The adsorptive immobilization of phospholipids D mediated by calcium ions

Immobilization of phospholipase D from cabbage was studied with the aim of stabilizing the enzyme for its use in synthesis of phospholipids. It was shown that phospholipase D can be immobilized by adsorption to polymeric carriers containing long chain anchor groups such as octadecyl, octyl, or other alkyl residues. Starting from the crude enzyme, phospholipase D activity is preferentially bound (up to 100%) in competition with contaminating proteins. A prerequisite of high binding rates is the presence of calcium ions, which play a mediating role in the adsorption process. The maximum activity of the carrier-enzyme complexes depends upon the calcium concentration in the immobilization process and the carrier material (>/=10mM CaCl(2) with octadecyl-Si40, >/=40 mM CaCl(2) with octyl-sepharose and butyl-fractogel). Immobilization of phospholipase D to octyl-sepharose was shown to result in a distinctly increased storage stability and an enlarged pH-optimum range for the catalytic activity. Operational stability of different phospholipase D-carrier complexes was compared. (c) 1993 Wiley & Sons, Inc.     

Determination of phosphatidylcholine in a flow injection system using immobilized enzyme reactors

Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow-injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow-injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first method.     

Enzymatic hydrolysis by bacterial phospolipases C and D of immobilized radioactive sphingomyelin and phosphatidylcholine

An assay system for phospholipases C has been described with sphingomyelin immobilized to octyl-Sepharose CL-4B as substrate. The immobilization procedure was further developed and used with [14 C-choline]-sphingomyelin and [14C-choline] phosphatidylcholine (lecithin). These immobilized radioactive phospholipids made the enzymatic assays easier to perform and made it possible to increase the sensitivity. Furthermore, since release of the choline part instead of the phosphate part of the substrate molecule was measured, it was possible to use this assay for phospholipase D as well. The enzyme characteristics of phospholipase D from Corynebacterium ovis were compared in this test system with those of three phospholipases C (from Clostridium perfringens, Bacillus cereus and Staphylococcus aureus) with respect to hydrolysing capacities and optimal ion concentrations.     

Phospholipase D production using immobilized cells of Streptoverticillium cinnamoneum

Summary Production of phospholipase D (PLD) by Streptoverticillium cinnamoneum immobilized within porous particles was investigated in repeated batch fermentation. The enzyme productivity in repeated batch fermentation was 2.2-fold that obtained in batch fermentation without immobilization, since many of the immobilized cells could be utilized as seed cells for each subsequent batch cycle.     

D301树脂固定化假丝酵母脂肪酶

本研究选择7种吸附和离子交换树脂进行了假丝酵母脂肪酶(Candida sp.lipase)的固定化试验,通过测定固定化后各脂肪酶的酶活,筛选出固定化效果较好的弱碱性阴离子交换树脂D301;并通过扫描电镜将D301与脂肪酶Novozym 435的表面形貌做比较,进一步选定D301树脂作为载体,并对其采用戊二醛交联固定化,研究并优化了其固定化条件。结果表明,5%戊二醛溶液的加入量为8mL,处理时间为5h,酶液浓度为1.0g/L,磷酸缓冲盐溶液pH6.0,固定化处理10h效果最好,获得的固定化酶活力可达35U/mg,酶的固定化效率约为3.5U/(mg·h)。     

Repeated batch and continuous operations for phosphatidylglycerol synthesis from phosphatidylcholine with immobilized phospholipase D

Summary The repeated batch and continuous operations for transphosphatidylation reaction were carried out for phosphatidylglycerol (PG) synthesis from phosphatidylcholine (PC) with the help of immobilized cabbage phospholipase D (PLD) in the presence of glycerol. The biphasic reaction system was used which included the aqueous phase containing immobilized PLD along with high concentrations of glycerol (30%–50%) and buffer, whereas the main part of substrate (PC) and products (mainly PG) formed were in the organic phase (diethyl ether).Octyl-Sepharose CL-4B having a hydrophobic octyl group was chosen for the PLD immobilization. Both immobilization yield and activity yield of immobilized enzyme were 100%. The effects of solvents, temperature and glycerol concentrations on the immobilized PLD were examined. Repeated batch conversion of PC (15 g/l) to PG was examined with the immobilized PLD in 30% glycerol. In all five batch cycles examined, 100% selectivity was obtained and there was no significant decrease in the fractional conversion of PC to PG (98%–99%) in the first three batch cycles. In the cases of a packed-bed reactor (PBR) and a continuous stirred-tank reactor (CSTR) used for continuous synthesis of PG with the immobilized PLD, the operational stabilities of the immobilized enzyme were almost the same (half life=14 h at 30°C) when purified PC was used, while in the case of partially purified PC in CSTR the half life increased more than five times.Abbreviations used PC phosphatidylcholine - PG phosphatidylglycerol - PA phosphatidic acid - PLD phospholipase D - PBR packed bed reactor - CSTR continuous stirred tank reactor Studies on enzymatic conversion of phospholipids (III)     

吸附-交联法固定D-泛解酸内酯水解酶的研究

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化。通过实验对固定化条件进行了优化,得出最佳的固定化条件为：加酶量6U／g树脂、吸附pH7．5、吸附时间4h、吸附温度30℃、交联剂戊二醛终浓度0．1％、交联时间2h。实验表明在此条件下制得的固定化酶有很好的稳定性：固定化酶在连续20次的底物水解反应后,剩余酶活达到71％。当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60％以上。游离酶的pH稳定性范围为pH7～8,而固定化酶为pH6．5～8．5。     

Several properties of cotton seed phospholipase D]

Properties of phospholipase D were studied using purified enzyme preparation from cotton seeds. The results obtained differ from those described in literature. It has been shown that the promoting action is exerted not only by diethyl ether and sodium dodecyl sulfate commonly used as initiators, but by some organic solvents in the presence of calcium ions as well. The activation of phospholipase D is also possible in the presence of other bivalent cations, e.g. Sr2+, Ba2+, Mn2+ and Mg2+. It is assumed that the enzyme activation occurs only in the presence of the stable heterogenous system: water-soluble enzyme--phospholipid--non-aqueous phase. Another important factor is the type of modification of the surface of the phospholipid phase, responsible for the enzyme adsorption and its subsequent activation. Comparison is made of the properties of phospholipases D isolated from cotton seeds and some other sources.     

Bioconversion of phospholipids by immobilized phospholipase A2

Phospholipase A₂ selectively hydrolyses the ester linkage at the sn-2 position of phospholipids forming lysocompounds. This bioconversion has importance in biotechnology since lysophospholipids are strong bioemulsifiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A₂ from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex^® support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A₂ was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A₂ was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V_max 883.4 μmol mg⁻¹ min⁻¹ and a K_m 12.9 mM for soluble form and V_max = 306 μmol mg⁻¹ min⁻¹ and a K_m = 3.9 for immobilized phospholipase A₂) were in agreement with the immobilization effect. The results obtained with CM-Sephadex^®-phospholipase A₂ system give a good framework for the development of a continuous phospholipid bioconversion process.     

Influence of immobilization stress on the phospholipid composition of alveolar surfactant and lungs in rats

The influence of immobilization stress on the lipid composition of alveolar surfactant and lungs in rats immobilized for 12 and 24 hours, the effects of phospholipase A2, and lipid transfer activity in alveolar surfactant were investigated. The results indicate that alveolar surfactant phospholipids underwent more significant alterations compared to lung phospholipids. Furthermore, phospholipase A2 and lipid transfer activity were reduced in alveolar surfactant of immobilized rats. The reported data suggest that the lower lipid transfer activity might be responsible for the reduced phospholipids in the surfactant system.     

5-Aminosalicylate stimulates phospholipase D activity in macrophages

5-Aminosalicylate, which is considered to be the active moiety of sulfasalazine, is one of the most widely used agents for treatment of inflammatory bowel disease. However, its mechanism of action is unclear. In this report, we provide evidence that the phospholipase D pathway is a target for this drug in macrophages. Activation of phospholipase D leads to the generation of important second messengers such as phosphatidic acid, lysophosphatidic acid and diacylglycerol, all of which can regulate cellular responses involved in inflammation. Murine peritoneal macrophages were labeled with [(3)H]myristate, incubated with various drugs, agonists, or inhibitors, and phospholipase D activity was assayed. 5-Aminosalicylate or sulfasalazine stimulated phospholipase D in a time- and concentration-dependent manner. Chelation of extracellular Ca(2+) inhibited phospholipase D activation by either of these drugs whereas pretreatment of macrophages with the tyrosine kinase inhibitor genistein had no effect. Downregulation of protein kinase C by prolonged incubation with phorbol ester completely blocked the activation of phospholipase D. Pertussis toxin decreased the activation of phospholipase D. The levels of inositol 1,4,5-trisphosphate increased by 260% after treatment of macrophages with 5-aminosalicylate. A phosphoinositide-specific phospholipase C inhibitor U73122 blocked phospholipase D activation completely. Interestingly, long-term preincubation of the macrophages with a relatively low concentration of 5-aminosalicylate that did not stimulate phospholipase D activity by itself, potentiated the effect of phorbol ester-induced activation of phospholipase D. Taken together, these results show that 5-aminosalicylate activates phospholipase D via a pathway involving inositol 1,4,5-trisphosphate generation, calcium fluxes, and Gi/Go. Although the mechanisms by which phospholipase D activation by 5-aminosalicylate or sulfasalazine might attenuate inflammatory responses in the intestine remain to be defined, these results highlight a novel potential mechanism of action for these drugs.     

Recovery of uranium by immobilized microorganisms

Summary Some attempts were made to recover uranium from sea and fresh water using immobilized Streptomyces viridochromogenes and Chlorella regularis cells. The cells immobilized in polyacrylamide gel have the most favorable features for uranium recovery; high adsorption ability, good mechanical properties, and applicability in a column system. The adsorption of uranium by the immobilized cells is not affected by the pH values between 4 and 9. These results show that uranium adsorption becomes independent of pH after immobilization. The amounts of uranium adsorbed by the immobilized cells increased linearly with temperature, suggesting that the adsorption of uranium by the immobilized cells is an endothermic reaction. The immobilized cells can recover uranium almost quantitatively from both fresh and sea water containing uranium, and almost all uranium adsorbed is desorbed with a solution of Na₂CO₃. Thus the immobilized cells of Streptomyces and Chlorella can be used repeatedly in adsorption-desorption process.Studies on the Accumulation of Heavy Metal Elements in Biological Systems. XXI     

Immobilization of <Emphasis Type="Italic">Streptomyces</Emphasis> phospholipase D on a Dowex macroporous resin

The immobilization of phospholipase D produced by Streptomyces sp. YU100 was evaluated to see it would be practical for industrial applications. To accomplish this, the purified enzyme, which contained 53 unit/mg of protein, was subjected to immobilization on various matrices. When immobilization supports including calcium alginate gel, polyacrylamide gel, and macroporous resin were evaluated, the highest enzyme retention ratio (> 42%) was observed on a Dowex MSA-2 macro-porous resin. This may have occurred as a result of the ability of the hydrophobic domain of phospholipase D to interact with the polystyrene backbone of the resin, as well as the ability of the dimethylethanolamine group of the MSA-2 resin to retain the enzyme by forming hydrogen bonds with the acidic residues of the enzyme. Upon the operation of a reactor packed with enzyme that had been immobilized on a Dowex MSA-2 resin, greater than 80% of the initial enzyme activity was retained for 16 days. During the reaction, phosphatidylcholine became bound to the immobilized resin and interfered with the enzyme reaction, therefore, the resin was washed with ethyl ether every 2 h. A process for recovering excessive l-serine from phospholipids using the Dowex MR-3 resin was designed, and the separated l -serine was employed again after replacing the amount that was used.     

Phospholipase D and membrane traffic: Potential roles in regulated exocytosis,membrane delivery and vesicle budding

It is now well-established that phospholipase D is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors in mammalian cells. Over the last 5 years, a tremendous effort has gone to identify the major intracellular regulators of mammalian phospholipase D and to the cloning of two mammalian phospholipase D enzymes (phospholipase D1 and D2). In this chapter, we review the physiological function of mammalian phospholipase D1 that is synergistically stimulated by ADP ribosylation factor, Rho and protein kinase Cα. We discuss the function of this enzyme in membrane traffic, emphasising the possible integrated relationships between consumption of vesicles in regulated exocytosis, membrane delivery and constitutive membrane traffic.     

Phospholipase D and membrane traffic. Potential roles in regulated exocytosis, membrane delivery and vesicle budding.

It is now well-established that phospholipase D is transiently stimulated upon activation by G-protein-coupled and receptor tyrosine kinase cell surface receptors in mammalian cells. Over the last 5 years, a tremendous effort has gone to identify the major intracellular regulators of mammalian phospholipase D and to the cloning of two mammalian phospholipase D enzymes (phospholipase D1 and D2). In this chapter, we review the physiological function of mammalian phospholipase D1 that is synergistically stimulated by ADP ribosylation factor, Rho and protein kinase Calpha. We discuss the function of this enzyme in membrane traffic, emphasising the possible integrated relationships between consumption of vesicles in regulated exocytosis, membrane delivery and constitutive membrane traffic.     

Interleukin-1 beta, tumor necrosis factor and forskolin stimulate the synthesis and secretion of group II phospholipase A2 in rat mesangial cells

Treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin resulted in the release of phospholipase A2 activity in the culture medium. Essentially all of this phospholipase A2 activity was bound to immobilized monoclonal antibodies raised against rat liver mitochondrial 14 kDa group II phospholipase A2. Gelfiltration confirmed the absence of higher molecular weight phospholipases A2 in the culture medium. Immunoblot experiments showed the virtual absence of this 14 kDa group II phospholipase A2 in unstimulated mesangial cells. The time-dependent increase of phospholipase A2 activity in both cells and culture medium upon stimulation with interleukin-1 beta plus forskolin is accompanied with elevated 14 kDa phospholipase A2 protein levels. These results indicate that the increased phospholipase A2 activity upon treatment of mesangial cells with these stimulators is due to increased synthesis of group II phospholipase A2. Over 85% of this newly synthesized phospholipase A2 appears to be secreted from the cells.     

Intermolecular association between caspase-mediated cleavage fragments of phospholipase D1 protects against apoptosis

Phospholipase D plays an anti-apoptotic role but little is known about dynamics of phospholipase D turnover during apoptosis. We have recently identified phospholipase D1 as a new substrate of caspases which generates the N-terminal and C-terminal fragment of phospholipase D1. In the present study, we tried to investigate whether association of the caspase cleavage fragments may be involved in regulation of apoptosis. Ectopically expressed C-terminal fragment, but not N-terminal fragment of phospholipase D1, is exclusively imported into the nucleus via a nuclear localization sequence; however, endogenous C-terminal fragment of phospholipase D1 from etoposide-induced apoptotic cells and Alzheimer's disease brain tissues with active caspase-3, was localized in the cytosolic fraction as well as the nuclear fraction. Intermolecular association between the two fragments of phospholipase D1 through hydrophobic residues within the catalytic motif inhibited nuclear localization of C-terminal fragment of phospholipase D1, and two catalytic motif and nuclear localization sequence regulated nuclocytoplasmic shuttling of phospholipase D1. Moreover, hydrophobic residues involved in the intermolecular association are also required for both its enzymatic activity and anti-apoptotic function. Taken together, we demonstrate that interdomain association and dissociation of phospholipase D1 might provide new insights into modulation of apoptosis.     

Self-quenched fluorogenic protein substrates for the detection of cathepsin D and other protease activities

Self-quenched fluorogenic substrates for proteolytic enzymes have been prepared by alkylation of thiol groups in reduced bovine serum albumin with iodoacetamidofluorescein or iodoacetamidoeosin. Substrates immobilized by adsorption onto nitrocellulose membranes or by incorporation into agarose gel slabs are suitable for fluorescence zymography after electrophoretic separation of catalytically active proteases, including cathepsin D.     

Cell adsorption and local accumulation of extracellular polysaccharide in an immobilized Acinetobacter calcoaceticus system

Acinetobacter calcoaceticus can be immobilized on Celite by adsorption. The salt concentrations suitable for immobilized cell fermentation are between 10 and 50 mM phosphate concentration. Low salt concentrations cause desorption of immobilized cells while high salt concentrations inhibit the adsorption of cells on Celite. It is also found that cell adsorption is better at lower pH than at higher pH. An airlift fermentation using immobilized cells at 300 g/L Celite loading shows that about 70% of the total polymer produced is accumulated in Celite pores at a concentration (15.4 g/L) almost threefold higher than that in the bulk liquid (5.7 g/L).     

Fas-mediated activation of phospholipase D is coupled to the stimulation of phosphatidylcholine-specific phospholipase C in A20 cells.

The activation of phospholipase D in murine B cell lymphoma A20 cells treated with anti-Fas monoclonal antibody has been investigated. Fas cross-linking resulted in a both dose- and time-dependent increases in phospholipase D activity. There was a nearly maximum saturated rise in phospholipase D activity at the dose of 200 ng/ml anti-Fas monoclonal antibody showing a fourfold increase within 3 h. Fas activation also caused an approximately twofold increase of phosphatidylcholine-specific phospholipase C activity and 1,2-diacylglycerol release, which could be blocked by 30 min pretreatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 (50 microgram/ml). Pretreatment of D609 also effectively inhibited the translocation of protein kinase C betaI and betaII from the cytosol to the membrane and the activation of phospholipase D induced by Fas cross-linking, suggesting that 1, 2-diacylglycerol released from the cellular phosphatidylcholine pool through phosphatidylcholine-specific phospholipase C plays a major role in protein kinase C/phospholipase D activation. Anti-Fas monoclonal antibody failed to elicit phosphoinositide-specific phospholipase C activation and any changes in the intracellular Ca2+ level in A20 cells, indicating that the phosphoinositide-mediated pathway is not involved in this Fas signaling. Therefore, these results suggest that Fas-mediated phospholipase D activation may be a consequence of primary stimulation of phosphatidylcholine-specific phospholipase C and that phospholipase D may play a role in Fas cross-linking signaling downstream from phosphatidylcholine-specific phospholipase C.