Study of bound phospholipase activities of fungal mycelia using an organic solvent system

It has been possible to demonstrate and characterize high phospholipase activities in mycelia ofRhizopus arrhizus andMucor javanicus by use of a system in which substrates were dissolved in diisopropyl ether. Such activities were associated with bound enzymes and would have been difficult to detect using aqueous assay systems. In both cases, phosphatidylcholine hydrolysis was by phospholipase A₁ (EC 3.1.1.32) activity followed by the action of lysophospholipase (EC 3.1.1.5). Phospholipase D (EC 3.1.4.4) activity was also detected. The methods used appear to be of general applicability for the detection and study of insoluble phospholipases.     

Directed evolution: From a staphylococcal lipase to a phospholipase

The lipase of Staphylococcus aureus (SAL) is able to degrade lipids and p‐nitrophenylesters but is not active on phospholipid substrates. Interestingly, the homologous lipase from Staphylococcus hyicus is highly active on phospholipids. In order to investigate the molecular basis for this difference in substrate specificity, phospholipase activity was introduced into SAL by directed evolution strategy. In this approach, sequential rounds of error‐prone PCR were performed in combination with a screening of the resulting mutant libraries. The screening was based on a high‐throughput plate assay and a subsequent chromogenic assay in 96‐well plate format to accurately determine the enzymatic activities in cell lysates of a selected number of clones. After 4 rounds of error‐prone PCR, two products were obtained, displaying a 7.8‐ and 9.2‐fold increase in absolute phospholipase activity and a 5.9‐ and 6.9‐fold increase in phospholipase/ lipase activity ratio. A final round of DNA shuffling with these two products and wildtype (WT)‐SAL was performed to combine beneficial mutations and to eliminate neutral or deleterious mutations. This procedure yielded a best variant containing 6 amino acid mutations displaying a 11.6‐fold increase in absolute phospholipase activity and a 11.5‐fold increase in phospholipase/lipase ratio as compared to the starting point. The character of the mutations and their possible effects on substrate specificity are discussed.     

Chromogenic assay for phospholipase D from Streptomyces chromofuscus: Application to the evaluation of substrate analogs

A rapid and convenient chromogenic assay for phospholipase D from Streptomyces chromofuscus (PLD_Sc) has been developed that converts the choline generated from the enzyme-catalyzed hydrolysis of phospholipids into a chromogenic dye. By quenching the reaction with EDTA at defined times, an initial rate curve is produced from which a k _cat and K _m can be readily derived. This assay has been applied to the biological evaluation of several substrate analogs, all of which appear to be activators rather than substrates or inhibitors of this enzyme. Performing the assay in 96-well microtiter plates allows for the easy screening of potential effectors of this enzyme.     

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay

Patatin is a family of glycoproteins that accounts for 30–40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl]PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V _m=9.8×10⁻³ μmol/min·μg protein, K _m=7.8 μM, V _m/K _m=1.3 min⁻¹·μg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.     

A micro enzymic method for determination of choline-containing phospholipids in serum and high density lipoproteins

A micro method is described for the assay of choline-containing phospholipids in serum and high density lipoproteins (HDL) using an automated microtiter plate reader. The method is adapted from the enzymic method of Takayama, Itoh, Nagasaki, and Tanimuzu (Clin. Chim. Acta 79, 93–98, 1977) using phospholipase D, choline oxidase, and peroxidase coupled with the color generating system phenol and 4-amino-antipyrine. The micro method requires 5 μL of serum or HDL sample, and 42 samples can be assayed in duplicate in one run using a 96-well flat-bottom microtiter plate. The reaction is linear up to 400 mg/dL and the lower limit of detection is 0.25 mg of choline-containing phospholipids per assay. The coefficient of variation within an assay is 0.86–0.79%, and day-to-day variation is 0.9–1.5%. Results obtained by the micro method are in excellent agreement with those obtained by the procedure of Takayamaet al. (r=0.997). The supernatant left after removal of low density lipoproteins and very low density lipoproteins from serum and precipitation with heparin/manganese chloride reagent can thus be conveniently used for the micro assay of choline phospholipids in HDL.     

Studies on phospholipase activities inneurospora crassa mycelia

Phospholipase A activity has been detected in mycelial homogenate ofNeurospora crassa. A submycelial fraction, obtained by differential centrifugation containing the highest specific activity of phospholipase A has been shown to contain ca. 66% phospholipase A₁ and 34% phospholipase A₂ activity along with lysophospholipase and degergent-stimulated phospholipase D activity. Phospholipase A activity bound toN. crassa mycelia also has been observed.     

Phospholipase D Activity in Relation to the Size of Substrate Micelles

The activity of phospholipase D (PLD) is strongly dependent on the aggregation state of its substrate. Artificial substrates, such as phosphatidyl‐p‐nitrophenol (PpNP), are preferably used in a mixture with Triton^® X‐100 (Serva) and sodium dodecyl sulfate (SDS) in the form of mixed micelles. In this paper the activities of PLD from cabbage (PLD_cab), which needs Ca²⁺ ions for its activity, and PLD from Streptomyces sp. (PLD_Str), which is independent of Ca²⁺ ions, are compared as a function of the detergent and substrate concentrations. While the variation of the Triton^® X‐100 and PpNP contents changed the activities of both enzymes in similar way, SDS showed activation effects on plant PLD but inactivation effects on microbial PLD. The activity decreased to 50% at a 7‐tenfold molar excess of Triton^® X‐100 related to PpNP. SDS induced a strong activation (up to fourfold) of PLD_cab but decreased the activity of PLD_Str with increasing concentration. Activity‐substrate profiles of both PLDs passed optima at 3–4 mM PpNP. In all experiments, the activity changes correlated with changes of the micelle sizes determined by dynamic light scattering. These results highlight the immense importance of the micelle structure, which is not considered in most studies.     

Synthesis and phospholipase C inhibitory activity of D609 diastereomers

The potassium xanthate D609 is widely accepted as a selective inhibitor of PC-specific phospholipase C (PC-PLC). The tricyclo[5.2.1.0^2,6]decane skeleton present in D609 can lead to four diastereomeric paris, but the diastereoselectivity of PC-PLC inhibition has never been reported. In this article, the synthesis of racemic D609 diastereomers and that of other xanthates, as well as their inhibitory effect on PC-PLC is reported. All xanthates obtained were competitive inhibitors of PC-PLC from Bacillus cereus (PLCBc). No significant differences were found in the activity of D609 diastereomers (K _i 13–17 μM), suggesting the absence of a diastereochemical control of the enzyme by xanthate inhibitors. This result was confirmed after obtaining other potassium xanthates differing from D609 in the aliphatic chain. Among them, the potassium O-n-decenylxanthate was the most active inhibitor of PLCBc (K _i 10 μM). These data indicate that the essential structural requirements for PLCBc in vitro inhibition by xanthates are the presence of a Zn-chelating dithiocarbonate head and a sufficiently hydrophobic aliphatic moiety.     

Enzymatic Synthesis of 1‐Alkyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate Using a Novel Lysoplasmalogen‐Specific Phopholipase D

Many phospholipase Ds (PLDs) are known to catalyze transphosphatidylation as well as hydrolysis of phospholipids. Transphosphatidylation of lysoplasmalogen (LyPls)‐specific phospholipase D (LyPls‐PLD), which catalyzes hydrolysis of ether lysophospholipids such as LyPls and 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine (Lyso‐PAF), still remains unclear. This study aims to reveal the transphosphatidylation activity of LyPls‐PLD, that is, the production of cyclic ether lysophospholipid. The enzymatic reaction is conducted in a buffer system, and the reaction products of a novel LyPls‐PLD from Thermocrispum sp. are investigated using mass spectrometry (MS). MS analyses demonstrate the reaction products to consist of 100% 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate (cLyPA) and choline from Lyso‐PAF; however, 1‐alkenyl‐2‐hydroxy‐sn‐glycero‐2,3‐cyclic‐phosphate from 1‐O‐1′‐(Z)‐octadecenyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine and 1‐O‐1′‐(Z)‐octadecenyl‐2‐hydroxy‐sn‐glycero‐3‐phosphoethanolamine is not produced. These results are expected to help in elucidating the catalytic mechanism of LyPls‐PLD, that is, the rate‐limiting step, and indicate LyPls‐PLD to be useful for the one‐pot synthesis of cLyPA. Practical Applications: A novel phospholipase D, LyPls‐PLD, can exclusively synthesize cLyPA from Lyso‐PAF using a one‐step enzymatic reaction without an organic solvent. cLyPA could be expected to show bioactivities similar to those of cyclic phosphatidic acid, which promotes normal cell differentiation, hyaluronic acid synthesis, antiproliferative activity in fibroblasts, and inhibitory activity toward cancer cell invasion and metastasis.     

Study of the Involvement of Phosphatidic Acid Formation in the Expression of Wound‐Responsive Genes in Cotton

Plants use phospholipase D (PLD, EC 3.1.4.4)/phosphatidic acid (PtdOH) for the transduction of environmental signals including those coming from wounding. Based on our previous findings suggesting that wound‐induced PLDα‐derived PtdOH can act as a local signaling molecule in cotton (Gossypium hirsutum), we show that wounding immediately increases local NADPH oxidase (NADPHox) and cellulose synthase A (CeSA) gene expression. After developing a novel fluorimetric assay for the investigation of n‐butanol inhibitory effect on PLD activity, we show that only NADPHox gene upregulation is reduced when n‐butanol is applied prior to wounding. This suggests that NADPHox is a possible downstream target of PLD function, while a different CeSA‐involving response system may exist in cotton. Overall, this study provides new knowledge on signal‐transduction mechanisms following wounding of cotton leaves.     

An evaluation of NBD-phospholipids as substrates for the measurement of phospholipase and lipase activities

Because most of the existing assays of phospholipase activity are quite laborious, the use of 1-acyl-2-[6-(7-nitro-1,3-benzoxadiazol-4-yl)amino]caproyl labeled phospholipids (NBD phospholipids) was investigated to determine whether they could be used as substrates in the routine assay of various phospholipases and lipases. NBD-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidic acid were evaluated. There was about a 50-fold increase in fluorescence upon hydrolysis of the NBD hexanoic acid from the NBD phospholipid, confirming an earlier report. This change in fluorescence was constant over the normal physiological pH range (pH 5–9). Detergents and bovine serum albumin interfered with the assay in a concentration dependent manner. An increase in fluorescence and a concomitant increase in NBD hexanoic acid was detected with the two phospholipase A₂ enzymes. Although a change in fluorescence was detected with a phospholipase C, careful evaluation revealed that the rate of increase in fluorescence was not proportional to the rate of production of diacylglycerol product. Neither of the two phospholipase D enzymes which were tested were able to cause an increase in fluorescence when incubated with NBD phospholipids. A small increase in fluorescence was detected with each of the four lipases. Of the five NBD lipids tested, the highest rates of hydrolysis were consistently obtained with NBD-phosphatidylglycerol followed by NBD-phosphatidylcholine.     

Determination of phospholipase D-mediated hydrolysis of phosphatidylethanolamine

While phospholipase D-mediated hydrolysis of phosphatidylcholine is well documented, we have recently shown that phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) [Kiss, Z., and Anderson, W.B.,J. Biol. Chem. 264, 1483–1487 (1989);J. Biol. Chem. 265, 7345–7350 (1990)] is equally prominent. This made it necessary to define in detail the conditions required for the detection of agonist-stimulated PtdEtn hydrolysis. Using the [¹⁴C]ethanolamine-prelabeled rat-1 fibroblast model and 12-O-tetradecanoylphorbor 13-acetate (TPA) as a model compound with the known ability to stimulate phospholipase D, we demonstrated that optimal detection of TPA-induced ethanolamine release requires i) fractionation of water-soluble ethanolamine products; ii) addition of unlabeled ethanolamine to quench the phosphorylation of newly formed [¹⁴C]ethanolamine; and/or iii) prolonged preincubation of prelabeled cells in an isotope-free medium before the addition of TPA. This preincubation step reduces the cellular content of unincorporated¹⁴C-labeled ethanolamine metabolites and improves the signal-to-noise ratio.     

Metal ion stimulation of phospholipase D-like activity of isolated rat intestinal mitochondria

Presence of phospholipase D-like (PLD) activity in the intestinal mitochondria was identified using endogenous phospholipids as substrate. The enzyme had a pH optimum of 6.5, did not show trans-phosphatidylation activity in the presence of ethanol or butanol, and the product formed was phosphatidic acid (PA). This was confirmed by separation of reaction products by high-performance liquid chromatography and analysis of composition of the PA formed which gave phosphate/fatty acid ratio of 1∶2. PLD-like activity was further confirmed by the formation of ethanolamine and choline as products of enzyme action. This activity was stimulated by various metal ions; when stimulated by Mg²⁺ and Ba²⁺, it hydrolyzed both phosphatidylcholine and phosphatidylethanolamine, and when stimulated by Ca²⁺, it preferentially hydrolyzed phosphatidylethanolamine. There was no requirement for sodium oleate for the PLD-like activity in mitochondria. These results suggest that intestinal mitochondria have an active PLD-like enzyme which differs in certain properties from phospholipase D from other tissues.     

Adipocyte Fatty Acid-Binding Protein as a Novel Marker of Psoriasis and Clinical Response to Acitretin

Psoriasis is a systemic disease associated with metabolic syndrome and cardiometabolic diseases. Adipocyte fatty acid-binding protein (A-FABP, FABP4) is a relevant mediator of lipid metabolism and several comorbidities development. Aim of the study was to explore the possible role of FABP4 in psoriasis and assess its relationship with disease activity, inflammation or metabolic disturbances, and impact of systemic treatment. Fasting blood samples were obtained from 33 patients with active plaque-type psoriasis before and after 12 weeks of therapy and from 11 healthy volunteers. Serum FABP4 concentrations were analyzed by the enzyme-linked immunosorbent assay (ELISA) and statistically analyzed for their correlations with clinical outcomes and the treatment introduced. Serum FABP4 levels were significantly increased in psoriatics compared to controls (p = 0.03). No relationship between the protein and psoriasis severity expressed through psoriasis area and severity index (PASI) was noted (p = 0.57). FABP4 did not correlate with CRP (p = 0.41), lipid profile, and body mass index (BMI) nor the glucose level or liver enzyme activity. FABP4 significantly correlated with morphotic blood elements. After total therapy, FABP4 did not statistically change (p = 0.07), but significantly decreased after administering acitretin (p = 0.03). FABP4 is a potential marker of psoriasis and clinical outcome after therapy with acitretin. Adipocyte-type FABP may be related to hematological disorders or obesity-mediated comorbidities in psoriasis.     

Coexistence of phosphatidylcholine-specific phospholipase C and phospholipase D activities in rat cerebral cortex synaptosomes

DAG derived from phosphatidylcholine (PtdCho) acts as a lipid second messenger. It can be generated by the activation of phospholipase D (PLD) and the phosphatidic acid phosphohydrolase type 2 (PAP2) pathway or by a PtdCho-specific phospholipase C (PtdCho-PLC). Our purpose was to study PtdCho-PLC activity in rat cerebral cortex synaptosomes (CC Syn). DAG production was highly stimulated by detergents such as Triton X-100 and sodium deoxycholate. Ethanol and tricyclodecan-9-yl-xanthate potassium salt decreased DAG generation by 42 and 61%, respectively, at 20 min of incubation. These data demonstrate that both the PLD/PAP2 pathway and PtdCho-PLC contribute to DAG generation in CC Syn. PtdCho-PLC activity remained located mainly in the synaptosomal plasma membrane fraction. Kinetic studies showed K _m and V _max values of 350 μM and 3.7 nmol DAG × (mg protein × h)⁻¹, respectively. Western blot analysis with anti-PtdCho-PLC antibody showed a band of 66 KDa in CC Syn. Our results indicate the presence of a novel DAG-generating pathway in CC Syn in addition to the known PLD/PAP2 pathway.     

Comparison of the mechanical and biological properties of self-adhering materials

Purpose: The purpose of the present study was to compare the Vickers hardness numbers (VHNs), roughness numbers (RNs) and biological compatibility of glass carbomer (Glass Fill), resin-modified glass ionomer (Fuji II LC) and self-adhering flowable composite (Vertise Flow) materials. Materials and methods: Disc-shaped specimens of test materials (n = 15/group) were prepared, and VHNs and RNs were determined after 24 h. A direct contact test was used for cytotoxicity evaluation. Cell viability was measured for 24 h post-exposition with a photometric test (MTT assay; n = 16). Data were analysed using one-way analysis of variance with a post hoc Tukey’s test, the Kruskal–Wallis test and the Mann–Whitney U-test (p < 0.05). Results: Fuji II LC had the highest VHN. The VHN of Fuji II LC differed significantly from those of the other materials (p < 0.05). The RNs of Fuji II and Glass Fill were higher than that of Vertise Flow (p < 0.05). The self-adhering materials were not significantly cytotoxic compared with the control group (p > 0.05). Conclusions: The materials tested in this study showed a similar lack of cytotoxicity. The VHN of Fuji II LC was the highest, and the RN of Vertise Flow was the lowest.     

Characterization of phospholipase A2 from rabbit lung microsomes

A phospholipase A₂ activity associated with the microsomal fraction of rabbit lung homogenates was studied. The enzyme showed specificity for thesn ⁻² ester bond of phosphatidylcholine, had an alkaline pH optimum and required Ca²⁺ for activity. Other divalent cations were unable to support hydrolysis. In the absence of detergents, exogenous phosphatidylethanolamine was deacylated at a rate sevenfold higher than phosphatidylcholine. The activity toward both substrates could be enhanced by sodium deoxycholate or, more effectively, by sodium taurodeoxycholate. Phosphatidylethanolamine required higher detergent/phospholipid molar ratios than phosphatidylcholine. Under these conditions, the preference for the former substrate over the latter was nearly abolished. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and the nonionic detergent Triton X-100 were either ineffective (phosphatidylcholine) or inhibitory (phosphatidylethanolamine). Addition of KCl produced opposite effects on the activity depending on the bile salt used to disperse the substrate. The phospholipase A₂ activity was inhibited byp-bromophenacyl bromide but remained unaffected after treatment with diisopropylfluorophosphate or NaF. N-Ethylmaleimide, but not other thiol reagents, partially inhibited the activity. Presented in part at the 29th CFBS meeting, Guelph, Canada, June 1986, and at the symposium “25 Years Lipids and Biomembranes,” Utrecht, The Netherlands, June 1986.     

Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography

Using the spectrofluorimetric method described by Wittenaueret al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984)Biochem. Biophys. Res. Commun. 118, 894–901] for phospholipase A₂ (PLA₂) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutantrin tomatoes at their normal harvest time (mature green stage). This activity in Ailsa Craig tomatoes increased at the beginning of fruit ripening (green-orange stage) and then decreased slowly. The decrease in activity, however, was greater when ripening occurred after tomato picking at normal harvest time than when ripening occurred on tomato plants. This phospholipase activity was always higher inrin tomatoes than in normal ones. Thin-layer chromatography of compounds obtained after incubation of tomato extract demonstrated a decrease in the substrate 1-acyl-2-{6[(7-nitro-2,1,3, benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine (C₆-NBD-PC), and an increase in one product (NBD-aminohexanoic acid), but failed to detect the second product (1-acyl-sn-glycero-3-phosphocholine). We, therefore, developed a new one-step method for separation and quantification of a mixture of phospholipids and other lipids, using straight-phase-high-performance liquid chromatography with light-scattering detection. This method detected another fatty acid-releasing activity in enzyme extract from green-orange tomatoes. This lipolytic enzyme (or family of enzymes) slowly produced free fatty acids when 1-oleoyl-sn-glycero-3-phosphocholine was added as substrate. The production of fatty acids was stoichiometric and more rapid when 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-sn-glycerol were used as substrates. On the other hand, the same tomato extract was unable to hydrolyze 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycerol. Crude tomato extract exhibited lipid acyl hydrolase activity according to the definition of Galliard [Galliard, T. (1979), inAdvances in the Biochemistry and Physiology of Plant Lipids (Appelqvist, L.A., and Liljenberg, C. eds.), pp. 121–132, Elsevier, Amsterdam]. But in order to demonstrate whether tomato extract contains PLA₂ activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.     

Synthesis of 6-phosphatidyl-L-ascorbic acid by phospholipase D

Phospholipase D (EC 3.1.4.4) ofStreptomyces species was found to catalyze transphosphatidylation to L-ascorbic acid from phosphatidylcholine (PC) in a biphasic reaction system. The product was identified as 1,2-diacyl-sn-glycero-3-phospho-6′-L-ascorbic acid (PA-AsA) by mass spectrometry and nuclear magnetic resonance spectroscopy. The optimal pH of transphosphatidylation was 4.5 and the rate of PA-AsA formation increased as concentrations of L-ascorbic acid increased. The conversion of PC to PA-AsA was greater than 80%. PA-AsA was found to be more resistant to hydrolysis by phospholipase D than was PC.