Thrombin activates a membrane-associated calcium-independent PLA2 in ventricular myocytes

Activation of phospholipaseA₂(PLA₂) and accumulation oflysophosphatidylcholine contribute importantly to arrhythmogenesis during acute myocardial ischemia. We examined thrombinstimulation of PLA₂ activity inisolated ventricular myocytes. Basal and thrombin-stimulated cardiacmyocyte PLA₂ activity demonstrateda distinct preference for sn-1ether-linked phospholipids with arachidonate esterified at thesn-2 position. The majority ofPLA₂ activity was calcium independent and membrane associated. Thrombin stimulation ofmembrane-associated PLA₂ occurs ina time- and concentration-dependent fashion. An increase inPLA₂ activity was also observedusing the synthetic peptide SFLLRNPNDKYEPF (the tethered ligandgenerated by thrombin cleavage of its receptor). Bromoenol lactone, aselective inhibitor of calcium-independentPLA₂, completely blockedthrombin-stimulated increases inPLA₂ activity and arachidonic acidrelease. No significant inhibition of thrombin-inducedPLA₂ was observed followingpretreatment with mepacrine or dibucaine. These data confirm thepresence of high-affinity thrombin receptors on isolated cardiacmyocytes and demonstrate the specific activation of a uniquemembrane-associated, calcium-independentPLA₂ following thrombin receptorligation.

     

Effect of Mastoparan on Phospholipase A2 Activity in Potato Tubers Treated with Fungal Elicitor

Participation of phospholipase (PL) A₂ in signal trans-ductionhas been reported to elicitate a resistance reaction in potatocells by inoculation of an incompatible race of Phytophthorainfestans, the late blight fungus, or by treatment with fungalelicitor hyphal wall components (HWC). Mastoparan, a genericG protein activator, has been shown to activate PLA in a G protein-dependentmanner in animal cells. We analyzed the effects of mastoparanand the inactive analog Mas-17 on PLA₂ activity in potato tubers.In healthy potato tubers, the activation of PLA₂ by mastoparanwas detected in the soluble fraction, but not micro-somal fraction.However, in potato tubers treated with HWC, PLA₂ activity wasstimulated by mastoparan in both soluble and microsomal fractions.Pretreatment of the microsomal fraction with neomycin, a PLCinhibitor, and staurosporine, a protein kinase inhibitor, inhibitedthe mastoparan-induced activation of PLA₂. This suggested thatthe PLA₂ activation in potato tubers by mastoparan was mediatedby the PLC pathway and protein phospho-rylation. We also examinedthe accumulation of potato phytoalexin rishitin. Mastoparanstimulated rishitin accumulation induced by HWC, but did notinduce the accumulation. This indicated that mastoparan mightactivate the signal transduction pathway in the resistance reactionsinduced in potato tubers. (Received March 12, 1998; Accepted August 6, 1998)     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

Redistribution and abnormal activity of phospholipase A(2) isoenzymes in postinfarct congestive heart failure

Cardiacsarcolemmal (SL) cis-unsaturated fatty acid sensitivephospholipase D (cis-UFA PLD) is modulated by SLCa²⁺-independent phospholipase A₂(iPLA₂) activity via intramembrane release ofcis-UFA. As PLD-derived phosphatidic acid influences intracellular Ca²⁺ concentration and contractileperformance of the cardiomyocyte, changes in iPLA₂ activitymay contribute to abnormal function of the failing heart. We examinedPLA₂ immunoprotein expression and activity in the SL andcytosol from noninfarcted left ventricular (LV) tissue of rats in anovert stage of congestive heart failure (CHF). Hemodynamic assessmentof CHF animals showed an increase of the LV end-diastolic pressure withloss of contractile function. In normal hearts, immunoblot analysisrevealed the presence of cytosolic PLA₂ (cPLA₂)and secretory PLA₂ (sPLA₂) in the cytosol, withcPLA₂ and iPLA₂ in the SL. IntracellularPLA₂ activity was predominantly Ca²⁺independent, with minimal sPLA₂ activity. CHF increasedcPLA₂ immunoprotein and PLA₂ activity in thecytosol and decreased SL iPLA₂ and cPLA₂immunoprotein and SL PLA₂ activity. sPLA₂activity and abundance decreased in the cytosol and increased in SL in CHF. The results show that intrinsic to the pathophysiology of post-myocardial infarction CHF are abnormalities of SL PLA₂isoenzymes, suggesting that PLA₂-mediated bioprocesses arealtered in CHF.

     

Purification and Characterization of a Low-Molecular-Weight Phospholipase A2 from Developing Seeds of Elm

Phospholipase A₂ (PLA₂) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca²⁺ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA₂, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA₂s and, to our knowledge, is the first plant enzyme of this type to be described.     

Effect of auxin and other hormones on the metabolic response to wounding in sweet potato roots

In roots of sweet potato (Ipomoea batatas Lam. cv. Kokei 14),the metabolic response to wounding was remarkable only in theproximal side. We assumed that the polarity resulted from apolar movement of indole-3-acetic acid (IAA) produced in thecut surface (8). As the metabolic response was slight in thedistal side, the effect of IAA and the other plant hormoneson the development of various enzyme activities was examinedin this side. Increases in activities of L-phenylalanine ammonia-lyase,acid invertase, NADPHa₂ : cytochrome c oxidoreductase, peroxidase,cytochrome c : O₂ oxidoreductase and o-diphenol oxidase, whichdeveloped in response to wounding, were stimulated by the treatmentwith IAA. Gibberellic acid had a stimulative effect on the developmentof only acid invertase activity. Abscisic acid and kinetin hadlittle effect. The results strongly support our hypothesis thatIAA plays an important role in the metabolic response to wounding. (Received September 29, 1979; )     

Synthesis of intraspecific somatic hybrids of Solanum tuberosum: assessments of morphological, biochemical and nematode (Globodera pallida) resistance characteristics

In an attempt to produce novel agronomic traits, a number ofintraspecific somatic hybrid plants have been produced followingleaf mesophyll protoplast fusion between S. tuberosum dihaploidclones PDH 40 (possessing good tuber shape and yield) and PDH417 (possessing resistance to potato cyst nematode, G. pallida).PDH 417 protoplast-derived calli failed to regenerate plantsusing the described culture conditions preventing this parentaltype amongst the mass of regenerated fusion products. Initially,somatic hybrid plants were selected based on differential pigmentationin tuber sprouts and where possible on petal colour. Differentialmobility of patatin bands in electrophoresed tuber extractsfurther confinned hybridity. The intraspecific somatic hybridsalso showed different levels of resistance to G. pallida pathotypesPa2 and Pa3 in the somatic hybrid plants examined. Key words: Somatic hybridization, dihaploids, patatin, nematode resistance, Solanum tuberosum, potato     

Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation

The presentstudy was performed to characterize thrombin-stimulated phospholipaseA₂(PLA₂) activity and theresultant release of lysophospholipids from endothelial cells. Themajority of PLA₂ activity inendothelial cells was membrane associated,Ca²⁺ independent, and arachidonateselective. Incubation with thrombin increased membrane-associatedPLA₂ activity using bothplasmenylcholine and alkylacyl glycerophosphocholine substrates in theabsence of Ca²⁺, with no increasein activity observed with phosphatidylcholine substrate. The increasedPLA₂ activity was accompanied byarachidonic acid and lysoplasmenylcholine (LPlasC) release fromendothelial cells into the surrounding medium. Thrombin-induced changeswere duplicated by stimulation with the thrombin-receptor-directed peptide SFLLRNPNDKYEPF. Pretreatment with theCa²⁺-independentPLA₂ inhibitor bromoenol lactoneblocked thrombin-stimulated increases inPLA₂ activity, arachidonic acid,and LPlasC release. Stimulation of protein kinase C (PKC) increasedbasal PLA₂ activity and LPlasCproduction. Thrombin-stimulatedPLA₂ activity and LPlasC production were enhanced with PKC activation and completely prevented with PKC downregulation. Thus thrombin treatment of endothelial cellsactivates a PKC-activated, membrane-associated,Ca²⁺-independentPLA₂ that selectively hydrolyzesarachidonylated, ether-linked phospholipid substrates, resulting inLPlasC and arachidonic acid release.

     

Carbon Dioxide, Oxygen and Ethylene Effects on Potato Tuber Dormancy Release and Sprout Growth

The possible roles of oxygen and carbon dioxide treatments inthe presence or absence of ethylene on tuber dormancy releasein potato (Solanum tuberosumL.) were examined. Using two gascompositions (I: 60% CO₂–20% O₂–20% N₂and II: 20%CO₂–40% O₂–40% N₂), the phase of tuber dormancyand previous storage temperature were demonstrated to be importantparameters for dormancy release by these gas mixtures. Gas Icaused decreased abscisic acid (ABA) levels within 24 h regardlessof previous storage temperature, although this effect was reversible.Exogenous C₂H₄, an effective dormancy release agent, also causeddecreased ABA levels within 24 h. It also enhanced dormancyrelease and further promoted ABA losses by gas I. Gas II treatmentled to slight reductions in ABA levels that were further decreasedby C₂H₄. Sprout length was modelled successfully by multipleregression analysis in terms of glucose and ABA levels withinthe apical eye tissues of Russet Burbank tubers immediatelyafter, and regardless of, previous gas treatments or storagetemperatures. Solanum tuberosum,potato, abscisic acid, ethylene, carbon dioxide, oxygen, dormancy.     

A phosphatidyl choline exchange protein isolated from germinated castor bean endosperms

A phospholipid exchange protein (PLEP) functioning between theendoplasmic reticulum and the mitochondrion was purified fromthe cytosolic fraction of germinated castor bean endosperms.In the protein fraction eluted from Sephadex G-100 column, theexchange rate reached 7.3µg phospholipids exchanged/mgprotein/15 min, which was 60-fold that of pota to tuber PLEP.The lipid transfer by this protein was specific for phosphatidylcholine and the transfer rate from microsomes to mitochondriawas as high as that from mitochondria to microsomes. Castorbean PLEP transferred phospholipid from castor bean microsomesto mitochondria from other sources such as potato tubers, cauliflowerinflorescences, pumpkin hypocotyls and rat livers, and to liposomes,but not to Avena etioplasts. In addition, it transferred phospholipidfrom potato microsomes to potato mitochondria. (Received November 17, 1978; )     

Purification and Characterization of Thiol Proteinase as a Nitrate Reductase-Inactivating Factor from Leaves of Hordeum distichum L.

A thiol proteinase was purified 6,400-fold from leaves of Hordeumdistichum L. by a sequence of ammonium sulfate fractionation,gel filtration, ion exchange chromatography, hydrophobic chromatographyand chromatofocusing. This enzyme also had nitrate reductase(NR)-inactivating activity, which was associated with proteolyticactivity in almost constant proportions during the purificationprocedures. Its molecular weight was estimated as 74,000 bygel filtration, and it had an isoelectric point of 4.05 andan apparent K_m of 0.83 mg ml^–1 for azocasein. The respectiveoptimum pH for proteolytic and NR-inactivating activities were6.0 and 7.0. On electrophoresis, the proteinase gave a majorband that coincided with both activities; it also produced afaint band associated with no activity. Our purified thiol proteinase inactivated FMNH₂-NR and MVH-NRas well as NADH-NR, but it had only a slight effect on NADHcytochrome c reductase activity. This enzyme also inactivatedglutamine synthetase. (Received September 16, 1983; Accepted January 26, 1984)     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Inactivation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase,which catalyses the final step in the biosynthesis of ethylene,showed a non-linear time-course in vitro and activity decayedwith a half-life of around 14 min. This loss of activity wasstudied using tomato ACC oxidase purified from Escherichia coiltransformed with the cDNA clone pTOM13. Inactivation was notdue to end-product inhibition by dehydroascorbic acid or cyanide.Preincubatlon of enzyme in the combined presence of Fe²⁺ ascorbateand ACC, which together allowed catalytic turnover, resultedin almost total loss of ACC oxidase activity. Enzyme Inactivatedby catalysis could not be reactivated by passage through SephadexG-25 or by treating with combina tions of DTT and CO₂ A non-lineartime-course and inactivation in the presence of all substratesand cofactors was also shown for the enzyme assayed in vivowith melon fruit discs. Using the purified tomato enzyme a distinctascorbate-dependent inactivation was also observed, which occurredIn the absence of catalysis and was prevented, although notreversed, by catalase. This ascorbate-dependent inactivationmay thus be due to H₂O₂ attack on ACC oxidase. Key words: 1-aminocyclopropane-1-carboxylate (ACC) oxidase, catalase, catalytic inactivation, ethylene     

Partial purification and characterization of phosphatidic acid-specific phospholipase A1 in porcine platelet membranes

We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A₁ type (PA-PLA₁) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA₁ was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca²⁺ and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA₁ in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA₂ inhibitor, a Ca²⁺-independent phospholipase A₂ inhibitor, and a DG lipase inhibitor.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A2 (RVVA-PLA2-I) from Daboia russelli venom

An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24 h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.     

Characterization of Porphobilinogen Synthase from an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

Porphobilinogen synthase (formerly 5-aminolevulinic acid dehydratase,EC 4.2.1.24 [EC] ) was purified 7,405-fold from an aerobic photosyntheticbacterium, Erythrobacter sp. strain OCh 114. The molecular weightof the enzyme was determined to be 260,000 by Sephadex G-200gel filtration. The enzyme had a single pH optimum at 8.0 andshowed no requirement for metal ion and thiol compound for itsmaximum activity. The K_m value for 5-aminolevulinic acid was0.29 mM. 4,5-Dioxovaleric acid and levulinic acid were foundto be competitive inhibitors of the enzyme, with K_i values of0.65 and 0.80 mM, respectively. The enzyme was extremely labilein acidic pH and almost completely lost its activity within1 h at pH 6.0 and 30?C. This Erythrobacter enzyme seems to besimilar to the enzyme from the anaerobic photosynthetic bacteriumRhodobacter capsulatus in its molecular and catalytic properties. (Received February 17, 1988; Accepted May 9, 1988)     

Preliminary Characterization of Phospholipase A2 in Lagenidium giganteum

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A₂ in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A₂ (PLA₂) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA₂ activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA₂ activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-¹⁴C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ all enhanced PLA² activity, while Co²⁺, Fe²⁺, and Zn²⁺ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca²⁺ and Mn²⁺ than Mg²⁺, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA₂ activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA₂ hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA₂ activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA₂ activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

Positional Distribution of Fatty Acids in Glycerolipids of the Marine Red Alga, Porphyra yezoensis

The positional distribution of fatty acids in glycerolipidsfrom thalli of Porphyra yezoensis was studied by enzymatic hydrolysis.In monogalactosyl diacylglycerol, icosapentaenoic acid was amajor fatty acid at both the sn-1 and sn-2 positions of theglycerol moiety, whereas palmitic acid was a minor componentat both positions. In digalactosyl diacylglycerol and sulfoquinovosyldiacylglycerol, icosapentaenoic and palmitic acids were almostexclusively distributed at the sn-1 and sn-2 positions, respectively.In phosphatidylglycerol, palmitic and trans--13-hexadecenoicacid were exclusively located at the sn-2 position. In phosphatidylcholine,icosapentaenoic acid occurred in both the sn-l and sn-2 positions,whereas palmitic acid was confined to the sn-1 position. Itis suggested that monogalactosyl diacylglycerol in P. yezoensissynthesized in both the cytoplasmic and chloroplastic pathways,while the diacylglycerol moieties of the other chloroplast lipidsare virtually all derived from the chloroplastic pathway. (Received March 7, 1986; Accepted April 6, 1987)     

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A_1/2 (PLA_1/2) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA_1/2 and acyltransferase activities.