Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes.

Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.     

Hydrolysis of palm and olive oils by immobilised lipase using hollow fibre reactor

Lipase from Mucor miehei immobilised by adsorption on microporous, asymmetric hollow fibre membrane reactors was used to hydrolyse two different oils, namely, palm and olive oils. The hydrolysis reaction was carried out at a temperature of 40 °C, an average transmembrane pressure (TMP) of 115 mmHg and oil and aqueous flow rates of 2.5 and 3.0 ml min⁻¹, respectively. It was experimentally proven that adsorption of lipase increased with temperature and was higher on hydrophobic membranes than hydrophilic ones. The effluent concentrations of fatty acid products were measured using gas chromatograph with FID detector. Hydrolysis experimental results were fitted to a multisubstrate kinetic model derived from the Ping Pong Bi Bi mechanism. The final model expression is useful for predicting the free fatty acid profile of the enzymatic hydrolysis of palm and olive oils for different substrate flow rates and enzyme loading.     

Hydrolysis of olive oil catalyzed by surfactant-coated<Emphasis Type="Italic">Candida rugosa</Emphasis> lipase in a hollow fiber membrane reactor

In this study, we invetigated the hydrolysis of olive oil catalyzed by a surfactant-coatedCandida rugosa lipase in a hydrophilic polyacrylonitrile hollow fiber membrane reactor and then compared the results to those using the native lipase. The organic phase was passed through the hollow inner fibers of the reactor and consisted of either the coated lipase and olive oil dissolved in isooctane or the coated lipase dissolved in pure olive oil. The aqueous phase was pumped through the outer space. After 12 h and with conditions of 30°C, 0.12 mg enzyme/mL and 0.62 M olive oil, the substrate conversion of the coated lipase reached 60%. This was twice the conversion for the same amount of native lipase that was pre-immobilized on the membrane surface. When using pure olive oil, after 12 h the substrate conversion of the coated lipase was 50%. which was 1.4 times higher than that of the native lipase.     

Lipase-catalyzed hydrolysis of olive oil in chemically-modified AOT/isooctane reverse micelles in a hollow fiber membrane reactor

The hydrolysis of olive oil catalyzed by Candida rugosa lipase in sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane and the synthetic sodium bis(2-ethylhexyl polyoxyethylene)sulfosuccinate (MAOT)/isooctane reverse micellar systems was investigated in a polysulfone hollow fiber membrane reactor with recycle of the reaction mixture. Lipase was completely retained by the membrane while olive oil and oleic acid freely passed through. The retention of reverse micelles depended on W ₀ (molar ratio of water to surfactant). At an olive oil concentration of 0.23 mol l^–1 the final substrate conversion in the MAOT micellar system was about 1.4 times of that in the AOT micellar system.     

Effects of phospholipase A2 treatment of human erythrocyte membranes on the rates of spectrin-actin dissociation

This work examines the extent to which alterations in the composition of the phospholipid bilayer of the erythrocyte membrane influences the stability of the association of the ‘cytoskeletal network’ to the rest of the membrane. Rates of spectrin-actin dissociation at low ionic strength were used as a measure of the stability, and composition of the phospholipid bilayer was altered by the action of the enzyme phospholipase A₂. Hydrolysis of all the phosphatidylcholine of the outer leaflet of the bilayer had no effect on dissociation rates, whether or not the hydrolysis products were extracted with albumin. Hydrolysis of inner leaflet phospholipids increased the rates by up to 2-fold if the hydrolysis products were not extracted; for ?50% hydrolysis, the rates were unaffected if the hydrolysis products were extracted. The moderate magnitudes of the increases in dissociation rates indicate that interactions between the ‘cytoskeletal network’ and the phospholipid bilayer are not a decisive factor in maintaining the stability of the membrane, at least under low ionic strength conditions.     

Sec-dependent preprotein translocation in bacteria

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multi-subunit protein complex termed translocase, which consists of the integral membrane heterotrimer SecYEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with SecYEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles. Received: 4 August 1995 / Accepted: 9 October 1995     

Enzymatic hydrolysis of olive wastewater for hydroxytyrosol enrichment

Aspergillus niger broth culture on wheat bran was assessed for olive wastewater (OW) hydrolysis in order to release hydroxytyrosol (HT). The enzyme profiles of this culture broth gave essentially (IU/L): 3000 β-glucosidase and 100 esterase. Hydrolysis activity of A. niger enzyme preparation was evaluated by using three substrates: raw OW, phenolic fraction extracted from OW by ethyl acetate and its corresponding exhausted fraction. Large amounts of free simple phenolics were released from exhausted fraction and raw OW after enzymatic treatment. HPLC analyses show that HT was the main phenolic compound. One step of ethyl acetate extraction of hydrolysed OW allowed the recovery of 0.8 g of HT per litre of OW. The antioxidant activity of extracts from OW and exhausted fraction, measured by DPPH method, was drastically enhanced after hydrolysis treatment. This study demonstrates that hydrolysed OW is a potential source of bioactive phenolic compounds with promising applications in food and pharmaceutical industries.     

The role of membrane proteins and phospholipids in the interaction of ribosomes with endoplasmic reticulum membranes.

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.     

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.     

Entrapment of lipase into K-carrageenan beads and its use in hydrolysis of olive oil in biphasic system

The porcine pancrease lipase was immobilized by entrapment in the beads of K-carrageenan and cured by treatment with polyethyleneimine (PEI) in the phosphate buffer. The retention of hydrolytic activity of lipase and compressive strength of the beads were examined. The activity of free and immobilized lipase was assessed by using olive oil as the substrate. The immobilized enzyme exhibited a little shift towards acidic pH for its optimal activity and retained 50% of its activity after 5 cycles. When the enzyme concentration was kept constant and substrate concentration was varied the K_m and V_max were observed to be 0.18 × 10⁻² and 0.10, and 0.10 × 10⁻² and 0.09 respectively, for free and for entrapped enzymes. When the substrate concentration was kept constant and enzyme concentration was varied, the values of K_m and V_max were observed to be 0.19 × 10⁻⁷ and 0.41, and 0.18 × 10⁻⁷ and 0.41 for free and entrapped enzymes. Though this indicates that there is no conformational change during immobilization, it also shows that the reaction velocity depends on the concentration. Immobilized enzyme showed improved thermal and storage stability. Hydrolysis of olive oil in organic–aqueous two-phase system using fixed bed reactor was carried out and conditions were optimized. The enzyme in reactor retained 30% of its initial activity after 480 min (12 cycles).     

Effect of various additives on enzymatic hydrolysis of castor oil

Hydrolysis of castor oil using lipase enzyme is carried out in a batch reactor at room temperature (35–40 °C). In order to reduce the cost of enzyme catalyzed reaction, water in oil emulsion and a 3:1 ratio of oil to water is selected. The concentration of enzyme in the reaction mixture is optimized. The effect of various additives like solvent and salt which can enhance the rate of reaction is studied. It is found that the glycerol has no effect on the hydrolysis of oil. The reusability of the lipase enzyme has also been tested. The yield of enzymatic hydrolysis of castor oil is compared with those of coconut oil and olive oil.     

Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins

The effect of different dietary fats on peritoneal macrophage plasma membrane fluidity, intracellular cyclic AMP (cAMP) production, GTP hydrolysis and TNF binding and TNF-induced IL1 and IL6 production was investigated. After a four week period, fluidity, as determined by both fluorescence recovery after photobleaching (FRAP) and anisotropy was lowest and highest in animals fed corn and fish oil respectively. After eight weeks feeding, lateral membrane movements were decreased substantially in fish, olive and coconut oil fed dietary groups, whereas an increase in the corn oil fed group was observed, no effect was observed in macrophages from the butter fed group. However, an increase in the packing was observed in macrophages from all dietary groups except in the olive oil fed group. GTPase values for the coconut oil and butter groups were higher than in any other dietary group. After receiving the diet for 8 weeks these differences between the groups were no longer apparent. Exposure of macrophages to TNF had no effect on the rate of GTP hydrolysis. A major enhancement of cAMP production became apparent between weeks 4 and 8 of dietary treatment. After 4 weeks on the diet, values were significantly higher from cells of animals fed corn and olive oils than from animals fed fish oil. After 8 weeks, while there was a general enhancement of production, further differences became apparent. Feeding corn and coconut oils resulted in the highest values and olive oil and chow in the lowest. It is proposed that fats rich in n-3 fatty acids (fish oils) alter membrane fluidity, decrease TNF binding affinity, GTPase activity and cAMP production which appears not to modify cytokine production after short term dietary supplementation. However, after long term feeding it appears that increases in the sensitivity of the TNF receptors plays a major role in modifying cytokine production. (Mol Cell Biochem 166: 135-143, 1997)     

The modulation of Ca-ATPase activity and protein-lipid interactions in the sarcoplasmic reticulum by ATP

The time-course of ATP hydrolysis by Ca-ATPase of purified sarcoplasmic reticulum is biphasic with an initial rate over 1 to 2 min exceeding the subsequent rate. Hydrolysis of GTP and p-nitrophenylphosphate (pNPP) occurs at a slower but constant rate. Arrhenius plots of GTP, p-nitrophenylphosphate and initial rates of ATP hydrolysis all exhibit a discontinuity at about 20-24 degrees C; no breaks are observed in plots of the slower phase of ATP hydrolysis. The effect of substrate hydrolysis on the disposition of the enzyme in the membrane was examined by monitoring the quenching of tryptophan fluorescence by pyrene present in the hydrophobic domain of the membrane. The presence of ATP, but not GTP, prevents a temperature-dependent decrease in fluorescence quenching suggesting that ATP binding causes a change in the protein domain in contact with the membrane lipids.     

Differential effects of oleuropein, a biophenol from Olea europaea, on anionic and zwiterionic phospholipid model membranes

Oleuropein (Ole) is the major phenolic constituent of the olive leaf (Olea europaea) and it is also present in olive oil and fruit. In the last years several compounds from olive tree, oleuropein among them, have shown a variety of biological activities such as antimicrobial or antioxidant. A phospholipid model membrane system was used to study whether the Ole biological effects could be membrane related. Ole showed a significant partition level in phospholipid membranes, i.e. 80%, at lipid-saturating conditions. Moreover, fluorescence quenching experiments indicated a shallow location for Ole in membranes. Ole promoted weak effects on zwiterionic phospholipids such as phosphatidylcholine or phosphatidylethanolamine. In contrast, differential scanning microcalorimetry, light scattering and fluorescence anisotropy pH titration studies revealed strong effects on anionic phospholipids such as phosphatidylglycerol at physiological pH and salt conditions. These effects consisted on perturbations at the phospholipid membrane surface, which might involve specific molecular interactions between Ole and the negatively charged phosphate group and therefore modify the phospholipid/water interface properties. It is proposed that Ole induces lipid structures similar to the gel-fluid intermediate phase (IP) described for PG membranes, in a similar way than low ionic strength does. These effects on phosphatidylglycerol may account for the antimicrobial activity of Ole.     

Insecticidal activity of strains of Bacillus thuringiensis on larvae and adults of Bactrocera oleae Gmelin (Dipt. Tephritidae).

The olive fly, Bactrocera oleae, is the key pest on olives in the Mediterranean area. The pest can destroy, in some cases, up to 70% of the olive production. Its control relies mainly on chemical treatments, sometimes applied by aircraft over vast areas, with their subsequent ecological and toxicological side effects. Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects, including the orders of Lepidoptera, Diptera, and Coleoptera and other invertebrates. The aim of this study was to search for isolates toxic to B. oleae. Several hundred B. thuringiensis isolates were obtained from olive groves and olive presses in different areas of Greece, Sardinia (Italy), and Spain and from cooperating scientists throughout the world. Some isolates were found toxic only to adults or larvae and some to both stages of the olive fly. In addition, the most toxic isolates were assayed on Opius concolor Szepl. (Hym. Braconidae), the most important parasitoid of the olive fruit fly. Only 3 isolates out of 14 gave significant mortality against this parasitoid. Several of the most toxic crystalliferous isolates may contain novel toxins since they gave no PCR products when probed with primers specified for 39 known toxin genes.     

Evolution of a rippled membrane during phospholipase A2 hydrolysis studied by time-resolved AFM

The sensitivity of phospholipase A(2) (PLA(2)) for lipid membrane curvature is explored by monitoring, through time-resolved atomic force microscopy, the hydrolysis of supported double bilayers in the ripple phase. The ripple phase presents a corrugated morphology. PLA(2) is shown to have higher activity toward the ripple phase compared to the gel phase in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes, indicating its preference for this highly curved membrane morphology. Hydrolysis of the stable and metastable ripple structures is monitored for equimolar DMPC/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-supported double bilayers. As shown by high-performance liquid chromatography results, DSPC is resistant to hydrolysis at this temperature, resulting in a more gradual hydrolysis of the surface that leads to a change in membrane morphology without loss of membrane integrity. This is reflected in an increase in ripple spacing, followed by a sudden flattening of the lipid membrane during hydrolysis. Hydrolysis of the ripple phase results in anisotropic holes running parallel to the ripples, suggesting that the ripple phase has strip regions of higher sensitivity to enzymatic attack. Bulk high-performance liquid chromatography measurements indicate that PLA(2) preferentially hydrolyzes DMPC in the DMPC/DSPC ripples. We suggest that this leads to the formation of a flat gel-phase lipid membrane due to enrichment in DSPC. The results point to the ability of PLA(2) for inducing a compositional phase transition in multicomponent membranes through preferential hydrolysis while preserving membrane integrity.     

Continuous glycerolysis of olive oil by Chromobacterium viscosum lipase immobilized in liposome in reversed micelles

Chromobacterium viscosum lipase which has adsorbed on liposome and solubilized in microemulsion droplets of glycerol containing a little amount of water could catalyze the glycerolysis of olive oil. Studies on the continuous glycerolysis of olive oil by the immobilized enzyme was done at 37 degrees C in continuous stirred vessel bioreactor with polysulfone membrane. The effect of the flow rate of substrate (olive oil) in isooctane on the conversion and composition of the outlet was investigated using high-performance liquid chromatography (HPLC). The conversion increased with decrease in the flow rate. And we studied the effect of water content in the glycerol-water-lipase solution on the glycerolysis reaction. The conversion to desirable products, mono- and di-olein, was improved without a substantial production of oleic acid at lower water concentrations, i.e., below 8.0% (w/v) which corresponds to a w(o) value of 0.97. At water concentration higher than 8.0% (w/v), the amount of free fatty acid was dramatically increased. Higher operational stability of the enzyme reactor, and the half-line of the enzyme continuous reaction was about 7 weeks.     

Isolation of a synaptic membrane fraction enriched in cholinergic receptors by controlled phospholipase A2 hydrolysis of synaptic membranes.

A procedure is described for the isolation of synaptic membrane fragments that retain such functionally important proteins as acetylcholine receptors, acetylcholinesterase, 3',5'-cyclic nucleotide phosphodiesterase, and (Na+ + K+)-ATPase. The method is based on the observation, made in brain slices, that junctional membranes are more resistant to phospholipase A2 attack than mitochondrial or plasma membranes. Hydrolysis by phospholipase A2 was controlled by addition of fatty acid-free bovine serum albumin. The membrane fraction obtained represents approximately a 15-fold enrichment of the postsynaptic marker proteins muscarinic and nicotinic acetylcholine receptor and 3',5'-cyclic nucleotide phosphodiesterase over an ordinary synaptic plasma membrane preparation, and is devoid of mitochondrial and microsomal contaminations. The membranes appear on the electron micrographs as rigid fragments (average length 2500-4000A), which do not form vesicles.     

Kinetics of olive oil hydrolysis in an enzyme membrane reactor. Bond graph network model of reactor performance (part 1)

Hydrolyses of olive oil were performed in a reactor with lipase immobilized on a laboratory ultrafiltration poliamide-6 membrane. The reactor consisted of two circulating phases of olive oil and buffer solution. For the characterization of the reactor performance, a model of the hydrolysis process was developed. It was created by means of thermodynamic network representation of both the chemical processes and the transport of the reactants. According to an estimated bond graph network, the model is represented quantatively by a set of thirty-three differential equations representing the time derivatives of the particular species concentration. The parameters of the model were estimated based on experimental data and/or literature notations. Close agreement of numerical estimations of the product concentrations with experimental data was gained. The model enabled an extended analysis of the influence of different reaction parameters, enzyme inhibition and concentration of the reactants on reactor performance.     

Nonisothermal bioreactors in the treatment of vegetation waters from olive oil: laccase versus syringic acid as bioremediation model

Laccase from Trametes versicolor was immobilized by diazotization on a nylon membrane grafted with glycidil methacrylate, using phenylenediamine as spacer and coupling agent. The behavior of these enzyme derivatives was studied under isothermal and nonisothermal conditions by using syringic acid as substrate, in view of the employment of these membranes in processes of detoxification of vegetation waters from olive oil mills. The pH and temperature dependence of catalytic activity under isothermal conditions has shown that these membranes can be usefully employed under extreme pH and temperatures. When employed under nonisothermal conditions, the membranes exhibited an increase of catalytic activity linearly proportional to the applied transmembrane temperature difference. Percentage activity increases ranging from 62% to 18% were found in the range of syringic acid concentration from 0.02 to 0.8 mM, when a difference of 1 degrees C was applied across the catalytic membrane. Because the percentage activity increase is strictly related to the reduction of the production times, the technology of nonisothermal bioreactors has been demonstrated to be an useful tool also in the treatment of vegetation waters from olive oil mills.