Activity Of Pancreatic Phospholipase A2 Research Articles

Triggering of the activity of phospholipase A2 by an electric field.

In this paper we show that the action of phospholipase A2 can be triggered by applying an electric field across a 1,2-didodecanoyl-sn-3-phosphoglycerol monolayer residing between an alkylated silicon surface and water. When the silicon wafer served as a cathode, rapid activation of porcine pancreatic phospholipase was observed and did depend on the magnitude of the applied potential. The degree of activation was different for the pancreatic phospholipase A2 and snake and bee venom enzymes. Maximally, a 7-fold activation of pancreatic phospholipase A2 was observed when the applied potential was 75 V. The effective field over the lipid film could be estimated to be approximately 25-175 mV, i.e., in the range of membrane potentials found in cells. On the basis of these results, we suggest that changes in membrane potential might be an important factor in the regulation of the action of intracellular phospholipases A2 in vivo.

Synthesis of prodan-phosphatidylcholine, a new fluorescent probe, and its interactions with pancreatic and snake venom phospholipases A2.

A new fluorescent probe, prodan-PC, was synthesized by incubating thio-PC, a thiol ester analogue of phosphatidylcholine [1,2-bis(decanoylthio)-1,2-dideoxy-sn-glycero-3-phosphocholine], with acrylodan, a fluorescent thiol-reactive reagent [6-acryloyl-2-(dimethylamino)naphthalene], in the presence of phospholipase A2, which served to generate lysothio-PC in situ. Prodan-PC (PPC) showed maximum absorption in ethanol at 370 nm. The fluorescence emission spectrum showed maximum emission at 530 nm in water and at 498 nm in ethanol. In the presence of a saturating amount of phospholipase A2, the emission maximum shifted to about 470 nm. PPC showed a critical micellar concentration around 5 microM, with evidence of premicellar aggregation above 1 microM. Binding of PPC to Crotalus adamanteus phospholipase A2 was evidenced by an increase in emission at 480 nm and an increase in fluorescence anisotropy. An apparent dissociation constant of 0.323 microM was calculated for this enzyme complex. Binding was dependent on the presence of calcium ion and was abolished by blocking the active site with p-bromophenacyl bromide. Binding was also followed by energy transfer from tryptophan in the enzyme to PPC. Apparent dissociation constants for PPC complexes with phospholipases A2 from Naja naja naja and porcine pancreas and the prophospholipase A2 from porcine pancreas were 0.509, 0.107, and 0.114 microM, respectively. PPC was shown to inhibit the activity of pancreatic phospholipase A2 in thio-PC-sodium cholate mixed micelles. Inhibition studies were complicated because PPC can also serve as an activator of the snake venom enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors.

Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase. A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein. These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma. Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated. Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I. Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein. Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity. Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta. The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase. The mechanism by which these proteins inhibit phospholipase A2 activity was investigated. Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful. However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the [3H]oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay. Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E. coli membranes. The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate.

Dexamethasone inhibits prostaglandin release from rabbit coronary microvessel endothelium

The effects of dexamethasone on prostaglandin secretion by cultivated rabbit coronary microvascular endothelial (RCME) cells were investigated. Incubation of RCME cells with dexamethasone resulted in a time- and concentration-dependent decrease in prostaglandin accumulation in the culture media and reduced basal and A23187-stimulated prostaglandin (PG) E2 and 6-keto-PGF1 alpha release. The maximal effects of dexamethasone (50-80% inhibition) were achieved after 16-18 h of incubation with the steroid at a final concentration of 10(-7) M. The effects of dexamethasone treatment were partially reversed 24 h after removal of the steroid from the culture media. Dexamethasone treatment did not reduce arachidonic acid-stimulated prostaglandin synthesis, indicating that the level of inhibition was proximal to that of cyclooxygenase. The inhibitory effects of dexamethasone could be prevented by pretreatment of the RCME cells with actinomycin D or cycloheximide, suggesting a requirement for protein synthesis in the inhibitory action of dexamethasone. Conditioned media from dexamethasone-treated cells contained a factor that inhibited porcine pancreatic phospholipase A2 (PLA2) in vitro. Transfer of conditioned media from dexamethasone-treated cells to untreated cells did not reduce basal or stimulated prostaglandin release; in contrast, a stimulatory action was consistently observed. Adherence of rabbit peripheral polymorphonuclear leukocytes (PMN) to RCME cells was reduced when the leukocytes were pretreated with 10(-7) M dexamethasone (4 h). However, dexamethasone pretreatment of the RCME cells did not significantly effect granulocyte adhesion. Thus coronary microvascular endothelial cell prostaglandin production is regulated by glucocorticoids, and glucocorticoid-pretreated microvascular endothelial cell release an inhibitor of PLA2 activity into the culture media.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of a phospholipase A2 inhibitor in porcine serum.

Porcine pancreatic phospholipase A2 (PLA2) was immobilized to Sepharose 4B and porcine serum was passed through this affinity column. Bound substances were eluted by an EDTA-containing buffer and fractionated in a Sepharose 6B column. A single protein peak of the eluate from the latter column was found to inhibit PLA2 activity in a dose-dependent manner in an assay system using radioactive lecithin as a substrate and porcine pancreatic PLA2 as the enzyme source. The serum fraction containing the PLA2 inhibitory protein(s) (PIP) appeared inhomogeneous on SDS-polyacrylamide gel electrophoresis with two major bands close to each other, corresponding to a molecular weight of approximately 60,000. It was concluded that PIP might act as a protective principle against autodigestion in acute pancreatitis and other inflammatory diseases as well as playing a regulatory role in prostaglandin metabolism.

Adsorption of pancreatic (pro)phospholipase A2 to various physiological substrates.

The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.

Hydrolysis of mixed monomolecular films of phosphatidylcholine/triacylglycerol by pancreatic phospholipase A2.

We studied the effect of glycerides on pancreatic phospholipase A2 hydrolysis of mixed monomolecular films of trioctanoylglycerol/1,2-didodecanoyl-sn-glycero-3-phosphocholine with the technique of Piéroni and Verger [(1979) J. Biol. Chem. 254, 10090-10094]. The quantity of enzyme adsorbed to the interface was concomitantly determined with [3H]amidinated phospholipase. At phospholipid packing above the critical penetration pressure, triacylglycerol stimulates phosphatidylcholine hydrolysis to a great extent. On the other hand, the activity of pancreatic phospholipase A2 on a mixed film is inhibited by the action of pancreatic lipase. Interface binding of phospholipase A2 to the lipid substrate does not imply activity.

Relationship between the activity of pancreatic phospholipase A2 and the physical state of the phospholipid substrate.

When pancreatic phospholipase A2 is mixed with small unilamellar vesicles made from dipalmitoyl phosphatidylcholine at or above the phase transition temperature (tm), the time course of hydrolysis exhibits a distinct lag period. On the other hand, if the enzyme is preincubated with substrate for a short period of time below the transition temperature (during which time only limited hydrolysis occurs) and then assayed at high temperature, no lag period is observed. This phenomenon does not appear to be the result of product formation. Instead, the reported results lend substantial support to the hypothesis that a relatively slow substrate-enzyme organizational step is required as part of an activation process and this is most rapid when the substrate is in the gel state. However, the intrinsic activity (after the initial step of activation) is maximal when the substrate is in the liquid-crystalline state.

Relationship between structure and activity of pancreatic phospholipase A2.

Phospholipase A2 (EC 3.1.1.4) hydrolyzes exclusively the 2­acyl ester linkage of all types of naturally occurring phosphogly­cerides (1). The enzyme acts highly stereospecific and only 3-sn­phosphoglycerides are hydrolyzed, while the stereoisomeric 1-sn­-phosphoglycerides are competitive inhibitors (2). The pancreatic phospholipase A2 has a nearly absolute requirement for Ca2+ ions which bind in a 1:1 molar ratio to the enzyme at the catalytic or high-affinity site. Ba2+ and Sr2+ ions are competitive inhibitors, while Mg2+ ions are indifferent (3). So far only Gd3+ and Tb3+ ions can replace Ca2+ ions with retention of some activity (4).

Regulation of phospholipase A2 activity by different lipid-water interfaces.

The activity of pancreatic phospholipase A2 (EC 3.1.1.4) is controlled not only by the architecture of the catalytic site, but is also strongly dependent on the penetrating power of the interface recognition site and the packing density of the lipid-water interface. The influence of the latter two factors on the interface activity has been investigated using chemically modified phospholipases A2 in which the NH2-terminal L-Ala8 has been replaced by DL-[3-13C]Ala, or in which the polypeptide chain has been elongated with DL-[3-13C]Ala. The [DL-(3-13C)Ala8]phospholipase A2 could be resolved into the pure diastereoisomers, [D-(3-13C)Ala8]phospholipase A2 and [L-(3-13C)Ala8]phospholipase A2 by elution on Sephadex G-100 in the presence of a micellar lipid-water interface, as well as by conventional ion exchange chromatography on carboxymethylcellulose. Similar procedures did not effect, however, a separation of DL-[3-13C]Ala7-phospholipase A2 into their respective diasteroisomers, indicating the strategic role of the NH2-terminal L-Ala8 residue in the interaction process between the enzyme and lipid-water interfaces. Kinetic experiments using various micellar short chain lecithins revealed the apparent absence of an interface recognition site in [D-(3-13C)Ala8]- and DL-[3-13C]Ala7-phospholipase A2, while these proteins still possess considerable enzymatic activity toward monomeric substrates. In contrast, however, kinetic experiments using monomolecular surface films, allowing a continuous change in surface density of the substrate molecules, revealed that [D-(3-13C)Ala8]- and DL-[3-13C]Ala7-phospholipase A2 at low surface pressure possess about 60 and 30% of the interface activity of native phospholipase A2, respectively. These results therefore suggest that the modified phospholipases A2 do possess an interface recognition site although less powerful as compared to that of the native enzyme, enabling the estimation of the surface density of micellar short chain lecithins.