Intracellular phospholipase activities of Tetrahymena pyriformis

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.     

Characterization of the adenosine triphosphatase of avian myeloblastosis virus.

The ATPase of avian myeloblastosis virus (AMV) is not a recognizable cellular enzyme. It hydrolyzes ATP, GTP, ITP, UTP, and dCTP at equal rates, is inhibited by high concentrations of dithiothreitol, and is partially inhibited by 1 × 10^?5mp-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzene sulfonate acid (PCMBS). The inhibition by the mercurials is reversed by increasing the concentration of PCMB or PCMBS to 1 × 10^?3m. The enzyme requires phospholipid for activity. Incubation with phospholipase C inhibits activity and subsequent addition of lecithin-containing saturated fatty acids partially restores activity, whereas lecithin-containing unsaturated fatty acids further inhibit activity.     

A long-lasting facilitation of hippocampal neurotransmission via a phospholipase A2 signaling pathway.

Phospholipase A2, which is linked to a protein kinase C pathway, hydrolyzes phosphatidylcholine into cis-unsaturated free fatty acids and lysophosphatidylcholine (lysoPC). The present study investigated the effect of the free fatty acids, such as arachidonic, oleic, linoleic, and linolenic acid, and lysoPC on neurotransmission by monitoring population spikes (PSs) from the granular cell layer of rat hippocampal slices. All the free fatty acids and lysoPC examined here gradually increased PS amplitude to a different extent, the effect being evident 60 min after treatment. No significant synergistic enhancement in the PS amplitude was not induced by arachidonic acid following oleic acid, linoleic acid or lysoPC. The results of the present study, thus, demonstrate that phospholipase A2-linked free fatty acids and lysoPC are employed in the sustained facilitation of hippocampal neurotransmission, suggesting a significant role of a phospholipase A2 signaling pathway in the neuroplasticity.     

Effect of trypsin, phospholipases, and membrane-impermeable reagents on the uptake of palmitic acid by isolated rat liver cells

Rat liver cells isolated by the collagenase-hyaluronidase perfusion method were treated with membrane-impermeable protein reagents (7-diazonium, 1–3-naphthalene disulfonate, diazotized sulfanilic acid, 8-anilino-naphthalene disulfonate), trypsin, phospholipase A, phospholipase C, and phospholipase D. The treated cells were incubated with [1-¹⁴C]palmitate and the ¹⁴CO₂ produced was taken as a measure of fatty acid uptake by the cells. ¹⁴CO₂ production by the cells was not inhibited after treatments with the membrane-impermeable protein reagents or phospholipase D. Treatments with small amounts of trypsin or phospholipases A or C caused inhibition of CO₂ production from tracer amounts of palmitate. The inhibition by trypsin was partially, and that by phospholipase A was fully, reversed by increasing the amount of palmitic acid in the incubation medium. The oxidation of shorter-chain fatty acids such as octanoic acid was not decreased but increased after treating the cells with trypsin or phospholipase A. The membrane-impermeable reagents inhibited the oxidation of palmitate to CO₂ by liver cells isolated by mechanical dispersion. These reagents also inhibited the long-chain acyl CoA ligase activity of liver microsomes. From these results it is suggested that the inhibition of CO₂ production by intact liver cells from palmitate after enzyme treatments, is due to partial removal or modification of a normal transport component for long-chain fatty acids on the plasma membrane. The possibility of proteins (or lipoproteins) buried below the surface layer of plasma membrane in fatty acid uptake by liver cells is indicated.     

Studies on the phospholipases of rat intestinal mucosa

1. Subcellular distribution and characteristics of different phospholipases of rat intestinal mucosa were studied. 2. The presence of free fatty acid was necessary for the maximal hydrolysis of lecithin (phosphatidylcholine), but there was no accumulation of lysolecithin (1 or 2-acylglycerophosphorylcholine);lysolecithin accumulated when the reaction was carried out in the presence of sodium deoxycholate and at or above pH8.0. 3. The fatty acid-activated phospholipase B as well as lysolecithinase showed optimum activity at pH6.5, whereas for the phospholipase A it was about pH8.6. 4. The bulk of the phospholipase A was present in the microsomal fraction, whereas the phospholipase B and lysolecithinase activities were distributed between the microsomal and soluble fractions of the mucosal homogenate. 5. Phospholipase A was equally distributed between the brush border and brush-border-free particulate fraction, with the brush border having highest specific activity, whereas the other two activities were distributed between the brush-border-free particulate and soluble fractions. 6. Various treatments showed marked differences between the phospholipase A and phospholipase B activities, but not between phospholipase B and lysolecithinase activities. 7. By using (beta[1-(14)C]-oleoyl) lecithin it was shown that the mucosal phospholipase A was specific for the beta-ester linkage of the lecithin molecule.     

Effect of unsaturated fatty acids and Ca2+ on phosphatidylinositol synthesis and breakdown

CDP-diglyceride : inositol transferase was inhibited by unsaturated fatty acids. The inhibitory activity decreased in the following order: arachidonic acid greater than linolenic acid greater than linoleic acid greater than oleic acid greater than or equal to palmitoleic acid. Saturated fatty acids such as myristic acid, palmitic acid, and stearic acid had no effect. Calcium ion also inhibited the activity of CDP-diglyceride : inositol transferase. In rat hepatocytes, arachidonic acid inhibited 32P incorporation into phosphatidylinositol and phosphatidic acid without any significant effect on 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Ca2+ ionophore A23187 also inhibited 32P incorporation into phosphatidylinositol. However, 32P incorporation into phosphatidic acid was stimulated with Ca2+ ionophore A23187. Phosphatidylinositol-specific phospholipase C was activated by unsaturated fatty acids. Polyunsaturated fatty acids such as arachidonic acid and linolenic acid had a stronger effect than di- and monounsaturated fatty acids. Saturated fatty acids had no effect on the phospholipase C activity. The phospholipase C required Ca2+ for activity. Arachidonic acid and Ca2+ had synergistic effects. These results suggest the reciprocal regulation of phosphatidylinositol synthesis and breakdown by unsaturated fatty acids and Ca2+.     

Control of arachidonic acid accumulation in bone marrow-derived macrophages by acyltransferases

The turnover of phospholipid fatty acid moieties of bone marrow-derived macrophages was analyzed by separate determination of degrading and acylating activities. Acylating activities were followed in intact cells by incubation with excess arachidonic acid and degradation of phospholipids was followed in cells prelabeled with fatty acids. Significant phospholipase A2 activity was detectable only if the reutilization of liberated fatty acid was inhibited , e.g. by p-chloromercuribenzoate. It was of interest that the divalent cation ionophore A 23187 and various antiphlogistic drugs like indomethacin, diclofenac, and acetylsalicylic acid were found to inhibit the acylation reaction. These compounds led to increased levels of free arachidonic acid in stimulated, as well as in unstimulated cells. Increased activities of phospholipase A2 were achieved by treatment with the bivalent cation ionophore A 23187 and with zymosan. The effect of zymosan obtained from various sources was found to be exclusively due to contamination of tee zymosan particles with phospholipase A2 activity. Even when the cellular phospholipase activity was increased by the addition of exogenous phospholipase activity contained in the zymosan particles, degradation of cellular phospholipids was not measurable unless the reacylation was inhibited. These results suggest that in the cells studied, the level of free arachidonic acid is mainly controlled by the activity of the lysophosphatide acyltransferase.     

Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin and its metabolites, ceramide and ceramide phosphate

Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the level of inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids on the phospholipase A and cholesterol esterification reactions of the enzyme were similar, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Incorporation of ceramide into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However, these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response.     

Fatty acids and lysophospholipids as potential second messengers in auxin action. Rapid activation of phospholipase A2 activity by auxin in suspension-cultured parsley and soybean cells

Activation of cytosolic phospholipase A₂ is a typical signal transduction reaction in animal cells and occurs in plants in response to auxin, elicitors and wounding. Exogenously added fluorescent bis-BODIPY-phosphatidylcholine was taken up and hydrolysed by a cellular phospholipase A₂. Rapid activation of a phospholipase A₂ by auxin in suspension-cultured parsley ( Petrosilenum crispum L.) and soybean ( Glycine max L.) cells was shown by detection and quantification of fluorescent reaction products of phospholipase A₂. Hormone-triggered fluorescent fatty acid accumulation could be detected as early as 5 min. Auxins at 2 μM or higher concentrations activated phospholipase A₂ and fluorescent fatty acids accumulated 1.1- to threefold after 90–120 min, depending on the auxin concentration. Fluorescent lysolipid did not accumulate up to 150 μM auxin. Known inhibitors of phospholipase A₂ inhibited hormone-dependent fluorescent fatty acid accumulation in cell cultures and, previously, elongation growth in etiolated zucchini hypocotyl segments ( Scherer & Arnold (1997 ) Planta 202, 462–469). When lipids were labeled by [¹⁴C]-choline and [¹⁴C]-ethanolamine the corresponding lysophospholipids could be quantified in cell extracts. Radioactive lysophospholipids accumulated as rapidly as 1–2 min after auxin treatment but only at concentrations well above 100 μM auxin. We hypothesize that phospholipase A₂ activation is an early intermediate step between receptor and downstream responses. We hypothesize that fatty acid(s) could be second messengers in several auxin functions, especially in cell elongation. Lysophospholipids seem to be indicators or second messengers for stress caused by high auxin concentrations or may have different auxin-linked functions and are also known to accumulate during elicitor action.     

Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

Differential inhibition of thromboxane B2 and leukotriene B4 biosynthesis by two naturally occurring acetylenic fatty acids

The seed oil of the plant Ixiolaena brevicompta is a rich source of crepenynic acid (octadec-cis-9-en-12-ynoic acid), which has been linked with extensive sheep mortalities in Western New South Wales and Queensland, Australia. A number of acetylenic fatty acids have been found to interfere with lipid and fatty acid metabolism and inhibit cyclooxygenase and lipoxygenase enzymes in a variety of tissues. We have investigated the effects of crepenynic acid and ximenynic acid (octadec-trans-11-en-9-ynoic acid) on leukotriene B4 and thromboxane B2 production in rat peritoneal leukocytes and compare them with non-acetylenic compounds linoleic and ricinoleic acids. In concentrations ranging from 10 to 100 microM linoleic acid and ricinoleic acid had only minimal effects on leukotriene B4 and thromboxane B2 production in ionophore-stimulated cells. Ximenynic acid gave dose-dependent inhibition of leukotriene B4, thromboxane B2 and 6-ketoprostaglandin F1 alpha production. Ximenynic acid appears to be a more effective inhibitor of leukotriene B4 than crepenynic acid with an IC50 of 60 microM compared to 85 microM. On the other hand, crepenynic acid is a much more effective inhibitor of the cyclooxygenase products, having an IC50 for thromboxane B2 of less than 10 microM. Both acetylenic fatty acids inhibited phospholipase activity in these cells by 40-50% at a concentration of 100 microM but had no inhibitory effect at 10 microM. These results indicate that crepenynic acid and ximenynic acid differentially inhibit the cyclooxygenase and lipoxygenase products of stimulated leukocytes, and that at high doses of these fatty acids the effect on these products may be partially due to inhibition of phospholipase A2.     

Isolation and properties of a phosphatidylcholine-specific phospholipase C from bull seminal plasma

A phospholipase C which hydrolyzes [14C]phosphatidylcholine has been purified 1782-fold from 70% ammonium sulfate extract of bull seminal plasma. Purification steps included acid precipitation, chromatography on DEAE-Sephacel, concanavalin A, octyl-Sepharose 4B and Ultrogel AcA 34. The final step provided homogeneous phospholipase C as determined by polyacrylamide gel electrophoresis. The enzyme comprised two subunits, Mr 69,000 and Mr 55,000, respectively. The enzyme had an optimum at pH 7.2 and pI 5.0. EDTA, Cd2+, Pb2+, Ni2+, Fe2+, and Zn2+ inhibited phospholipase C activity. Km and Vmax on p-nitrophenyl phosphorylcholine and phosphatidylcholine substrates were 20 mM and 17 mumol/min/mg of the purified enzyme and 100 microM and 18 mumol/min/mg of the purified enzyme, respectively. The enzyme appeared to be localized in the acrosome as judged by the binding of anti-phospholipase C to the acrosome. This phospholipase C, unlike other known phospholipases (C), did not hydrolyze [1-14C]phosphatidylinositol. The testicular extract of the guinea pig contained inactive phospholipase C which was activated on incubation with acrosin and trypsin but not chymotrypsin.     

In vivo evidence for the involvement of phospholipase A and protein kinase in the signal transduction pathway for auxin-induced corn coleoptile elongation

Auxin-induced elongation of com coleoptiles is accompanied by cell wall acidification, which depends upon H⁺-pump activity. We tested the hypothesis that phospholipase A and a protein kinase are involved in the pathway of auxin signal transduction leading to H⁺ secretion, and elongation of corn coleoptiles. Initially, the pH of the bath solution at 50–100 μm from the surface of a coleoptile segment (pH_o) ranged between 4.8 and 6.6 when measured with an H⁺-sensitive microelectrode. Twenty or 50 μM lysophosphatidylcholine, 50 μM linolenic acid or 50 μM arachidonic acid induced a decline in pH_o by 0.3 to 2.1 units. The effect was blocked by 1 mM vanadate, suggesting that lysophosphatidylcholine or linolenic acid induced acidification of the apoplast by activating the H⁺-pump. Lysophosphatidylcholine and linolenic acid also accelerated the elongation rate of the coleoptiles. While linolenic acid and arachidonic acid, highly unsaturated fatty acids, promoted pH_o decrease and coleoptile elongation, linoleic acid, oleic acid, and stearic acid, fatty acids with a lesser extent of unsaturation, had no such effects. The effects of lysophosphatidylcholine, linolenic acid, and arachidonic acid on H⁺ secretion were not additive to that of indoleacetic acid (IAA), suggesting that lysophospholipids, fatty acids and auxin use similar pathways for the activation of the H⁺-pump. The phospholipase A₂ inhibitors, aristolochic acid and manoalide, inhibited the IAA-induced pH_o decrease and coleoptile elongation. The general protein kinase inhibitors, H-7 or staurosporine, blocked the IAA- or lysophosphatidylcholine-induced decrease in pH_o. H-7 also inhibited the coleoptile elongation induced by IAA or lysophosphatidylcholine. These results support the hypothesis that phospholipase A is activated by auxin, and that the products of the enzyme, lysophospholipids and fatty acids, induce acidification of the apoplast by activating the H⁺-pump through a mechanism involving a protein kinase, which in turn promotes com coleoptile elongation.     

Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids

We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.     

Relation of mitochondrial phospholipase A activity to mitochondrial swelling

Assays of mitochondrial phospholipase A activity and mitochondrial swelling demonstrated that the phospholipase A activity is related to the swelling under the experimental conditions used. Both were stimulated by added free fatty acid and CaCl(2), not affected greatly by the addition of monoacyl phosphoglycerides, and inhibited by EDTA. The amount of fatty acid hydrolyzed from endogenous phosphatidyl ethanolamine and phosphatidyl choline during swelling was calculated to be 20-30 times less than the amount of added free fatty acid that gave comparable swelling. Under the experimental conditions about 4% of the phospholipid was hydrolyzed. Mitochondrial swelling was studied by electron microscopy and turbidity measurements. The results found were in agreement, whether oleic acid was present or not, except for those values obtained after very brief incubation (1 min) and after incubation for longer than 35 min. The lack of direct proportion between swelling and the concentration of lysosomes present indicated that the swelling is related mainly to mitochondrial phospholipase A, although swelling due to contaminating lysosomes cannot be excluded entirely. The temperature dependence of spontaneous, fatty acid-induced, or CaCl(2)-induced swelling suggested that enzymatic activities are responsible for swelling.     

A novel fluorogenic substrate for the measurement of endothelial lipase activity

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.     

Enzymatic properties of human cytosolic phospholipase A(2)gamma

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.     

Evidence for a phosphatidylinositol anchor in glycolipid antigens of Leishmania major

The structure of the membrane anchor of three Leishmania major surface antigenic glycolipids was analyzed. Phosphatidylinositol-specific phospholipase C treatment and nitrous acid deamination indicated the presence of a phosphatidylinositol anchor linked to the glycan through a non-N-acetylated hexosamine. An ester linkage on the C-2 of glycerol was revealed by phospholipase A2 hydrolysis. This fatty acyl substitution was not found on the phosphatidylinositol anchor of the Leishmania lipophosphoglycan.     

Purification of lysosomal phospholipase A and demonstration of proteins that inhibit phospholipase A in a lysosomal fraction from rat kidney cortex

Phospholipase A has been isolated from a crude lysosomal fraction from rat kidney cortex and purified 7600-fold with a recovery of 9.8% of the starting activity. The purified enzyme is a glycoprotein having an isoelectric point of pH 5.4 and an apparent molecular weight of 30,000 by high-pressure liquid chromatography gel permeation. Naturally occurring inhibitors of lysosomal phospholipase A are present in two of the lysosomal-soluble protein fractions obtained in the purification. They inhibit hydrolysis of 1,2-di[1-14C]oleoylphosphatidylcholine by purified phospholipase A1 with IC50 values of 7-11 micrograms. The inhibition is abolished by preincubation with trypsin at 37 degrees C, but preincubation with trypsin at 4 degrees C has no effect, providing evidence that the inhibitors are proteins. The results suggest that the activity of lysosomal phospholipase A may be regulated in part by inhibitory proteins. Lysosomal phospholipase A from rat kidney hydrolyzes the sn-1 acyl group of phosphatidylcholine, does not require divalent cations for full activity, and is not inhibited by ethylenediaminetetraacetic acid. It has an acid pH optimum of 3.6-3.8. Neither p-bromophenacyl bromide, diisopropyl fluorophosphate, nor mercuric ion inhibits phospholipase A1. In contrast to rat liver, which has two major isoenzymes of acid phospholipase A1, kidney cortex has only one isoenzyme of lysosomal phospholipase A1.     

On the substrate specificity of rat liver phospholipase A1

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.