A convenient spectrophotometric assay for phospholipase D usingp-nitrophenyl-phosphocholine as substrate

A simple assay for plant phospholipase D is described. The enzyme hydrolyzesp-nitrophenyl-phosphocholine top-nitrophenyl phosphate, which in the presence of an acid phosphatase generatesp-nitrophenol (a chromogenic compound). Thus,p-nitrophenylphosphocholine can be used for assay of phospholipase D in sources that do not also contain high levels of phospholipase C. This work was supported by a U.S. Public Health Service Research Grant (GM15053).     

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipaseA₂ in catalysis and substrate binding, using site-directed mutagenesis.A mutant was constructed containing Phe at position 69. Kineticcharacterization revealed that the Phe-69 mutant has retainedenzymatic activity on monomeric and micellar substrates, andthat the mutation has only minor effects on k_cat and K_m. Thisshows that Tyr-69 plays no role in the true catalytic eventsduring substrate hydrolysis. In contrast, the mutation has aprofound influence on the stereospecificity of the enzyme. Whereasthe wild-type phospholipase A₂ is only able to catalyse thedegradation of sn-3 phospholipids, the Phe-69 mutant hydrolysesboth the sn-3 isomers and, at a low (1–2%) rate, the sn-1isomers. Despite the fact that the stereospecificity of themutant phospholipase has been altered, Phe-69 phospholipasestill requires Ca²⁺ ions as a cofactor and also retains itsspecificity for the sn-2 ester bond. Our data suggest that inporcine pancreatic phospholipase A₂ the hydroxyl group of Tyr-69serves to fix and orient the phosphate group of phospholipidmonomers by hydrogen bonding. Because no such interaction canoccur between the Phe-69 side-chain and the phosphate moietyof the substrate monomer, the mutant enzyme loses part of itsstereospecificity but not its positional specificity.     

Structural modifications of rat serum high density lipoprotein by pancreatic phospholipase A2

An important and unusual aspect of the high density lipoprotein (HDL) in the rat is its tendency to undergo marked alterations in structure in response to physiological perturbations. In this study, the role of the surface lipids for maintenance of HDL integrity were investigated. Hydrolysis by pancreatic phospholipase A₂ of the phospholipids of rat HDL in the presence of the d>1.21 g/ml fraction of rat serum results in an increase in the particle diameter and an uptake of apo-E and apo A-IV from the lipoprotein-free fraction; augmentation of the albumin concentration in the incubation mixture intensified the observed changes, probably due to enhancement of the compositional changes brought about by phospholipase treatment. Phospholipase A₂ treatment of the d<1.21 g/ml fraction of rat serum produces only minor changes in the properties of the isolated HDL. These data suggest that changes in apoprotein content reflect an uptake of A-IV and E by the rat HDL, rather than a net loss of apo A-I. Likewise, titration of the action of pancreatic phospholipase A₂ on HDL apoprotein composition showed that initially a modest increase in apo A-IV content occurred, but with more extensive phospholipolysis there was a considerably greater increase in the apo-E content of the particle. The data suggest that hydrolysis of phospholipids such as occurs physiologically through the action of lecithin: cholesterol acyl transferase and hepatic lipase may alter the HDL structure independently from changes effected in the neutral lipid core.     

Hydrolysis of intralipid by pancreatic lipase and phospholipase A2-gel filtration studies

Intralipid was incubated with pancreatic lipase (EC 3.1.1.3) and/or phospholipase A₂ (EC 3.1.1.4) at two bile salts/phosphatidylcholine molar ratios and at two different triglyceride hydrolysis rates using various amounts of lipase. Incubations were studied by gel filtration. Results show: (i) During lipase action, three phases of lipids coexist: an emulsified phase, a micellar phase and an intermediate heavy phase sized between the two others. The equilibrium between each phase is dependent upon the bile salts concentration. (ii) Under these conditions, pancreatic lipase was at 60% bound to the emulsified phase, whereas pancreatic phospholipase A₂ was bound at 94% to the micellar phase.     

Phosphatidylcholine synthesis in the rat: The substrate for methylation and regulation by choline

Two lines of evidence led us to reexamine the possibility that methylation of phosphoethanolamine and its partially methylated derivatives, in addition to methylation of the corresponding phosphatidyl derivatives, plays a role in mammalian phosphatidylcholine biosynthesis: (a) Results obtained by Salerno and Beeler with rat [Salerno, D. M. and Beeler, D. A. (1973)Biochim. Biophys. Acta 326, 325–338] appear to strongly support such a role for methylation of phosphobases; (b) Such reactions have recently been shown to play major roles in phosphatidylcholine synthesis by higher plants [see Datko, A. H. and Mudd, S. H. (1988)Plant Physiol. 88, 854–861 and references therein]. We found that, following coninuous labeling of rat liver with L-[methyl-³H]methionine for 10.4 min (intraperitoneal administration) or for 0.75 min (intraportal administration), virtually no³H was detected in methylated derivatives of phosphoethanolamine, but readily detectable amounts of³H were present in the base moiety of each methylated derivative of phosphatidylethanolamine. Thus, there was no indication that phospho-base methylation makes a significant contribution. Studies of cultured rat hepatoma cells showed definitively for the first time in a mammalian system that choline deprivation up-regulates the rate of flow of methyl groups originating in methionine into phosphatidylethanolamine and derivatives. Even under these conditions, methylation of phosphoethanolamine bases appeared to play a negligible role.     

Is intestinal villus phospholipase A2/lysophospholipase bound pancreatic carboxylester lipase?

Similarities in substrate specificity, localization and molecular weight between villus membrane phospholipase A₂/lysophospholipase and carboxylester lipase of pancreatic origin suggested their possible identity. To test this, a preparation of the phospholipase A₂/lysophospholipase released from brush border vesicles by papain was compared to authentic, pancreatic carboxylester lipase. Susceptibility of both activities to the inhibitor, diisopropylfluorophosphate, was consistent with their identity, but inconclusive. It also indicated that two populations of phospholipase A₂ species may be present in the papain-released preparation. However, comparison of binding of the activities to Sepharose-coupled, anti-carboxylester-lipase IgG indicates that they are immunologically distinct.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 63–74 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' – 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 59–70 of RNase were inserted into angiogenin[Harper and Vallee (1989) Biochemistry, 28, 1875–1884].Thus, the external loop in residues 63–74 of RNase A appearsto behave, at least in part, as an interchangeable ‘module’that influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein.     

重组人脂蛋白相关磷脂酶A2的原核表达及其纯化

目的原核表达并纯化重组人脂蛋白相关磷脂酶A2(lipoprotein-associated phospholipase A2,LP-PLA2),并初步确定其保存条件。方法将重组表达菌pCold TF-LP-PLA2-BL21(DE3)和pET28-HRV 3C-BL21(DE3)分别经IPTG诱导表达,表达的重组蛋白经SDS-PAGE鉴定后,经Ni-Sepharose 6FF金属螯合层析粗提重组蛋白,经HRV 3C蛋白酶酶切去除融合标签后,用Blue Sepharose誖6 Fast Flow(蓝胶)染料亲和层析及HiPrep 16/60 Sephacryl S-100 HR分子筛进行分离纯化;通过人LP-PLA2临床检测金标准ELISA试剂盒验证重组蛋白;将重组LP-PLA2置于含10 mmol/L CHAPS和0.5 mmol/L PMSF的PBS溶液中,分别于4℃、室温、37℃保存1周以及-20、-80℃冻存4个月后,确立重组蛋白的保存条件。结果重组LP-PLA2相对分子质量约100 000,以可溶性形式表达,包涵体无表达;纯化后的重组LP-PLA2蛋白含量为3.68 mg/ml,蛋白回收率为21%,纯度可达95%,可被ELISA试剂盒识别,且具有良好的线性关系(R2=0.996 4);重组LP-PLA2于4℃保存1周,-20、-80℃冻存4个月,均无降解现象。结论成功表达、纯化了重组LP-PLA2,并初步确定了其保存条件。     

Preparation of therapeutic phospholipids through porcine pancreatic phospholipase A2-mediated esterification and lipozyme-mediated acidolysis

Highly unsaturated fatty acid-containing phospholipids (HUFA-PL), which serve to increase the deformability of human red blood cells, were prepared through porcine phospholipase A₂-mediated esterification of the lysophosphatidylcholine, which was derived from soy phosphatidylcholine (PC), and by Lipozyme-mediated acidolysis. Through these processes, phospholipase A₂, with formamide as a water mimic, enhanced the incorporation of HUFA into positionsn-2 of PC and suppressed hydrolysis of the synthesized PL. On the other hand, Lipozyme-mediated acidolysis between positionsn-1 of soy PC and HUFA was enhanced by a combination of water and propylene glycol. Simultaneously, the recovered PL products showed decreased hydrolysis of newly synthesized health-beneficial HUFA-PL.     

The binding mode of cladocoran A to the human group IIA phospholipase A(2)

The molecular basis for human group IIA phospholipase A(2) inactivation by the marine natural product cladocoran A (CLD A) has been studied in order to elucidate its relevant anti-inflammatory properties. Indeed, secretory phospholipases A(2) are well-known to be implicated in the pathogenesis of inflammation, such as rheumatoid arthritis, septic shock, psoriasis and asthma, thus the understanding of their inactivation mechanism could be useful for the development of new chemical classes of selective inhibitors. Our results, collected by a combination of biochemical approaches, advanced mass spectrometry and molecular modeling, suggest a competitive inhibition mechanism guided by a noncovalent molecular recognition event, and disclose the key role of the CLD A γ-hydroxybutenolide ring in the chelation of the catalytic calcium ion inside the enzyme active site. Moreover, CLD A is able to react selectively with Ser82, although this covalent event seems to play a secondary role in terms of enzyme inhibition.     

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A₂ (PLA₂) islocated at the entrance to the active site. To study the roleof residue 31 in PLA₂, six mutant enzymes were produced by site-directedmutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Seror Gly. Direct binding studies indicated a three to six timesgreater affinity of the Trp31 PLA₂ for both monomeric and micellarsubstrate analogs, relative to the wild-type enzyme. The otherfive mutants possess an unchanged affinity for monomers of theproduct analog n-decylphosphocholine and for micelles of thediacyl substrate analog rac-l,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine.The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholinewere decreased 9–20 times for these five mutants. Kineticstudies with monomeric substrates showed that the mutants haveV_max values which range between 15 and 70% relative to the wild-typeenzyme. The V_max values for micelles of the zwitterionic substratel,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3–50times. The K_m values for the monomeric substrate and the k_mvalues for the micellar substrate were hardly affected in thecase of five of the six mutants, but were considerably decreasedwhen Trp was present at position 31. The results of these investigationspoint to a versatile role for the residue at position 31: involvementin the binding and orientating of monomeric substrate (analogs),involvement in the binding of the enzyme to micellar substrateanalogs and possibly involvement in shielding the active sitefrom excess water.     

D-Tyrosine as a chiral precusor to potent inhibitors of human nonpancreatic secretory phospholipase A2 (IIa) with antiinflammatory activity

Few reported inhibitors of secretory phospholipase A(2) enzymes truly inhibit the IIa human isoform (hnpsPLA(2)-IIa) noncovalently at submicromolar concentrations. Herein, the simple chiral precursor D-tyrosine was derivatised to give a series of potent new inhibitors of hnpsPLA(2)-IIa. A 2.2-A crystal structure shows an inhibitor bound in the active site of the enzyme, chelated to a Ca(2+) ion through carboxylate and amide oxygen atoms, H-bonded through an amide NH group to His48, with multiple hydrophobic contacts and a T-shaped aromatic-group-His6 interaction. Antiinflammatory activity is also demonstrated for two compounds administered orally to rats.     

Role of the substrate state in electrochemical nucleation

The possibility to obtain information about the substrate state and the energetic characteristics of small clusters is discussed in the case of electrochemical nucleation on a foreign substrate. Conclusions are drawn about the character of the supercritical clusters location on the electrode surface.     

Energy state of InGaAs quantum dots on SiO2-patterned vicinal substrate

The optical properties of In_0.8Ga_0.2As self-assembled quantum dots (SAQDs) grown on GaAs wire structures formed by utilizing SiO₂-patterned exact and 5°-off (001) GaAs substrates have been studied with micro-photoluminescence (μ-PL). Single PL peak was occurred for In_0.8Ga_0.2As SAQDs grown on SiO₂-patterned exact (001) GaAs, whereas double PL peaks were showed for SAQDs grown on 5°-off (001) GaAs substrates as the width of the opening windows increased. The power-dependent μ-PL spectra show that the first and second peaks of these double peaks were originated from the well-defined ground and excited state, respectively. These results demonstrated that In_0.8Ga_0.2As SAQDs selectively grown by utilizing SiO₂-patterned 5°-off (001) GaAs substrates have well-defined zero-dimensional quantum states.     

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.     

Effect of ethanol on platelet phospholipase A2

Platelet aggregation is known to be inhibited by ethanol, and this has been suggested to be one of the attenuating effects of ethanol in cardiovascular disease. Recent studies have implicated an inhibition of phospholipase A₂ induced arachidonic acid release, since the production of prostanoids that are formed from arachidonic acid and are involved in the aggregation process has been shown to be diminished by ethanol. Phospholipase A₂ is found in platelets in both a cytosolic form, from where it may translocate to the plasma membrane to release arachidonic acid, and in a secretory form which is released extracellularly upon activition. In the present study, the effect of ethanol on the secretion of phospholipase A₂ and on its activity was determined. It was found that ethanol inhibited trast, the activity of the cytosolic form of phospholipase A₂ was inhibited by ethanol.     

Effect of substrate temperature on the physical properties of co-evaporated Sn2S3 thin films

We report the deposition of tin sulfide (Sn₂S₃) thin films by co-evaporation technique at different substrate temperatures. The influence of substrate temperature on the structural and optical properties of the thin films is investigated. X- ray diffraction (XRD) analysis and Micro-Raman studies confirm the formation of Sn₂S₃ phase. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) are used to examine the surface morphology. The transmission spectra of the deposited Sn₂S₃ thin films have been recorded in the wavelength range of 200–3000 nm using UV–vis-NIR spectrometer. Film thickness (d) and optical constants such as refractive index (n), extinction coefficient (k), real (ε₁) and imaginary (ε₂) parts of the dielectric constants of thin films are estimated from the optical transmittance. The optical band gaps of the deposited films at different substrate temperatures are in the range of 1.46–1.64 eV. Hall effect measurements confirm the n-type nature of the as-prepared Sn₂S₃ thin films.     

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.     

Phosphorothiolate analogues of phosphatidylinositols as assay substrates for phospholipase C

Accurate measurement of phosphatidylinositol-specific phospholipase C (PI-PLC) activity is important in view of the key role of this enzyme in signal-transduction pathways. In this work we synthesized enantiomerically pure phosphorothiolate analogues of all natural PI-PLC substrates, including those of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), 4-phosphate (PI-4-P), 5-phosphate (PI-5-P) and unphosphorylated PI, in both long- and short-chain versions. The enzymatic cleavage of these substrates produces thiol analogues of diacyl glycerol, which can be quantified by UV absorbance after treatment with dipyridyl disulfide. The monodisperse dihexanoyl derivatives are suitable substrates for PI-PLC assay: they give rise to high enzyme activity, and provide excellent linear kinetic responses. For all substrates, we found a good linear correlation between the reaction rate and the amount of enzyme; this indicated the suitability of this assay for enzyme quantification. The short-chain substrates enable the enzyme specificity with variously phosphorylated inositol head groups to be established--unobstructed by substrate aggregation, "scooting" kinetics on micelles, or surface dilution effects. The kinetic results indicated allosteric behavior of PLC for all substrates tested. We found that substrates phosphorylated at the inositol 4-position (phosphorothiolate analogues of PI-4,5-P2 and PI-4-P) displayed very similar kinetic properties, and were cleaved with approximately 20- to 30-fold higher activity than the 4-nonphosphorylated substrates (analogues of PI-5-P and PI). Hence it appears that interactions between the enzyme and the 4-phosphate group of the substrate, but not its 5-phosphate group, is important for PI-PLC catalysis. In addition, the binding affinities of all four substrate types were found to be quite similar; this indicates that the energy of enzyme interaction with the 4-phosphate group is directed almost entirely to catalysis.