Effect of increasing the level of ω-3 fatty acids on rat skeletal muscle sarcoplasmic reticulum

The effect of dietary supplementation with fish oil as compared to corn oil on the lipid dynamics and calcium ATPase activity of rat skeletal sarcoplasmic reticulum was examined. After four-week supplementation with fish oil, the levels of eicosapentaenoic (20∶5ω3), docosapentaenoic (22∶5ω3) and docosahexaenoic (22∶6ω3) acids in the total lipids were 5.3, 5.5 and 28.1% of the total fatty acids, respectively. In contrast, with corn oil only 22∶6 was found (8.9%). The level of these fatty acids in phosphatidylethanolamine from the membranes of animals fed fish oil was 4.2 (20∶5), 5.4 (22∶5) and 49.1% (22∶6); and for phosphatidylcholine it was 5.4 (20∶5), 4.6 (22∶5) and 17.4% (22∶6). Again, in corn oil fed animals, only 22∶6 was found in appreciable amounts, namely 28.3% in phosphatidylethanolamine and 1.8% in phosphatidylcholine. The steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess lipid order and was found to be only slightly less for membranes from animals supplemented with fish oil (0.120) as compared to those supplemented with corn oil (0.124). The calcium ATPase was found to be unaffected by supplementation consistent with the observed modest changes in lipid order as well as with suggestions that the enzyme is relatively insensitive to the level of unsaturation. It could be argued that if large increases in fatty acyl polyunsaturation in mammalian cell membranes would lead to marked alterations in bulk membrane lipid motional properties, this may not be in the interest of preserving physiological function. The complex mixture of phospholipid molecular species present in natural membranes may buffer against this by a type of passive adaptation, without the expenditure of metabolic energy, thus providing a homeoviscous environment able to optimally support membrane protein function.     

Protein kinase C-dependent stimulation of phospholipase D in phospholipase C-treated fibroblasts

Treatment of [¹⁴C]choline- or [¹⁴C]ethanolamine-labeled NIH 3T3 fibroblasts withBacillus cereus phosphatidylcholine-specific phospholipase C (PLC) enhanced phospholipase D (PLD)-mediated hydrolysis of the respective¹⁴C-labeled phospholipids. PLD activity was stimulated by 1.5 U/mL of POLC and by 100 nM of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) to similar extents. Treatment of¹⁴C]palmitic acid-labeled fibroblasts with PLC in the presence of ethanol also enhanced PLD-mediated formation of phosphatidylethanol; the effects of PLC and PMA were nonadditive. PLC had no effect on PLD activity in fibroblasts in which PKC was down-regulated by prolonged (24 h) treatment with 300 nM PMA. These data indicate that treatment of fibroblasts with exogenous PLC results in PKC-dependent activation of PLD.     

Fatty acid composition of sarcoplasmic reticulum phospholipids from red and white muscles of the rabbit

The fatty acid composition of the phospholipids of sarcoplasmic reticulum preparations from rabbit psoas (white) and soleus (red) muscles was determined. The sarcoplasmic reticulum from psoas muscle was lower in stearic and oleic acids and higher in palmitic and linoleic acids than that from soleus muscle, and contained a greater proportion of polyunsaturated fatty acids. However most of the differences in fatty acids were small.     

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.     

磁性磷脂酶D交联酶聚集体的制备及其酶学性质

以磁性Fe3O4为载体,采用吸附-聚集-交联的方法固定来源于Streptomyces chromofuscus的磷脂酶D （scPLD）。该方法制备的磁性磷脂酶D交联酶聚集体（MCLEA）酶活回收率可达72.89%,酶活为（437±6.60）U/g。与游离酶相比,MCLEA在不同温度和pH下的稳定性都得到了一定程度的提升,但由于与底物的亲和力降低,需要更高浓度的磷脂酰胆碱（PC）和Ca2+作为反应底物与MCLEA结合。另外,还研究了MCLEA的有机溶剂耐受性,发现乙醇、四氢呋喃、叔丁醇、乙酸乙酯、乙醚和甲苯等有机溶剂对MCLEA的酶促反应具有促进作用,此发现为scPLD催化转磷脂酰作用生成磷脂酰丝氨酸（PS）过程中构建双相体系时有机溶剂的选择提供了参考。MCLEA在进行连续13次酶促反应后,酶活仍可保留在76%以上,其半衰期为331.67 min,说明所选择的固定化方案可以提高酶的稳定性,从而提高其在催化反应过程中的重复利用性。     

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.     

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.     

Isolation and characterization of phospholipase D from fababeans

An enzyme activity in crude extract of fababeans hydrolyzed phosphatidylcholine-U-¹⁴C to produce choline and phosphatidic acid. This enzyme, phospholipase D, was stable at 50 C in the presence of 5 mM DTT but was inactivated at 55 C. The enzyme was precipitated with cold acetone, concentrated between 30% saturation to 40% saturation with ammonium sulphate, absorbed on calcium phosphate gel and eluted with 0.2 M phosphate buffer. This procedure resulted in a 20-fold increase in specific activity. The activity of fababean phospholipase D was much higher when assayed at 38 C than that at room temperature. There was an obligatory requirement for calcium, and for maximal activity 40 mM calcium was required. A narrow pH optimum of about pH 5.7 was observed. The enzyme activity was extremely dependent on substrate dispersion. When 5 mM phosphatidylcholine (PC) was sonicated with increasing levels of sodium dodecyl sulphate (1 mM to 4 mM), the enzyme activity kept increasing. By using equimolar concentrations of PC and sodium dodecyl sulphate (1 mM to 5 mM), the Michaelis constant (Km) was estimated to be 1.74 mM. Addition of choline and serine at 10 mM concentration reduced phospholipase D activity by 31% and 22%, respectively.     

A structural model of the complex formed by phospholamban and the calcium pump of sarcoplasmic reticulum obtained by molecular mechanics

Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids that modulates the activity of the reticular Ca(2+) ion pump. We recently solved the three-dimensional structure of chemically synthesized, unphosphorylated, monomeric PLN (C41F) by high-resolution nuclear magnetic resonance spectroscopy in chloroform/methanol. The structure is composed of two alpha-helical regions connected by a beta turn (Type III). We used this structure and the crystallographic structure of the sarcoplasmic reticulum calcium pump (SERCA) recently determined by Toyoshima and co-workers and modeled into its E(2) form by Stokes (1KJU) or by Toyoshima (1FQU). We applied restrained and unrestrained energy optimizations and used the AMBER molecular mechanics force field to model the complex formed between PLN and the pump. The results indicate that transmembrane helix 6 (M6) of the SERCA pump is energetically favored, with respect to the other transmembrane helices, as the PLN binding partner within the membrane and is the only one of these helices that also permits contact between the N-terminal residues of PLN and the critical cytosolic binding loop region of the pump. This result is in agreement with published biochemical data and with the predictions of previous mutagenesis work on the membrane sector of the pump. The model reveals that PLN does not span the entire width of the membrane, that is, its hydrophobic C-terminal end is located near the center of the transmembrane region of the SERCA pump. The model also shows that interaction with M6 is stabilized by additional contacts made by PLN to M4. The contact between the N-terminal portion of PLN and the pump is stabilized by a number of salt and hydrogen-bond bridges, which may be abolished by phosphorylation of PLN. The contacts between the cytosolic portions of PLN and the pump are only observed in the E(2) conformation of the pump. Our model of the complex also offers a plausible structural explanation for the preference of protein kinase A for phosphorylation of Ser16 of PLN.     

Characterization of phospholipase D activity in bovine photoreceptor membranes

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2³H]glycerol-3-phosphocholine (PC), or [¹⁴C]ethanol. Using [³H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [³H]phosphatidic acid and phosphatidylethanol [³H]PtdEt; whereas with [¹⁴C]ethanol or [³H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [¹⁴C]PtdEt or [³H]phosphatidylglycerol, respectively). The use of varying concentrations of [³H]PC and 400 mM of ethanol gave an apparent K _m value for PC of 0.51 mM and a V _max value of 111 nmol × h⁻¹ × (mg protein)⁻¹. The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent K _m of 202 mM and a V _max value for ethanol of 125 nmol × h⁻¹ × (mg protein)⁻¹. A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca²⁺ or Mg²⁺ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Effects of dietary n−3 polyunsaturated fatty acids on phospholipid composition and calcium transport in mouse cardiac sarcoplasmic reticulum

The effects of dietary n−3 and n−6 polyunsaturated fatty acids on the fatty acid composition of phospholipid, Ca⁺⁺· Mg⁺⁺ ATPase and Ca⁺⁺ transport activities of mouse sarcoplasmic reticulum were investigated. Mice were fed a 2 weight percent fat diet containing either 0.5 weight percent ethyl esters of 18∶3n−3, 20∶5n−3 or 22∶6n−3 as a source of n−3 polyusaturated fatty acid or 0.5 weight percent safflower oil as a cource of n−6 polyunsaturated fatty acid for 10 days. Olive oil (2 weight percent) was used as a control diet. Although feeding n−6 polyunsaturated fatty acid induced very little modifications of the phospholipid sarcoplasmic reticulum fatty acid composition, feeding n−3 polyunsaturated fatty acid altered it markedly. Inclusion of 18∶−3, 20∶5n−3 or 22∶6n−3 in the diet caused an accumulation of 22∶6n−3, which replaced 20∶4n−6 and 18∶2n−6 in phospholipid sarcoplasmic reticulum. The saturated fatty acids were significantly increased with a concurrent reduction of 18∶1n−9. These changes in the fatty acid composition resulted in a decrease in the values of the n−6/n−3 polyunsaturated fatty acid ratio and a decrease in the ratio of 20 carbon to 22 carbon fatty acids esterified in the phospholipid sarcoplasmic reticulum. This was associated with a decrease in Ca⁺⁺ uptake by n−3 polyunsaturated fatty acid enriched sarcoplasmic reticulum vesicles as compared with n−6 fatty acid and control diet sarcoplasmic reticulum vesicles. However, neither the affinity for Ca⁺⁺ nor the maximal velocity of ATP hydrolysis activity of Ca⁺⁺·MG⁺⁺ ATPase were altered by the different diets. The data suggest that the incorporation of 22∶6n−3 and/or the decrease of 20∶4n−6 plus 18∶2n−6 in the phospholipid sarcoplasmic reticulum may affect the membrane lipid bilayer structure and make it more permeable to Ca⁺⁺.     

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.     

Synthesis of phosphatidylglycerol from soybean lecithin with immobilized phospholipase D

Phosphatidylglycerol (PG) was synthesized from soybean lecithin with the help of phospholipase D (PLD) that was immobilized with calcium alginate gel-enveloped polyethyleneimine-glutarald ehyde. The immobilization conditions were optimized with response surface analysis: Ca-alginate 1.39%, polyethyleneimine 7.78%, glutaraldehyde 1.22%. The conversion of phosphatidylglycerol was 87%. The properties of the immobilized PLD were: optimal temperature 25–30°C, optimal pH 8.2, ratio of ether phase and water phase 1.5–2.5:1 (vol/vol), repeated 15 batches operating with stabilizing conversion percentage of phosphatidylglycerol.     

Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol

In this study, the effect of temperature on the stereoselectivity of phospholipase D (PLD) toward the two primary hydroxyl groups of glycerol in the transphosphatidylation reaction of phosphatidylcholine to phosphatidylglycerol (PtdGro) was investigated. For this purpose, PLD from bacteria (Streptomyces septatus TH-2, S. halstedii subsp. scabies K6, and Actinomadura sp.) and cabbage were tested. At the reaction temperatures employed (0–60°C), the proportions of the two PtdGro diastereomers, namely, 1,2-dioleoyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-dioleoyl-sn-glycero-3-phosphol-1′-sn-glycerol (R,S configuration), which were produced with PLD from Streptomyces TH-2 and Actinomadura sp., changed gradually from 50% R,R and 50% R,S at 50–60°C to 70% R,R and 30% R,S at O°C. These alterations suggested that the stereoselectivity of the bacterial PLD toward the two primary hydroxyl groups of prochiral glycerol was significantly influenced by reaction temperature. PLD from Streptomyces K6 showed relatively little effect of temperature on stereoselectivity, giving 65–69% R,R in the temperature range of 60–10°C examined. The plots of In ([R,R]/[R,S]) vs. 1/T gave good linear fits for these three bacterial PLD. No temperature effect was observed for cabbage PLD, which gave an almost equimolar mixture of the R,R and R,S diastereomers in the range from 0 to 40°C. The temperature-dependent change in enantiomeric selectivity of the bacterial PLD promises potentially profitable commercial exploitation.     

磷脂酶D的紫外诱变选育及发酵条件的优化

磷脂酶D在催化磷脂酰基交换反应中具有重要的应用价值。本文对产磷脂酶D野生链霉菌株进行紫外诱变,筛选得到一株产酶活力提高42.5%的变异株。通过摇瓶培养,对发酵条件进行探究。确定的最佳培养基组成为：葡萄糖10.0 g/L,牛肉膏和蛋白胨各5.0 g/L,MgSO4?7H2O 1.0 g/L,CaCl2 3.0 g/L,NaCl 2.0 g/L;表面活性剂Tween80对产酶有促进作用,其适宜浓度为0.60 g/L。在上述条件下,摇瓶发酵产酶活力达3.23 U/mL。     

The triggering signal dictates the effect of docosahexaenoic acid on lymphocyte function in vitro

Docosahexaenoic acid (DHA) is an n-3 fatty acid beneficial to several human conditions including inflammation and autoimmune disease. To better understand the effect of DHA on immunity, we monitored the rise in cytosolic free calcium, interleukin 2 receptor (IL2R) expression, and proliferation of splenic lymphocytes triggered with three different stimuli in the presence or absence of DHA. We found that 10 μg DHA/mL suppressed concanavalin A-induced mitogenesis and the mixed lymphocyte reaction while concurrently enhancing proliferation stimulated with anti-Thy-1 antibodies. Proliferation, as measured by [³H]thymidine incorporation after 2 to 5 d of culture, was affected by DHA, but earlier activation effects such as elevation of cytosolic free calcium and IL2R expression were not altered. These results imply that DHA incorporated into membrane phospholipids differentially affects the activity of distinct membrane-bound receptors and signaling molecules. This result suggests that DHA may be used to modulate immune responses selectively, e.g., to suppress, undesired autoimmunity while maintaining protective immunity.     

The effect on selectivity of the macroscopic distribution of the components in a dual function catalyst

This paper is concerned with the effect on selectivity of the macroscopic distribution of the components X and Y of a dual function catalyst. Three catalyst formulations are considered: (1) catalyst I where catalyst X 1: distributed at the pore mouth, (2) catalyst II where catalyst Y is distributed at the pore mouth, and (3) nonporous catalyst III where catalysts X and Y are mixed together. The selectivities of each catalyst are compared for the consecutive reaction scheme A_X→^k₁B_Y→^k₂C_X→^k₁D and factors influencing the optimum catalyst composition are discussed. The results show that under certain circumstances the macroscopic distribution of catalyst functions greatly affects the yields of intermediate products and suggest that the macroscopic distribution of functions can be an important variable in catalyst design.     

Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.     

Production and Scale-up of phosphatidyl-tyrosol catalyzed by a food grade phospholipase D

Highly purified phosphatidyl-tyrosol was obtained by using a food grade phospholipase D from Actinamadure sp. transphosphatidylation in a GRAS (Generally Recognized As Safe) biphasic medium. The reaction medium, comprised of an aqueous phase and ethyl butyrate, has been considered as an alternative to other biphasic systems previously reported utilizing more harmful organic solvents. The purpose of the present study was to purify phosphatidyl-tyrosol from a transphosphatidylation reaction mixture by using a procedure readily scalable to obtain a new valuable food ingredient. Initially, phosphatidyl-tyrosol was purified via semi-preparative HPLC equipment to be used as analytical standard. The best results of the transphosphatidylation reaction were obtained for two different PC concentrations, namely 83 and 166 mmol/L, with PC conversion of ca. 97 and 94% (w/w) and a final phosphatidyl-tyrosol concentration of 81 and 157 mmol/L, respectively. Finally, the procedure was scaled-up and 40 g of highly purified phosphatidyl-tyrosol (97% (w/w)) were readily purified by centrifugation without involving the utilization of organic solvents.