Purification of a phospholipase from Botrytis and its effects on plant tissues

A phospholipase from Botrytis cinerea, grown in pure culture, was purified more than 1000-fold. Whilst it possessed no acyl hydrolase activity toward a variety of p-nitrophenyl fatty acyl derivatives, phosphatidyl choline (lecithin) acted as a substrate; the enzyme being of either type ‘A’ or ‘B’ specificity. When the purified enzyme was applied to washed beetroot discs, betacyanin leakage was induced. Loss of substances which absorb at 260 nm also occurred when washed potato tuber discs were incubated with the enzyme. Incubation with a lysosome-enriched fraction from potato sprout tissues resulted in increased acid phosphatase activity in the incubation supernatant. The phospholipase had no macerating effect on a variety of plant tissues, nor did it cause disruption of isolated protoplasts from these tissues. Studies with mitochondria from mung beans revealed no effects of the enzyme upon the respiratory activity of these organelles. The result suggested that a major site of action of the B.cinerea phospholipase was the lysosomes.     

Catabolism of sulphoquinovosyl diacylglycerol by an enzyme preparation from Phaseolus multiflorus

An enzyme which will deacylate sulphoquinovosyl diacylglycerol (SQDG) has been partially purified from the leaves of runner bean (Phaseolus multiflorus). No monoacyl intermediate was observed and the acyl hydrolase was more active towards unsaturated molecular species of SQDG than towards saturated species. The major peak of activity of SQDG acyl hydrolase, separated on both DEAE-cellulose and Sephadex columns, also contained galactolipid acyl hydrolase activity. The distribution of these activities together with substrate competition and inhibitor experiments indicated that at least part of the SQDG acyl hydrolase activity was due to an enzyme that also hydrolysed galactolipids.     

Membrane-degrading enzymes in the leaves of Solanum tuberosum

When homogenates of potato leaves were prepared under conditions which are typical for organelle isolation (pH 7.5 and 4°), membrane lipids underwent rapid hydrolysis (17% of phosphatidylcholine was hydrolysed in 2 hr). Leaves of 41 potato cultivars were surveyed for phospholipase activity to determine whether certain cultivars might be more suitable for the preparation of organelles. Phospholipase activities ranged from 1.04 to 11.60, μmol/min · g fr. wt and p-nitrophenyl palmitate hydrolase activity ranged from 0.0119 to 0.0502,μmol/min · g fr. wt. These phospholipase values were several hundred-fold higher than previously reported for potato leaves and nearly as high as in potato tubers. Most of the phospholipase activity in leaves was soluble and not membrane-associated as previously reported.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Wax ester formation catalysed by isoenzymes of lipolytic acyl hydrolase

Wax ester formation by esterification of a long chain fatty acid (palmitic acid) with a long chain fatty alcohol (octadecanol) was enzymically catalysed by acetone dried powder preparations of potato tubers. The enzyme responsible for wax ester formation had multiple isoenzymic forms and was identical with lipolytic acyl hydrolase, a lipid deacylating enzyme. Tubers from different varietiees of potato (Solanum tuberosum) demonstrated markedly different levels of activity and electrophoretic patterns for both wax ester formation and lipid deacylation.     

The enzymic degradation of lipids resulting from physical disruption of cucumber (Cucumis sativus) fruit

Homogenization of fresh tissue from cucumber fruits results in a loss of endogenous lipid catalysed by acyl hydrolase enzymes. Deacylation of lipids is not accompanied by accumulation of free fatty acids. The levels of both saturated (mainly palmitic) and polyunsaturated (linoleic and linolenic) fatty acids in the lipids are reduced. Losses of the major acyl lipid constituents of cucumber (triacylglycerols and phospholipids) are mainly responsible for the observed hydrolysis. Triacylglycerol acyl hydrolase (lipase), phospholipase D and polar lipid acyl hydrolase enzyme activities were demonstrated. It is suggested that hydrolytic attack on endogenous lipids is the initial event on disruption of cucumber tissue, in the formation of lipid degradation products, amongst which are the volatile carbonyl compounds responsible for the characteristic flavour of cucumber.     

Regulation of Photosystem I electron flow activity by phosphatidylglycerol in thylakoid membranes as revealed by phospholipase treatment

Thylakoid membranes were treated by potato lipolytic acyl hydrolase, phospholipases A₂ from pancreas and snake venom, and by phospholipase C from Bacillus cereus under various conditions. The changes in the uncoupled rates of electron transport through Photosystem I (PS I) and in lipid composition were followed during these treatments. Pancreatic phospholipase A₂ which destroyed all phospholipids in thylakoid membranes stimulated the NADP⁺ reduction supported by reduced 2,6-dichlorophenolindophenol. This stimulation concerned only the dark but not the light reactions of this pathway. The main site of action of pancreatic phospholipase A₂ may be located on the donor side of PS I; the hydrolysis of phospholipids at this site caused an increased ability of reduced 2,6-dichlorophenolindophenol and ascorbate alone to feed electrons into PS I. A second site may be located on the acceptor side of PS I, probably between the primary acceptor and the ferredoxin system. When thylakoid membranes were first preincubated with or without lipolytic acyl hydrolase at 30°C (pH 8), the NADP⁺ photoreduction was inhibited whilst the methyl viologen-mediated O₂ uptake was stimulated. A subsequent addition of pancreatic phospholipase A₂ (which had the same hydrolysis rates for phosphatidylglycerol but not for phosphatidylcholine) further stimulated the O₂ uptake and restored NADP⁺ photoreduction. The extent of this stimulation, which depended on the presence of lipolytic acyl hydrolase, was ascribed partly to the hydrolysis of the phospholipids and partly to the generation of their lyso derivatives but not to the release of free fatty acids. On the contrary, phospholipase C which destroyed only phosphatidylcholine failed to restore this activity. It is suggested that phosphatidylglycerol is the only phospholipid associated with thylakoid membrane structures supporting PS I activities and that this lipid may play a physiological role in the regulation of these activities.     

Phospholipids and Lipid Acyl Hydrolase (Phospholipase) in Leaf Galls (Hymenoptera: Cynipidae of Black Oak [Quercus robor L.])

Phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and cardiolipin are the major phospholipids in young leaves of black oak (Quercus robor L.). Except for phosphatidylcholine, young, developing cynipid-galls on black oak leaves, i.e. the insect-transformed tissues, contain less phospholipid than normal leaf tissues. Lipid acyl hydrolase activity determined by the cleavage of free fatty acids from a labeled phospholipid substrate is higher in the tissue extracts from galls than from leaves. The increase in enzyme activity and the altered phospholipid composition are discussed in relation to expected membrane modifications and transport phenomena in insect-transformed tissues.     

Cloning,screening and characterization of ester hydrolases with enantioselectivity in typical bacteria

A broad exploitation of ester hydrolases from 7 typical bacteria was reported in this study. Thirty-two predicted esterases and hydrolases were cloned based on published genomic information. The catalytic activity of obtained clones was tested with p-nitrophenyl esters at various temperatures and pH values. The results indicated that eight enzymes presented with typical esterase activity on p-nitrophenyl butylate and caprylate. The result also showed that despite their great sequence difference, the eight enzymes shared similar properties (substrate specificity, optimal pH and temperature) with each other. Phylogenetic analysis revealed a close relationship between these eight enzymes and “true esterases”. As there was no information on enantioselectivity of these enzymes reported, the enantioselectivity of these enzymes to various chiral substrates was investigated for the first time. In comparison with commercial enzyme, Candida rugosa lipase (CRL), enzymes E12, E14, E18, E21 and E24 presented with equal or higher activity and enantioselectivity to the substrates. Furthermore, enzyme E14 (predicted carboxylesterase from Rhodobacter sphaeroides), E21 (S-formylglutathione hydrolase from Pseudomonas putida) and E24 (carboxylesterase from P. putida) presented with enantioselectivity in the resolution of methyl mandelate, 1-phenyethyl acetate and 2-octanol. These findings suggested that the novel ester hydrolases with high activity and enantioselectivity could be obtained from alpha/beta hydrolase family.     

Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A2

The thioesterase activity of porcine pancreatic phospholipase A₂ has been investigated with non-phospholipid substrates. The acyl-CoA hydrolase activity towards acyl-CoA derivatives is specific for long chain fatty acids (14 C, 16 C) but is unable to hydrolyze short chain acyl-CoA compounds (below 8 C). The same enzyme also shows protein deacylase activity liberating [³H]palmitic acid from [³H]palmitoyl-acyl carrier protein.     

Substrate specificity of potato lipoxygenase

The substrate specificity of potato lipoxygenase was examined using a partially purified enzyme preparation from tubers of a potato variety with low lipolytic acyl hydrolase activity. Potato lipoxygenase is fully active only on free linoleic acid or linolenic acid, and only acts directly on more complex glyceride moieties in the absence of any significant endogenous lipolytic acyl hydrolase activity.     

Properties of acyl hydrolase enzymes from Phaseolus multiflorus leaves

The properties of acyl hydrolase enzymes purified from the leaves of Phaseolus multiflorus have been studied. Hydrolase I which deacylates phosphatidylcholine and oleoylglycerol had a pH optimum towards phosphatidylcholine of 5.3. Hydrolase II which deacylates glycosylglycerides and oleoylglycerol showed pH optima of 7.3 (monogalactosyldiglyceride, MGDG) and 4.3 (sulphoquinovosyldiglyceride, SQDG). Both enzymes showed activity peaks towards oleoylglycerol at pH 6.8 and 8.8. Unesterified fatty acids and Triton X-100 inhibited the rate of SQDG hydrolysis while bovine serum albumin increased activity. An apparent K_m for SQDG of 0.15 mM was found. Hydrolase II catalysed transmethylation of liberated fatty acids during the hydrolysis of oleoylglycerol when methanol was included in the assay system. A number of salts inhibited SQDG hydrolysis but their effect on oleoylglycerol was less consistent. The position of ester cleavage of oleoylglycerol was determined by the use of H₂¹⁸O. Cell-free extracts from P. multiflorus leaves degraded SQDG as far as sulphoquinovose.     

Purification and properties of two enzymes from Dichomitus squalens which exhibit both cellobiohydrolase and xylanase activity

Two fractions (Ex I and II), exhibiting activity towards p-nitrophenyl β-cellobioside (pNPC) have been isolated by chromatofocusing of the proteins obtained from the supernatant solution of a cellulose-containing culture of the white-rot fungus Dichomitus squalens. They were further purified up to 16.0- and 14.2-fold by chromatography on Phenyl-Sepharose CL-4B and ion-exchange chromatography on DEAE-Trisacryl. Each purified enzyme gave a single peak for protein and activity on chromatography on Ultrogel AcA-54 and a single protein band in disc gel electrophoresis, in the absence and presence of sodium dodecyl sulphate, and on isoelectrofocusing. The mol. wts. of Ex I and II were 39,000 and 36,000, respectively, and their isoelectric points were 4.6 and 4.5, respectively. Maximum activity towards pNPC was shown at pH 5.0 and 60°, and each enzyme was stable over the pH range 4.0–8.0, and up to 70° and 60° for Ex I and II, respectively. The enzymes cleaved pNPC, released mainly cellobiose from cellulose, were especially active towards xylan and o-nitrophenyl β-d-xylopyranoside, and exhibited strong transglycosylating activities.     

Isoenzymes of lipolytic acyl hydrolase and esterase in potato tuber

Five varieties of potato (Solanum tuberosum) were shown by gel- and free-flow-electrophoresis to exhibit multiple forms of lipolytic acyl hydrolase (LAH) and esterase enzymes. The electrophoretic patterns of LAH and esterase activities and protein differed with the variety and were characteristic for a given variety. In the variety (Golden Wonder) with the highest LAH activity (p-nitrophenylpalmitate as substrate), this was 200-fold greater than the esterase activity (p-nitrophenylacetate as substrate) and isoenzyme patterns for both enzymes were the most complex. In the variety with a very low LAH activity (Désirée), the LAH and esterase activities were similar and more simple isoenzyme patterns for these enzymes were observed.     

Acyl transfer reactions associated with cis Golgi apparatus of rat liver

Isolated Golgi apparatus, highly purified from rat liver, were found to contain an acyl transfer activity capable of restoring the acyl chains of the lysophospholipid products of the action of phospholipase A₂ on phosphatidylcholine. The activity was located primarily in cis and medial Golgi apparatus fractions, had a pH optimum of 6.0 to 7.5 and was stimulated by various acyl-CoA derivatives but not by fatty acids plus ATP. The activity, determined from the conversion of [¹⁴C]lysophosphatidylcholine to [¹⁴C]phosphatidylcholine, was unaffected by EGTA, inhibited by manoalide at high concentrations (0.2 mM), and temperature-dependent. Temperature dependency, however, showed no definite transition temperature over the range 15 to 37°C. The results demonstrated that cis Golgi apparatus membranes have the enzymatic capacity to restore fatty acids lost from phospholipids through the action of phospholipase A. The latter has been previously suggested to occur at the cis Golgi apparatus membranes based on analyses of cell-free transfer of radiolabeled phosphatidylcholine.     

Evidence that pancreatic lipase is responsible for the hydrolysis of cutin,a biopolyester present in mammalian diet,and the role of bile salt and colipase in this hydrolysis

The enzyme, which catalyzes hydrolysis of cutin, an insoluble biopolyester of hydroxy and epoxy fatty acids, was purified from porcine pancreas. With three different purification methods, previously used for the purification of pancreatic lipase, it is shown that cutin hydrolase is pancreatic lipase. This enzyme released oligomers and all types of monomers from the polymer with a pH optimum around 7.5. Taurodeoxycholate inhibited cutin hydrolysis by lipase and colipase reversed this inhibition. Evidence is presented which suggests that bile salt stabilizes the enzyme at the surface of the insoluble substrate and that the interaction of the polymer surface with the lipase-colipase-bile salt system is similar to that previously observed with triglycerides. Diethyl-p-nitrophenyl phosphate inhibited cutin hydrolysis by lipase but the hydrolysis was insensitive to diisopropyl fluorophosphate.     

Purification and characterization of novel cutinase from nasturtium (Tropaeolum majus) pollen.

Cutinase from pollen grains of Tropaeolum majus was purified by Sephadex G-100 gel filtration, QAE-Sephadex chromatography, and isoelectric focusing. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be 40,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cutinase was found to be a glycoprotein containing about 7% carbohydrate and the isoelectric point of this enzyme was 5.45. It catalyzed hydrolysis of p-nitrophenyl esters of C₂ to C₁₈ fatty acids with similar K_m and V. The purified cutinase showed an optimum pH of 6.8 with cutin as the substrate, whereas with p-nitrophenyl esters of fatty acids the optimum pH was 8.0. This enzyme did not show any metal ion requirement. Unlike the previously studied fungal cutinases, the present pollen enzyme was strongly inhibited by thiol-directed reagents such as N-ethylmaleimide and p-hydroxymercuribenzoate whereas it was totally insensitive to the active serine-directed reagent, diisopropylfluorophosphate. The purified pollen cutinase showed preference for primary alcohol esters, but it did not catalyze hydrolysis of tripalmitoyl or trioleyl glycerol at significant rates. The properties of the pollen enzyme are, in general, in sharp contrast to those of the fungal cutinase, and the present results strongly suggest that the pollen enzyme belongs to a new class of cutinases. Another esterase which preferentially hydrolyzed p-nitrophenyl acetate was also found in the extracellular fluid. This enzyme, separated from cutinase, showed a pI of 5.6 and it was sensitive to diisopropylfluorophosphate, but not to SH-directed reagents.     

Purification and properties of an extracellular esterase from a cold-adapted <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis>

An extracellular esterase, EstK, was purified from the psychrotrophic bacterium Pseudomonas mandelii grown at 25°C. Prior to harvest, cells were treated with 0.2 M MgCl₂ to precipitate lipopolysaccharides in the outer membranes, which otherwise form aggregates with the secreted enzymes. EstK was purified to homogeneity using standard procedures. It had substrate specificity towards esters of short-chain fatty acids, particularly, p-nitrophenyl acetate. Optimum activity of EstK was at 40°C; at 4°C the activity was ~50% of its maximum. EstK has a unique substrate preference for p-nitrophenyl acetate and remains active at low temperatures.     

Translucent Tissue Defects in Solanum tuberosum L. : II. Alterations in Lipolytic Acyl Hydrolase, Lipoxygenase, and Morphology of Mitochondria and Amyloplasts

We analyzed a physiological defect that involved translucent-like tissue which occurred randomly in potato tubers (Solanum tuberosum L., cv Kennebec) after 8 months of storage. The translucent areas had reduced lipoxygenase (0.73-fold) and lipolytic acyl hydrolase (0.27-fold) activities. The effect(s) of these reduced enzyme activities in vivo is uncertain, but they may have influenced composition, turnover and permeability of membranes because potato lipid is primarily membranous in nature. Electron micrographs of the translucent tissue revealed a discernible decrease in the number of starch granules compared to normal/healthy tissue. A few remaining amyloplasts, which still contained starch granules, possessed large electrondense areas (stroma) within the organelle. Mitochondria in translucent tissue appeared to be present in increased numbers, were aggregated, had fewer but swollen cristae, and, morphologically, were of irregular size and shape suggestive of division. The result of this tuber defect appeared, in part, to be an exaggerated or accelerated form of senescence.     

Activation of a specific phospholipid fatty acyl hydrolase in Dunaliella salina microsomes during acclimation to low growth temperature

Dunaliella salina microsomes, but not chloroplasts, contain a fatty acyl hydrolase with high activity towards endogenous or exogenous phosphatidylglycerol and phosphatidylethanolamine. There is relatively little activity towards other phospholipids or added monogalactosyldiacylglycerol. Lipolysis is most active in the presence of 10 mM Ca²⁺ and is enhanced by added calmodulin. Microsomal acyl hydrolase activity in 30°C-grown cells is low when measured in vitro at 12°C but increases rapidly after cells are chilled to 12°C, finally attaining more than 13-times the pre-chilling value. The changing capacity for lipolysis may explain the key role of microsomes in the acclimation of Dunaliella to low temperature.