Isolation, characterization, and surface chemistry of a surface-active fraction from dog lung

A procedure is described for the isolation of a surface-active fraction from dog lung. This material meets the established criteria for pulmonary surfactant. The fraction was shown to contain lipid, protein, and carbohydrate. The predominant lipid present was dipalmitoyl phosphatidylcholine. Surface chemistry studies indicated the surface properties of the fraction could not be explained solely from a consideration of the properties of dipalmitoyl phosphatidylcholine. Electron microscopic studies demonstrated the presence of intact osmiophilic bodies as well as other myelin forms in the surface-active fraction. It is speculated that, in situ, the alveolar lining layer is similar to a structured gel.     

Methyl branching in short-chain lecithins: are both chains important for effective phospholipase A2 activity?

Several seven-carbon fatty acyl lecithins with varied acyl chain branching have been synthesized and characterized as potential phospholipase A2 substrates. Micellar bis(4,4-dimethylpentanoyl) phosphatidylcholine, bis(5-methylhexanoyl)phosphatidylcholine, bis(3-methylhexanoyl)phosphatidylcholine, and bis(2-methylhexanoyl)phosphatidylcholine are poor substrates for phospholipase A2 (Naja naja naja). These branched lecithins also inhibit the hydrolysis of diheptanoylphosphatidylcholine by the enzyme with Ki values comparable to or smaller than the apparent Km of the linear compound. The terminally branched lecithins are excellent substrates for another surface-active hydrolytic enzyme, phospholipase C from Bacillus cereus. When only one acyl chain bears a methyl group, the hybrid lecithins 1-heptanoyl-2-(2-methylhexanoyl)phosphatidylcholine and 1-(3-methylhexanoyl)-2-heptanoylphosphatidylcholine are substrates comparable to diheptanoylphosphatidylcholine. Analysis of micellar structure and dynamics by 1H and 13C NMR spectroscopy, quasi-elastic light scattering, and comparison of critical micellar concentrations indicates little significant difference in the conformation and dynamics of these seven-carbon fatty acyl lecithin micelles, even when the methyl groups are adjacent to the carbonyls. Phospholipase A2 UV difference spectra induced by phospholipid binding imply different enzyme conformations or aggregation states caused by linear-chain and asymmetric-chain lipids compared to bis(methylhexanoyl)phosphatidylcholines. The differences in hydrolytic activity of phospholipase A2 against the branched-chain micellar lecithins can then be attributed to an enzyme-lipid interaction at the active site. The species with both fatty acyl chains branched bind to phospholipase A2 but are not turned over rapidly. Since poor enzymatic activity only occurs for lecithins with both chains methylated, the interaction of both chains with the enzyme must be important for catalytic efficiency.     

Analysis of a specific oxygenation reaction of soybean lipoxygenase-1 with fatty acids esterified in phospholipids

Soybean lipoxygenase was reacted with phosphatidylcholine (at pH 9, with 10 mM deoxycholate), and the oxygenation products were analyzed by high-pressure liquid chromatography, UV, gas chromatography-mass spectrometry (GC-MS), and NMR. The structures of the intact glycerolipid products were established by GC-MS of diglycerides recovered by phospholipase C hydrolysis and by proton NMR of the intact phosphatidylcholine. These analyses, together with analyses of the transesterified fatty acids, indicated that arachidonyl and linoleoyl moieties in the phosphatidylcholine were converted exclusively to the 15(S)-hydroperoxy-5(Z),8(Z),11(Z),13(E)-eicosatetraenoate and 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate analogues, respectively. Control experiments proved that the intact phospholipid (and not hydrolyzed/reesterified fatty acid) was the true substrate of the oxygenation reaction. Phosphatidylethanolamine and phosphatidylinositol lipids were also substrates for specific oxygenation by the soybean lipoxygenase. The results provide concrete evidence that fatty acids esterified in phospholipid can be subject to highly specific oxygenation by a lipoxygenase enzyme.     

Localization and characterization of phospholipase A2 in mouse mammary gland-derived cells

Phospholipase A2 (PLA2) can participate in the regulation of eicosanoid biosynthesis via PLA2-mediated control of the release of arachidonic acid from phospholipids. Arachidonoyl-hydrolyzing PLA2s were examined in cells from normal mouse mammary glands and mammary carcinomas. Tumor-derived cells exhibited significant PLA2 activity(ies) with arachidonoyl containing phosphatidylcholine and phosphatidylethanolamine as substrates in cell-free assays. In contrast, arachidonoyl containing phosphatidylinositol was a poor substrate. When phosphatidylcholines with varying sn-2 fatty acyl groups were tested as substrates, activity was highest with the arachidonoyl containing lipid. The pH profiles for hydrolysis of phosphatidylcholine and phosphatidylethanolamine differed; all other aspects of PLA2-mediated hydrolysis of these two substrates were similar including a Ca2+ requirement for activity. Moreover, Ca2+ affected the subcellular localization of the enzyme activity. Activity was predominately in the supernatant fraction when cells were harvested in an EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid) containing buffer and largely in the particulate fraction when cells were harvested in a buffer containing free Ca2+. The localization of activity could be modulated from the supernatant fraction to the particulate fraction by recentrifugation in the presence of Ca2+. Normal gland-derived cells contained a PLA2 activity with properties similar to those of the tumor-derived cells. There was a significant difference in the level of activity in the normal versus tumor cells, the normal gland-derived cells had less than half the PLA2 activity of the carcinoma-derived cells.     

Transformation of Phospholipids by Cabbage Phospholipase D in Mixed Micelles Containing 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

We compared the activities of cabbage phospholipase D during hydrolysis and transesterification of phosphatidylcholine in mixed micelles of surface-active compounds with various physicochemical properties. Mixed micelles of phospholipids and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (ratio, 1 : 2) were among the best substrates. Hydrolysis and transphosphatidylation were studied in micelles containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Mixed micelles of phosphatidylcholine and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate may serve as a new substrate for the measurement of phospholipase D activity and preparation of phospholipids using this enzyme.     

Transformation of phospholipids by cabbage phospholipase D in mixed micelles containing 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate

We compared the activities of cabbage phospholipase D during hydrolysis and transesterification of phosphatidylcholine in mixed micelles of surface-active compounds with various physicochemical properties. Mixed micelles of phospholipids and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (ratio, 1:2) were among the best substrates. Hydrolysis and transphosphatidylation were studied in micelles containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Mixed micelles of phosphatidylcholine and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate may serve as a new substrate for measurements of phospholipase D activity and preparative isolation of phospholipids using this enzyme.     

Effect of surfactant and particle size reduction on hydrolysis of deinking sludge and nonrecyclable newsprint

Disposal of sludge from deinking mills represents a significant proportion of operating costs. Bioconversion of the cellulosic fraction of deinking sludge (DIS) to ethanol greatly reduces disposal costs while producing an environmentally friendly fuel. In this study, the cellulosic fraction of newsprint and deinking sludge was hydrolysed to produce fermentable sugars. For newsprint, a particle size of 1 to 1.5 mm provided optimal reaction rates in batch reactors over practical hydrolysis times, and reducing sugar concentrations as high as 35 g/L could be achieved using a fed-batch reactor configuration. For both newsprint and DIS, the hydrolysis rate increased nonlinearly with enzyme loading. Tween-80 only marginally improved sugar production but was able to release sugars from cellulosic substrates in the absence of lytic enzymes, in an amount proportional to the surfactant concentration and the substrate particle size. DIS was relatively recalcitrant to enzymatic hydrolysis, possibly due in part to inhibition by hydrophobic constituents. (c) 1995 John Wiley & Sons, Inc.     

Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase

Degradation of phosphatidylcholine to lysophosphatidylcholine occurs during oxidative modification of low density lipoproteins (LDL). In this study, we have shown that this phospholipid hydrolysis is brought about by an LDL-associated phospholipase A2 that can hydrolyze oxidized but not intact LDL phosphatidylcholine. The chemical nature of the oxidized phospholipids that can act as substrates for this enzyme was not fully characterized, but we hypothesized that the specificity of the enzyme for oxidized LDL phosphatidylcholine might be explained by fragmentation of polyunsaturated sn-2 fatty acyl groups in LDL phosphatidylcholine during oxidation. To facilitate characterization of this enzyme, we therefore selected a fluorescent phosphatidylcholine substrate that had a short-chain, polar residue in the sn-2 position: 1-palmitoyl 2-(6-[7-nitrobenzoxadiazolyl]amino) caproyl phosphatidylcholine, (C6NBD PC). This substrate was efficiently hydrolyzed by LDL, but the dodecanoyl analogue of C6NBD PC, which differed only in that a 12-carbon rather than a 6-carbon acyl derivative was present in the sn-2 position, was not hydrolyzed. The phospholipase activity was heat-stable, calcium-independent, and was inhibited by the serine esterase inhibitors phenylmethylsulfonyl-fluoride and diisopropylfluorophosphate, but was resistant to p-bromophenacylbromide and dithiobisnitrobenzoic acid. The phospholipid hydrolysis could not be attributed to the action of lecithin:cholesterol acyltransferase or lipoprotein lipase. Nearly all of the activity in EDTA-anticoagulated normal plasma was physically associated with apoB-containing lipoproteins, but this apoprotein was not essential as enzyme activity was present in plasma from abetalipoproteinemic patients. These properties are very similar to those recently reported for human plasma platelet-activating factor (PAF) acetylhydrolase. In the present study, we found that acylhydrolase activity against C6NBD PC, PAF, and oxidized phosphatidylcholine copurfied through gel filtration and ion-exchange chromatography. Substrate competition was demonstrated between C6NBD PC, PAF, and oxidized 2-arachidonyl phosphatidylcholine, suggesting that a single enzyme was active against all three substrates. The enzyme had an apparent molecular weight of 40,000-45,000 by high pressure gel exclusion chromatography. Inhibition of this activity with disopropyfluorophosphate prior to oxidative modification of LDL prevented phospholipid hydrolysis but did not affect the production of thiobarbituric acid reactive compounds or the change in electrophoretic mobility. In addition, this inhibition of phospholipase did not prevent the rapid degradati     

Kinetic behavior of the pancreatic lipase-colipase-lipid system

Pancreatic lipase is a surface-active protein that binds avidly to interfaces comprised of the substrates and products of lipolysis. However, both lipase binding to substrate-containing particles and subsequent interfacial catalysis are inhibited by a number of amphipathic molecules. The most thoroughly studied of these, phosphatidylcholine, is a common constituent of membranes and intestinal lipid contents. Colipase, a surface-active cofactor of lipase, relieves inhibition by phosphatidylcholine in several ways. Through protein-protein interactions, colipase helps anchor lipase to surfaces and stabilizes it in the open conformation. Within the interface, colipase packs more efficiently with substrates and products of lipolysis than with phosphatidylcholine, thereby concentrating these reactants in the vicinity of colipase. This enrichment of lipase substrates and products in the vicinity of colipase enhances lipase-lipid interactions. The result is that colipase facilitates the adsorption of lipase to the interface and, possibly, increases the availability of substrate to the enzyme. Thus, the functional unit in intestinal lipolysis appears to be a lipase-colipase-reactant complex.     

Exploring the action and specificity of cobra venom phospholipase A2 toward human erythrocytes, ghost membranes, and lipid mixtures

We have investigated the action and substrate specificity of phospholipase A2 (EC 3.1.1.4) purified from cobra venom (Naja naja naja) toward intact and Triton-solubilized human erythrocytes, toward ghost membranes, and toward extracted ghost lipids in mixed micelles with Triton X-100. We have found that: (i) phospholipids in the outer surface of intact erythrocytes are extremely poor substrates for the phospholipase, (ii) phospholipids in ghost erythrocyte membranes and in Triton-solubilized erythrocytes are suitable substrates for the enzyme, (iii) in these latter systems which contain a mixture of lipids, phosphatidylethanolamine is preferentially hydrolyzed, whereas in model studies on individual phospholipid species in mixed micelles with Triton, phosphatidylcholine is the preferred substrate of the enzyme, and (iv) the preferential hydrolysis of phosphatidylethanolamine is also observed for extracted ghost lipid mixtures in mixed micelles. These results demonstrate a dependence of phospholipase A2 activity on the ghosting procedure and a dependence of substrate specificity on the presence of other lipids. The relevance of these findings to the interpretation of membrane lipid asymmetry studies utilizing phospholipases is considered in detail.     

Hydrolysis of 2-acyl-sn-glycero-3-phosphocholines in guinea pig heart mitochondria

Although both 2-acyl-sn-glycero-3-phosphocholine and 1-acyl-sn-glycero-3-phosphocholine may be produced from phosphatidylcholine hydrolysis, studies on the former have lagged behind that of the latter. In this study a lysophospholipase A2 that hydrolyses 2-acyl-sn-glycero-3-phosphocholine has been characterized in guinea pig heart mitochondria. The lysophospholipase A2 activity was not dependent on Ca2+ and was inhibited differentially by saturated and unsaturated fatty acids. This lysophospholipase A2 activity was able to discriminate among different molecular species of 2-acyl-sn-glycero-3-phosphocholines when they were presented individually or in pairs. The order of decreasing rates of hydrolysis of different molecular species of 2-lysophosphatidylcholines, when the substrates were presented singly, was 18:2 greater than 20:4 greater than 18:1 greater than 16:0. A differential inhibition of the rate of hydrolysis of the individual substrates was observed when the substrates were presented in pairs. The degree of inhibition was dependent on the molar ratio of the mixed substrates. The characteristics of the enzyme suggest that involvement in the selective release of fatty acids from mitochondrial phosphatidylcholine would depend on a high selectivity of phospholipase A1 for different molecular species of phosphatidylcholine. A lysophospholipase A1 activity was also characterized in the mitochondria with a distinct acyl specificity from the lysophospholipase A2. Other characteristics of the two lysophospholipases suggest that the two reactions are not catalysed by the same enzyme.     

Hydrolysis of membrane-associated phosphoglycerides by mitochondrial phospholipase A2

Conversion of membrane-bound substrates by membrane-associated enzymes can proceed in principle via intramembrane and intermembrane action. By using rat-liver mitochondria containing labeled phosphatidylethanolamine and inactivated phospholipase A2 as substrate source, and mitochondria containing unlabeled substrate and active enzyme, it is shown that hydrolysis of phosphatidylethanolamine by mitochondrial phospholipase A2 proceeds nearly entirely via intramembrane enzyme action. A study of the characteristics of this mode of enzyme action showed that all mitochondrial phosphoglycerides were hydrolyzed. Plots of approximate initial velocities of hydrolysis against the remaining amounts of each individual phospholipid, indicated that phosphatidylethanolamine was hydrolyzed fastest, with a rate about twice that for phosphatidylcholine and about 10-fold that for cardiolipin. The initial rates remained nearly constant in the initial phase of the hydrolysis, suggesting that the enzyme is surrounded by excess substrate.     

Mechanism of action of lipoprotein lipase on triolein particles: effect of apolipoprotein C-II

Triolein particles stabilized by a phosphatidylcholine monolayer were used to study the lipoprotein lipase (LpL) reaction. They were prepared in two different sizes and with triolein and phosphatidylcholine in the molar ratios of 0.9-1.2 : 1 (small particles) and 8-17 : 1 (large particles). The rate of hydrolysis by LpL of phosphatidylcholine on the surface of both lipid particles was only 1/20 as much as that of triolein, even if it was activated to the maximum by apolipoprotein C-II (apoC-II). Thus, the phospholipase activity of LpL was low enough to measure the initial rate of hydrolysis of triolein without causing a gross change of the surface of the lipid particle. When the hydrolysis of triolein by LpL was monitored, fatty acid was released at a constant rate until all of the triolein molecules were hydrolyzed. The enzyme required 220 +/- 17 and 66 +/- 9 nM apoC-II for its half-maximal activity (Km (apoC-II] with small and large particles as a substrate (1.15 mM triolein for small and 2.13 mM triolein for large particles), respectively, using various concentrations of LpL. The Km(apoC-II) values for these two substrates became similar when LpL activity was analyzed with respect to the density of apoC-II on the phosphatidylcholine monolayer at the surface of the particles (bound apoC-II/phosphatidylcholine). The concentration of substrate particles did not affect the Km(apoC-II) values. The presence of an adequate amount of apoC-II increased the maximal activity of LpL (Vmax(triolein)) from 0.48 +/- 0.21 to 6.81 +/- 0.45 and from 0.32 +/- 0.04 to 7.13 +/- 0.64 mmol/h/mg with a slight decrease in the apparent Michaelis constant (Km(triolein)) for small (from 90 to 54 microM triolein) and large (from 1.00 to 0.65 mM triolein) particles, respectively. Although the apparent Km for triolein in large particles was about ten times greater than that in small particles, the values became similar when they were corrected for the concentration of phosphatidylcholine (50-100 microM phosphatidylcholine), which corresponded to the surface area of the substrate particles. It was suggested that bound apoC-II molecules were transferred relatively slowly to other lipid particles while LpL molecules moved rapidly among the lipid particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Very low density lipoprotein. Fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis in vitro.

In this study we have determined the fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis of rat plasma very low density lipoprotein (rat VLDL). The experiment was carried out in vitro with lipoprotein lipase purified from bovine milk, VLDL labeled with [(14)C]palmitate, [(3)H]cholesterol, [(32)P]phospholipids, and (125)I-labeled apolipoprotein C and in plasma-devoid systems. Triglyceride hydrolysis ranged between 0 and 98.6%. [(32)P]Phospholipids, unesterified [(3)H]cholesterol, and (125)I-labeled apolipoprotein C were removed from the VLDL (d < 1.019 g/ml) during lipolysis. About one-third of the [(32)P]phosphatidylcholine was hydrolyzed to lysolecithin, and was transferred to the fraction d > 1.21 g/ml. The other two-thirds of the phospholipids were removed unhydrolyzed, mainly to the fraction d 1.04-1.21 g/ml. With the progression of the lipolysis, unesterified [(3)H]cholesterol was removed from VLDL at increasing rates, predominantly to the fraction d 1.04-1.21 g/ml. (125)I-Labeled apolipoprotein C removed from the VLDL partitioned between the fraction of d 1.04-1.21 g/ml and d > 1.21 g/ml. Negative-staining electron microscopy of the fraction d 1.04-1.21 g/ml (containing phospholipids, unesterified cholesterol, and apolipoprotein C) revealed many discoidal lipoproteins. [(3)H]Cholesteryl esters remained associated with the VLDL even when 70-80% of the triglycerides were hydrolyzed. These observations suggest that during in vitro lipolysis of VLDL, surface constituents leave the lipoprotein concomitantly with the hydrolysis of core triglycerides. The process of removal of surface constituents is independent of the presence of an acceptor lipoprotein and may occur in the form of a surface-fragment particle. -Eisenberg, S., and T. Olivecrona. Very low density lipoprotein. Fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis in vitro.     

Local anesthetic inhibition of pancreatic phospholipase A2 action on lecithin monolayers.

Using quantitative data previously reported for the penetration of local anesthetics into lecithin monolayers, the effects of surface and subphase concentrations of anesthetics on the inhibition of pancreatic phospholipase A2 action on didecanoyl phosphatidylcholine monolayers was investigated. Inhibition as a function of subphase concentration of anesthetic was in the order: dibucaine greater than tetracaine greater than butacaine greater than lidocaine = procaine. Inhibition as a function of surface concentration showed no obvious correlation; procaine inhibited at a very low surface concentration, followed by lidocaine at a somewhat higher concentration, and tetracaine, butacaine and dibucaine only at rather high concentrations. Ultraviolet difference spectroscopy indicated an interaction between lidocaine and enzyme in the subphase. Fluorescence studies showed that lidocaine is a competitive inhibitor of enzyme-lipid interface interaction. It is proposed that the more surface-active anesthetics inhibit by surface effects while the less surface-active anesthetics (lidocaine and procaine) inhibit by interaction with the enzyme in the subphase, which prevents enzyme penetration at the monolayer interface.     

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta. Effect of substrates and potassium

This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM     

Purification and properties of a lipid acyl-hydrolase from potato tubers.

1. A pure lipid acyl-hydrolase was prepared from potato tubers by acetone precipitation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography, and by electrofocusing. 2. The purified enzyme was an acidic protein of pI 5.0 and molecular weight of about 70 000. Km values were 0.38 mM for monogalactosyldiacylglycerol and 1.7 mM for phosphatidylcholine. 3. The hydrolytic activity of the enzyme on different substrates was determined. The relative rates were acylsterylglucoside greater than monogalactosyldiacylglycerol greater than monogalactosylmonoacylglycerol greater than digalactosyldiacylglycerol greater than diagalactosylmonoacylglycerol, while the rates for phospholipids were lysophosphatidylcholine greater than phosphatidylcholine greater than lysophosphatidylethanolamine greater than phosphatidylethanolamine. 4. Analyses of enzymatic hydrolysis products suggested that a single enzyme had both galactolipase and phospholipase activities, and for the phospholipids it showed activities similar to phospholipase B and glycerylphosphorylcholine diesterase. 5. A competitive relation was found between monogalactosyldiacylglycerol and phosphatidylcholine as substrates of the enzyme, indicating that the active sites for both substrates may be the same. 6. It was suggested that histidine and probably serine residues were important to the enzymic activity, and that a tyrosine residue might be involved in the activity as an accessory component.     

Purification of phosphatidylethanolamine N-methyltransferase from rat liver

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine.     

Lysosomal lipolytic enzymes,lipid peroxidation,and injury

Summary We have used highly purified lysosomes to investigate three models of hydrolytic injury by lysosomal phospholipases. Lysosomes, enriched up to 70-fold in marker enzyme activities, can be isolated from homogenized hepatic tissue by differential centrifugation and subsequent free flow electrophoresis. These organelles remain latent and can also be utilized to obtain lysosol, the soluble fraction of the lysosomes tissue containing acid active phospholipases. The first model investigated the effect of lysosol on non-lysosomal membranes. When this soluble fraction was incubated with plasmalemma (sarcolemma) from cardiac cells, selective hydrolysis of the phospholipids was observed: phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were the preferred substrates, and only lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in significant amounts. Hydrolysis of sphingomyelin was enhanced significantly by Triton-X-100. In the second model, when intact lysosomes were incubated at acid pH, hydrolysis of phospholipids by the endogenous lipases was observed. Once again this lipolysis was specific for phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin: significant amounts of lysophospholipids also accumulated in this model. Concurrent with these lipid changes, an increase in lysosomal permeability also occurred and pH 5.0 was optimal for this lipolytic activity. However, no phospholipase activity was detected when lysosomes were incubated at pH ranges found in acidotic tissue (pH 6.0 or higher). In the third model, lysosomes were incubated at pH 6.0 in the presence of exogenously generated free radicals (dihydroxyfumarate-FeADP). A rapid loss of membrane phospholipids was observed, and most of this loss could be contributed to peroxidation of membrane phospholipids; the production of malondialdehyde preceded loss of N-acetylglucosaminidase from the lysosome. However, significant accumulation of lysophospholipids, from 2% at control time to 6.6 and 8.7% at 10 and 20 minutes, suggested that lysosomal phospholipase were hydrolyzing lysosomal phospholipids. Thus, we hypothesize that this free radical-induced lipolysis is a result of peroxidized phospholipids serving as preferred substrate for phospholipases at pH 6.0.     

A mechanistic model for rational design of optimal cellulase mixtures

A model‐based framework is described that permits the optimal composition of cellulase enzyme mixtures to be found for lignocellulose hydrolysis. The rates of hydrolysis are shown to be dependent on the nature of the substrate. For bacterial microcrystalline cellulose (BMCC) hydrolyzed by a ternary cellulase mixture of EG2, CBHI, and CBHII, the optimal predicted mixture was 1:0:1 EG2:CBHI:CBHII at 24 h and 1:1:0 at 72 h, at loadings of 10 mg enzyme per g substrate. The model was validated with measurements of soluble cello‐oligosaccharide production from BMCC during both single enzyme and mixed enzyme hydrolysis. Three‐dimensional diagrams illustrating cellulose conversion were developed for mixtures of EG2, CBHI, CBHII acting on BMCC and predicted for other substrates with a range of substrate properties. Model predictions agreed well with experimental values of conversion after 24 h for a variety of enzyme mixtures. The predicted mixture performances for substrates with varying properties demonstrated the effects of initial degree of polymerization (DP) and surface area on the performance of cellulase mixtures. For substrates with a higher initial DP, endoglucanase enzymes accounted for a larger fraction of the optimal mixture. Substrates with low surface areas showed significantly reduced hydrolysis rates regardless of mixture composition. These insights, along with the quantitative predictions, demonstrate the utility of this model‐based framework for optimizing cellulase mixtures. Biotechnol. Bioeng. 2011;108: 2561–2570. © 2011 Wiley Periodicals, Inc.