Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p-nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p-nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.     

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.     

DNA-Breaking Action of 2-tert-Butyl-p-quinone

Phospholipase B from baker’s yeast (Saccharomyces cerevisiae) was purified by acid treatment of the crude extract, ammonium sulfate fractionation, and column chromatographies on DEAE-Sepharose CL-6B, Sepharose 4B, and Bio-Gel HTP. The purified preparation had lysophospholipase activity and phospholipase B activity in a ratio of 16:1. The optimum pH of both activities was 3.5 ~ 4.0. The enzyme was a glycoprotein and its molecular size was somewhat heterogeneous, ranged from about 280,000 to 420,000 by gel filtration. Phospholipase B activity was strongly stimulated by 0.1 % DOC, but lysophospholipase activity was completely inhibited by the detergent. Neither activity was stimulated by Ca²⁺ and both were inhibited by SDS, Triton X-100, and Fe³⁺. The enzyme hydrolyzed the acyl ester bonds of phosphatidylcholine sequentially, first the 2-acyl and then the 1-acyl groups. The Km values for phosphatidylcholine and lysophosphatidylcholine were 0.63 mm and 0.05 mm, respectively.     

β-N-Acetylhexosaminidases in the spleen of a patient with hairy-cell leukaemia

The spleen from a patient with hairy-cell leukaemia had β-N-acetylhexosaminidase activity that could be resolved into three isoenzymes by chromatography on phenyl boronate agarose. Two of these were the major forms, A and B, found in normal tissues but, in addition, there was an ‘extra’ form that accounted for 15% of total activity. The ‘extra’ form hydrolysed the synthetic substrate 4-methylumbelliferyl-β-N-acetylglucosamine 6-sulphate, indicating the presence of α-subunits. It was more acidic than A, was less heat-stable and showed no generation of B on denaturation under a variety of conditions. These findings and the immunoblot (Western blotting) analysis demonstrate that the ‘extra’ form is entirely composed of α-subunits, and most closely resembles S, the residual activity in Sandhoff's disease.     

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.     

The hydrolytic and transacylation activity of the phospholipase A1 purified from human post-heparin plasma.

This study demonstrated that the phospholipase A₁ purified from human post-heparin plasma catalyzes the same reactions (hydrolysis and transacylation) and utilizes the same substrates as the phospholipase A₁ obtained by heparin treatment of the plasmalemma of rat liver (Waite, M. and Sisson, P. (1973) $\text{J. Biol. Chem. 248}$, 7985). 1-acylglycerol was the preferred acyl donor and transacylation was the predominate reaction. The results strongly support our earlier conclusions that the phospholipase in plasma originates from the liver and that this enzyme is capable of using a variety of acyl acceptors, including water.     

Partial purification and characterization of phosphatidic acid-specific phospholipase A1 in porcine platelet membranes

We have shown previously that the phospholipase A (PLA) activity specific for phosphatidic acid (PA) in porcine platelet membranes is of the A₁ type (PA-PLA₁) [J. Biol. Chem. 259 (1984) 5083]. In the present study, the PA-PLA₁ was solubilized in Triton X-100 from membranes pre-treated with 1 M NaCl, and purified 280-fold from platelet homogenates by sequential chromatography on blue-Toyopearl, red-Toyopearl, DEAE-Toyopearl, green-agarose, brown-agarose, polylysine-agarose, palmitoyl-CoA-agarose and blue-5PW columns. In the presence of 0.1% Triton X-100 in the assay mixture, the partially purified enzyme hydrolyzed the acyl group from the sn-1 position of PA independently of Ca²⁺ and was highly specific for PA; phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) were poor substrates. The enzyme exhibited lysophospholipase activity for l-acyl-lysoPA at 7% of the activity for PA hydrolysis but no lipase activity was observed for triacylglycerol (TG) and diacylglycerol (DG). At 0.025% Triton X-100, the enzyme exhibited the highest activity, and PA was the best substrate, but PE was also hydrolyzed substantially. The partially purified PA-PLA₁ in porcine platelet membranes was shown to be different from previously purified and cloned phospholipases and lipases by comparing the sensitivities to a reducing agent, a serine-esterase inhibitor, a PLA₂ inhibitor, a Ca²⁺-independent phospholipase A₂ inhibitor, and a DG lipase inhibitor.     

Purification and Partial Characterization of Potato (Solanum tuberosum) Invertase and Its Endogenous Proteinaceous Inhibitor

Invertase plays an important role in the hydrolysis of sucrose in higher plants, especially in the storage organs. In potato (Solanum tuberosum) tubers, and in some other plant tissues, the enzyme seems to be controlled by interaction with an endogenous proteinaceous inhibitor. An acid invertase from potato tubers (variety russet) was purified 1560-fold to electrophoretic homogeneity by consecutive use of concanvalin A-Sepharose 4B affinity chromatography, DEAE-Sephadex A-50-120 chromatography, Sephadex G-150 chromatography, and DEAE-Sephadex A-50-120 chromatography. The enzyme contained 10.9% carbohydrate, had an apparent molecular weight of 60,000 by gel filtration, and was composed of two identical molecular weight subunits (M_r 30,000). The enzyme had a K_m for sucrose of 16 millimolar at pH 4.70 and was most stable and had maximum activity around pH 5. The endogenous inhibitor was purified 610-fold to homogeneity by consecutive treatment at pH 1 to 1.5 at 37°C for 1 hour, (NH₄)₂SO₄ fractionation, Sephadex G-100 chromatography, DEAE-Sephadex G-50-120 chromatography, and hydroxylapatite chromatography. The inhibitor appears to be a single polypeptide (M_r 17,000) without glyco groups. The purified inhibitor was stable over the pH range of 2 to 7 when incubated at 37°C for 1 hour.     

β-Glucosidase from Botrytis cinerea: Its Relation to the Pathogenicity of This Fungus

Cellulolytic enzyme activities in 11 strains of B. cinerea and their pathogenicity against apples, grapes, and lettuce were tested. A positive correlation was found between the β-glucosidase activity of B. cinerea cultured in PYA-medium and the pathogenicity, as expressed in the area of lesions. Further, this correlation was found to hold for the β-glucosidase activity of the apple fruit lesion caused by B. cinerea.

Since it was suggested that pathogenicity of B. cinerea depended on its β-glucosidase activity, the β-glucosidase was purified and characterized. The enzyme had its optimum pH at 3.0, and was very stable in a wide range of pH. These properties were extremely advantageous for B. cinerea to infect the apple fruit.     

Latency of Infection in Unripe Avocados by Colletotrichum gloeosporioides is Not Due to Inadequate Enzyme Potential

Colletotrichum gloeosporioides produced exo-pectin lyase and protease in a) liquid cultures with incorporated washed cell wall material from unripe or ripe avocado and b) autoclaved immature fruit. The activity of exo-pectin lyase and protease produced in liquid cultures incorporating washed cell walls from immature fruits was almost the same as when washed cell walls from ripe fruits were incorporated. Ripe fruit tissue rotted by the fungus contained exo-pectin lyase, endo-polygalacturonase (endo-PG) and protease. The endo-PG was found to be endogenous to avocado fruit, and had a pH optimum of 5.5. The pH optima of exo-pectin lyase and protease were 8.5 and 7.5 respectively in all three enzyme preparations. All these enzyme preparations completely macerated avocado fruit tissue discs in vitro in less than 3 h of incubation but not potato tuber discs. Neither immature nor ripe fruit contained substances, proteinaceous or otherwise, which could inhibit the exo-pectin lyase or protease activity of these preparations. The results indicated that C. gloeosporioides possesses sufficient enzyme potential to invade cell walls of unripe fruit and that the fruit tissue does not have a mechanism to inactivate such enzymes.     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Recent studies of the enzymic synthesis of ricinoleic Acid by developing castor beans

Oleate Δ¹²-hydroxylase activity was measured in extracts of developing castor bean seeds. Most of the hydroxylase activity is associated with microsomes. However, when microsomes are washed, the activity is completely lost. Some (50%) of the activity can be restored by addition of the 100,000g supernatant to the washed microsomes. Supernatant extracts (100,000g) of developing safflower seeds are able to restore all (100%) of the hydroxylase activity to the washed castor bean microsomes. In addition, purified mammalian catalase can restore some (25%) of the activity to the microsomes but is not as effective as either castor bean or safflower 100,000g supernatants. The K_m of the hydroxylase for oxygen is 4 micromolar. Inasmuch as the activity was not inhibited by high concentrations of either carbon monoxide or cyanide, neither the involvement of cytochrome P450 nor other cytochrome systems is suggested. The enzyme system was not saturated by oleoyl-CoA, even at concentrations as high as 200 micromolar. When [¹⁴C]oleoyl-CoA is supplied as a substrate, the acyl component is rapidly transferred to phosphatidylcholine (PC). Hydroxylation may occur on PC or on a lipid which receives its acyl component from PC. However, exogeneously added 2-[1-¹⁴C]oleoyl-PC was hydroxylated at a much lower rate than was [1-¹⁴C]oleoyl-CoA added as the primary substrate.     

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.     

Studies in the Physiology of Parasitism: XIX. On the Killing of Plant Cells by Enzymes from Botrytis cinerea and Bacterium aroideae

1. Enzyme preparations were obtained from culture filtratesof the soft-rot pathogens Botrytis cinerea Pers. and Bacteriumaroideae (Townsend) Stapp grown in simple synthetic nutrientmedia. Crude culture filtrates and preparations purified byacetone-precipitation and dialysis had three characteristicproperties. They (i) decreased viscosity of pectin and pectatesolutions, (ii) macerated parenchymatous tissues of higher plants,and (iii) killed cells of tissues so macerated. A parallelismwas demonstrated between activity estimated by these three criteria. 2. B. cinerea enzyme preparations were active from about pH3.5 to pH 6.0, activity decreasing rapidly from pH 6.o to nearlynil at pH 8.0. Conversely B. aroideae was most active abovepH 8.0, activity decreasing progressively to nearly nil at pH5.5. 3. Both enzymes lost much activity on prolonged dialysis againstdistilled water and this was not recovered on readdition ofdialysed salts. On dialysis against certain salts or salt mixturesreduced or negligible losses occurred. 4. Plasmolysing concentrations of salts or non-electrolytesgreatly retarded the killing action of the enzyme preparations,the effect being out of all proportion to that on macerationor on rate of pectin degradation. 5. Protoplasts were isolated in the plasmolysed condition fromcertain tissues. These were resistant to toxicity in similarmanner to those inside the tissue.     

Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation

Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.     

Role of the carbohydrate moiety of phospholipase B from Penicillium notatum in enzyme activity

Two types of phospholipase B from Penicillium notatum—the native enzyme and enzyme modified by endogenous protease (T. Okumura, S. Kimura, and K. Saito (1980) Biochim. Biophys. Acta, 617, 264–273)—were treated with endoglycosidase H (endo-β-N-acetylglucosaminidase H, Streptomyces griseus) to investigate the orientational change of the sugar chains associated with the lower activity of the modified enzyme. On measurement of release of sugar chains, by periodic acid-Schiff staining of endoglycosidase H-treated phospholipase B on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by direct sugar analysis of the isolated endoglycosidase H-treated phospholipase B, distinct curves were obtained for release of sugar chains from the native and modified enzymes with ultimately loss of about 30 and 55%, respectively, of the carbohydrate. Removal of sugar chains from the two enzymes resulted in similar increases in phospholipase B activity (phosphatidylcholine hydrolysis) and their phospholipase A₁ and A₂ activities in the presence of Triton X-100, but no change of lysophospholipase activity (lysophosphatidylcholine hydrolysis). The three former activities of the native and modified enzymes increased to almost 170 and 350%, respectively, of their initial values. However, little increase in phospholipase B activity was observed when the activity was assayed in the absence of Triton X-100, and none when it was assayed in the presence of sodium taurocholate. These findings suggest that the carbohydrate moiety of phospholipase B greatly influence the phospholipase B activity, especially in the presence of Triton X-100, and that the low phospholipase B activity of the modified enzyme is due to excess exposure of sugar chains on the surface of the molecule as a result of protease attack.     

Interactions between ribulose-1,5-bisphosphate carboxylase and stromal metabolites. II. Corroboration of the role of this enzyme as a metabolite buffer

The capacity of ribulose-1,5-bisphosphate carboxylase to bind reversibly chloroplast metabolites which are the substrates for both thylakoid and stromal enzymes was assessed using spinach chloroplasts and chloroplast extracts and with pure wheat ribulose-1,5-bisphosphate carboxylase. Measurements of the rate of coupled electron flow to methyl viologen in ‘leaky’ chloroplasts (which retained the chloroplast envelope and stromal enzymes but which were permeable to metabolites) and also with broken chloroplasts and washed thylakoids were used to study the effects of binding ADP and inorganic phopshate to ribulose-1,5-bisphosphate carboxylase. The presence of ribulose-1,5-bisphosphate carboxylase significantly altered the values obtained for apparent K_m for inorganic phosphate and ADP of coupled electron transport. The K_m (P_i) in washed thylakoids was 60–80 μM, in ‘leaky’ chloroplasts it was increased to 180–200 μM, while in ‘leaky’ chloroplasts preincubated with KCN and ribulose 1,5-bisphosphate the value was decreased to 40–50 μM. Similarly, the K_m (ADP) of coupled electron transport in washed thylakoids was 60–70 μM, in ‘leaky’ chloroplasts it was 130–150 μM and with ‘leaky’ chloroplasts incubated in the presence of KCN and ribulose 1,5-bisphosphate a value of 45–50 μM was obtained. The ability of ribulose 1,5-bisphosphate carboxylase to reduce the levels of free glycerate 3-phosphate in the absence of ribulose 1,5-bisphosphate was examined using a chloroplast extract system by varying the concentrations of stromal protein or purified ribulose 1,5-bisphosphate carboxylase. The effect of binding glycerate 3-phosphate to ribulose-1,5-bisphosphate carboxylase on glycerate 3-phosphate reduction was to reduce both the rate an the amount of NADPH oxidation for a given amount of glycerate 3-phosphate added. The addition of ribulose 1,5-bisphosphate reinitiated NADPH oxidation but ATP or NADPH did not. Incubation of purified ribulose-1,5-bisphosphate carboxylase with carboxyarabinitolbisphosphate completely inhibited the catalytic activity of the enzyme and decreased inhibition of glycerate-3-phosphate reduction. Two binding sites with different affinities for glycerate 3-phosphate were observed with pure ribulose-1,5-bisphosphate carboxylase.     

Regulation by Lipids of Plant Microsomal Enzymes: II. LIPID DEPENDENCE OF THE NADH-CYTOCHROME c REDUCTASE OF POTATO TUBERS

Microsomal membranes from potato tubers were treated with a phospholipase C extracted from Bacillus cereus. A positive correlation could be observed between the hydrolysis of membranous phospholipids and the decrease of the NADH-cytochrome c reductase activity. Addition of total lipid or phospholipid micelles to phospholipase C-treated microsomes partially restored the NADH-cytochrome c reductase activity, thus proving the lipid-dependence of this enzyme.     

Studies in the Physiology of Parasitism: XVI. Effect of the Condition of Potato Tissue, as modified by Temperature and Water-content, upon Attack by Certain Organisms and their Pectinase Enzymes

1.Certain bacteria which are normally termed saprophytic, viz.Bacillus subtilis and B. megatherium, are able to parasitizeliving potato tissue at a suitably high temperature or whenthe tissue is injected with water. 2.Within the group of four bacteria tested, there is a correlationbetween capacity to attack potato tissue and amount of pectinaseenzyme excreted under standard conditions. 3.A qualitative difference between the pectinase enzymes ofBotrytis cinerea and Bacterium carotovorum has been demonstrated.Preparations of the bacterial enzyme, which when tested on turgidpotato discs of standard thickness were found to be weaker thanBotrytis enzyme, were able to attack normal (subturgid) potatotissue, whereas the Botrytis enzyme failed to do so. No explanationof this difference is yet forthcoming. It does not seem to restupon osmotic differences between the two enzymic preparations. 4.Rate of diffusion appears .to be a limiting factor in theattack of potato tissue by preparations of pectinase enzyme.