealed high-level expression of COX-2 in endothelial cells in L3.6pl xenografts that increased following therapy with celecoxib, whereas the tumor cells expressed uniformly low levels of COX- 2. Celecoxib did not decrease tumor-associated VEGF levels in orthotopic human L3.6pl xenografts, but the drug did decrease tumor microvessel density (MVD) and increase apoptosis in tumor-associated endothelial cells in a dose-dependent fashion. Together, our results demonstrate that the anti-angiogeneic effects of NSAIDs in human pancreatic cancer cells are exerted via direct effects on endothelial cells.

Atomic resolution (0.97 Å) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.47 degrees. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63,926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60-70 are ordered and have clear electron density.

A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A2 at 1.6 A resolution using sulfur-SAS at 1.54 A wavelength.

The crystal structure of the triple mutant K53,56,120M of bovine pancreatic phospholipase A(2) has been redetermined using sulfur single-wavelength anomalous scattering. The synchrotron data were collected at lambda = 1.54 A and the crystal diffracted to 1.6 A resolution. The program SOLVE was used to locate the heavy atoms and to estimate the initial phases and the resulting map was then subjected to RESOLVE. The output of 455 non-H atoms, including 12 S atoms, one calcium ion and one chloride ion, were then subjected to ARP/wARP followed by REFMAC. With the improved phases, the automatic model building successfully built more than 85% of the 123 residues, excluding the N- and C-terminal residues. The final crystallographic R factor is 17.7% (R(free) = 21.7%). The refined model consists of 954 non-H protein atoms, 165 water O atoms, three 2-methyl-2,4-pentanediol (MPD) molecules, one calcium ion and one chloride ion. The present work is yet another example that shows the utility of single-wavelength anomalous scattering data for solving a protein structure.

Investigating porcine pancreatic phospholipase A2 action on vesicles and supported planar bilayers using a quartz crystal microbalance with dissipation.

We present an investigation of the activity of porcine pancreatic phospholipase A2 towards phospholipids. The phospholipids are presented in three different ways, namely as tethered vesicles, intact surface-bound vesicles, and supported planar bilayers (SPBs). The process is followed using a quartz crystal microbalance which measures both the frequency shift and the energy dissipation factor. This technique is very sensitive not only to the mass of the material deposited on the crystal, but also to its viscoelasticity. The breakdown of the phospholipid vesicles and bilayers consequently gives rise to very large signal changes. Enzyme binding is separated from vesicle hydrolysis using nonhydrolyzable ether lipids. Intact and tethered vesicles give rise to the same profile, indicating that direct immobilization of the vesicles does not affect hydrolysis significantly. The data fit well to a Voight-based model describing the change in film structure with time. Initial enzyme binding to intact vesicles is accompanied by a significant increase in layer thickness as well as a decrease in viscosity and shear modulus. This effect, which is less pronounced in SPBs, is probably mainly due to the accumulation of hydrolysis products in the vesicle prior to rupture of the vesicles and release of bound water, since it disappears when lysolipid is included in the vesicles prior to hydrolysis.

Bromoacyl Analogues of Phosphatidylcholine with Intramolecular Fluorescence Quenching and Their Use as Substrates for Continuous Monitoring of Phospholipase A2Activity

Two new derivatives of phosphatidylcholine with intramolecular fluorescence quenching were obtained by substituting residues of pyrene butyric and bromine-containing fatty acids for acyl chains. The two compounds can be used for quantitative evaluation of catalytic activity of pancreatic phospholipase A2 in kinetic mode.

Origins of delays in monolayer kinetics: phospholipase A2 paradigm.

The interfacial kinetic paradigm is adopted to model the kinetic behavior of pig pancreatic phospholipase A(2) (PLA2) at the monolayer interface. A short delay of about a minute to the onset of the steady state is observed under all monolayer reaction progress conditions, including the PLA2-catalyzed hydrolysis of didecanoylphosphatidyl-choline (PC10) and -glycerol (PG10) monolayers as analyzed in this paper. This delay is independent of enzyme concentration and surface pressure and is attributed to the equilibration time by stationary diffusion of the enzyme added to the stirred subphase to the monolayer through the intervening unstirred aqueous layer. The longer delays of up to several hours, seen with the PC10 monolayers at >15 mN/m, are influenced by surface pressure as well as enzyme concentration. Virtually all features of the monolayer reaction progress are consistent with the assumption that the product accumulates in the substrate monolayer, although the products alone do not spread as a compressible monolayer. These results rule out models that invoke slow "activation" of PLA2 on the monolayer. The observed steady-state rate on monolayers after the delays is <1% of the rate observed with micellar or vesicles substrates of comparable substrate. Together these results suggest that the monolayer steady-state rate includes contributions from steps other than those of the interfacial turnover cycle. Additional considerations that provide understanding of the pre-steady-state behaviors and other nonideal effects at the surface are also discussed.

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.

Hydrolysis of nuclear phospholipids in relation with proliferative state in uterine stromal cells

The current study examined the metabolism of phospholipid (PL) in the whole cell homogenate and in the nuclear fraction in proliferative and non-proliferative uterine stromal cells (U III cells). Growth arrested cells were obtained either from contact-inhibited confluent cells or from proliferative cells treated with aristolochic acid (AR) for 2 days. Fatty acid composition and fatty acid amount of both total and nuclear PL were not significantly different between proliferative, confluent and AR-treated cells. In contrast, marked differences were observed in the incorporation of [ 3H]AA, with greater incorporation in proliferative cells than in confluent or AR-treated cells, particularly in nuclear PL. Considering endogenous level of arachidonic acid (AA) in total and nuclear PL, we found that AA turnover in nuclear PL was especially high compared to that in total PL and that this difference was accentuated in proliferative cells compared to non-proliferative cells. Interestingly, [ 3H]AA incorporation and AA turnover in proliferative, confluent and AR-treated cells vary accordingly to the expression, activity and/or content of pancreatic phospholipase A 2 (PLA 2-I) in the nuclear compartment of these cells that we reported in previous studies. The changes in metabolism of nuclear PL during cell proliferation are consistent with an enhanced PL hydrolysis that could involve PLA 2-I.

Surface pressure dependence of phospholipase A 2 activity in lipid monolayers is linked to interfacial water activity

The specific activity of pancreatic phospholipase A 2 (PLA 2) was studied in two disparate systems, one involving phosphatidylcholine monolayer at various surface pressures at the air–water interface and the other involving a solid-state system exposed to various equilibrium relative humidity (ERH). The results were examined in terms of thermodynamic activity of water in the interfacial region ( a w s∗ ) and in the hydrated solid phase ( a w). In both these physically different systems, the specific activity versus a w and a w s∗ profiles of PLA 2 were remarkably similar. In both cases, the specific activity exhibited a maximum at a w (or a w s∗ ) ∼ 0.3. These results suggested that the mechanism of control of PLA 2 activity at the lipid–water interface might involve modulation of the hydration state of the enzyme through control of the thermodynamic activity of water in the interfacial region. Extension of these results to biomembranes suggests that one of the functions of lipid bilayer might be the control of local water activity at the lipid–water interface. In biological membranes, localized subtle changes in interfacial water activity may occur as a result of local stretching or compression of the membrane facilitated by conformational changes in membrane-bound receptor proteins.

The sequence of Sol i 1 the cross-reactive allergen of imported fire ant venom

Rationale Sol i 1 is cross-reactive with phospholipases from vespid venoms. Methods The venom protein was isolated and the amino acid sequences of tryptic and endoproteinase Glu-C peptides were determined. The cDNA sequence was determined by RACE pcr based cloning. Three dimensional models were constructed using Swiss-Model with pancreatic lipoprotein lipase template structures. Results The sequence identified Sol i 1 as a lipase of the GX class, lipoprotein lipase superfamily, pancreatic lipase homologous family and RP2 subgroup phospholipases as are the vespid venom phospholipases. The 148 residues identified by amino acid sequencing represent about 48% of the translated cDNA sequence. Sol i 1 was 31-32% identical to yellow jacket phospholipases. The identical regions of sequence were clustered in the domain which forms the serine hydrolase active site. The conformation of this domain is very similar to that of vespid phospholipases consistent with the observed immunologic cross-reactivity. Conclusions Sol i 1 is a phospholipase of the pancreatic lipoprotein lipase RP2 subgroup with significant sequence similarity to vespid venom PL's and a domain of homologous 3-dimensional structure.

Dynamics of ordered water in interfacial enzyme recognition: bovine pancreatic phospholipase A2.

Slippery when wet: Water molecules at the interface between a protein (e.g. bovine pancreatic phospholipase A2) and a micellar substrate act as a lubricant and facilitate movement and binding of the protein at the surface (see picture). Studies of the hydration dynamics before and after complexation reveal that these interactions are crucial for enzymatic function.