Several properties of cotton seed phospholipase D]

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

Molecular and biochemical properties and physiological roles of plant phospholipase D

Recent advances have thrust the study of plant phospholipase D (PLD) into the molecular era. This review will highlight some of the recent progress made in elucidating the molecular and biochemical nature of plant PLDs as well as their roles in plant physiology.     

Molecular and biochemical properties and physiological roles of plant phospholipase D.

Recent advances have thrust the study of plant phospholipase D (PLD) into the molecular era. This review will highlight some of the recent progress made in elucidating the molecular and biochemical nature of plant PLDs as well as their roles in plant physiology.     

Immobilized phospholipase D]

Conditions of phospholipase D adsorption on silica gels have been studied. The immobilized phospholipase D is shown to differ from the soluble form in thermostability, pH optima and activation conditions. A question is discussed as to the connection of the use of activators and the adsorption immobilization. It is assumed that phospholipase D belongs to enzymes, functioning only in the immobilized state.     

Identification of caspase 3 motifs and critical aspartate residues in human phospholipase D1b and phospholipase D2a

Stimulation of mammalian cells frequently initiates phospholipase D-catalyzed hydrolysis of phosphatidylcholine in the plasma membrane to yield phosphatidic acid (PA) a novel lipid messenger. PA plays a regulatory role in important cellular processes such as secretion, cellular shape change, and movement. A number of studies have highlighted that PLD-based signaling also plays a pro-mitogenic and pro-survival role in cells and therefore anti-apoptotic. We show that human PLD1b and PLD2a contain functional caspase 3 cleavage sites and identify the critical aspartate residues within PLD1b that affect its activation by phorbol esters and attenuate phosphatidylcholine hydrolysis during apoptosis.     

Isolation and some properties of phospholipase D from Bacillus subtilis G-22]

A method of isolating highly purified phospholipase D from Bac. subtilis G-22 is described. It includes ammonium sulphate fractionation, thermal denaturation, chromatography on lipoprotein bound with sepharose 6B and AH-sepharose 4B. The enzyme is 130-fold purified, its yield exceeds 90.0%, its specific activity is 164 units per mg of protein. The homogeneity of the enzyme is demonstrated by polyacrylamide gel electrophoresis, ultracentrifugation, isoelectric focusing and N-terminal amino acid determination by means of dinitrophenylation and dancylation. Proline is found to be N-terminal amino acid. The molecular weight of the enzyme, as determined from gel filtration through Sephadex G-100, is 21500 +/- 300, its sedimentation constant is 1.4S, isoelectric point is at pH 4.2. The molecular weight calculated from amino acid composition, is 21000--22000. Polypeptide chain contains of 196--205 amino acid residues. Phospholipase D develops its maximal activity at pH 8.5 and does not contain free SH-groups. Benzylsulphofluoride does not inhibit the enzyme activity. Phospholipase D is activated by Cd2+, Co2+, Zn2+, Ca2+ and is inhibited by EDTA, pIi50 being about 2.6.     

Identification of phospholipase D from cabbage as N-terminally acetylated PLD2

Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli [Schäffner, I., Rücknagel, K.-P., Mansfeld, J., and Ulbrich-Hofmann, R. (2002). Eur. J. Lipid Sci. Technol. 104: 79–87]. Both enzymes are highly homologous (91% identity) and behave very similarly. Phospholipase D purified from white cabbage leaves (PLD_cab) is compared with the two recombinant enzymes in sodium dodecylsulfate and native polyacrylamide gel electrophoresis, isoelectric focusing, N-terminal sequencing, and mass spectrometry after tryptic digestion. As a result, PLD_cab clearly can be assigned to PLD2. In contrast to recombinant PLD2, however, PLD_cab is N-terminally acetylated.     

Identification of Ca2+-stimulated polyphosphoinositide phospholipase C in isolated plant plasma membranes

A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings. The enzyme preferentially hydrolysed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate and had a different phosphoinositide substrate profile from soluble phospholipase C. The enzyme activity was lower in plasma membranes isolated from light-grown shoots than from dark-grown ones, whereas no differences in activity between plasma membranes from light- and dark-grown roots were seen. Maximum activity of the membrane-bound enzyme was observed around pH 6. It was activated by micromolar concentrations of Ca2+, but not by GTP or GTP analogues. The enzyme may participate in signal transduction over the plant plasma membrane.     

Insights into the structure of plant alpha-type phospholipase D

Phospholipases D play an important role in the regulation of cellular processes in plants and mammals. Moreover, they are an essential tool in the synthesis of phospholipids and phospholipid analogs. Knowledge of phospholipase D structures, however, is widely restricted to sequence data. The only known tertiary structure of a microbial phospholipase D cannot be generalized to eukaryotic phospholipases D. In this study, the isoenzyme form of phospholipase D from white cabbage (PLDalpha2), which is the most widely used plant phospholipase D in biocatalytic applications, has been characterized by small-angle X-ray scattering, UV-absorption, CD and fluorescence spectroscopy to yield the first insights into its secondary and tertiary structure. The structural model derived from small-angle X-ray scattering measurements reveals a barrel-shaped monomer with loosely structured tops. The far-UV CD-spectroscopic data indicate the presence of alpha-helical as well as beta-structural elements, with the latter being dominant. The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein. From the near-UV CD signals and activity data as a function of the calcium ion concentration, two binding events characterized by dissociation constants in the ranges of 0.1 mm and 10-20 mm can be confirmed. The stability of PLDalpha2 proved to be substantially reduced in the presence of calcium ions, with salt-induced aggregation being the main reason for irreversible inactivation.     

Substrate-specific forms of human platelet phospholipase A2

Purification of human platelet phospholipase A2 (PLA2) from a particulate fraction by ion-exchange chromatography at 4 degrees C yielded a single peak of enzyme activity, which catalyzed the hydrolysis of arachidonic acid from the 2-position of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). The activity toward PtdCho and that toward PtdEtn differed in stability during storage, pH optimum, Ca2+ requirement, and affinity for the substrate; however, each activity preferred phospholipid with arachidonate at the 2-position. The two activities appeared to be eluted as an aggregate in a single peak from the ion-exchange column. When the column was run at 22 degrees C, an additional PLA2 activity peak specific for PtdEtn was resolved from the original PLA2 peak. But when the particulate fraction was briefly sonicated in 0.1% octylglucoside before chromatography at 22 degrees C, a different PLA2 activity peak, specific for PtdCho, was obtained. Resolution of the two specific forms of PLA2 under different conditions probably resulted from selective solubilization of the aggregate. The specific PLA2 activities thus isolated were very labile, whereas those in the aggregate were relatively stable. These findings suggest that human platelets contain at least two substrate-specific forms of PLA2, one for PtdCho and another for PtdEtn.     

Distinct Ca2+ binding properties of novel C2 domains of plant phospholipase dalpha and beta

Of the isoforms of plant phospholipase D (PLD) that have been cloned and characterized, PLDalpha requires millimolar levels of Ca(2+) for optimal activity, whereas PLDbeta is most active at micromolar concentrations of Ca(2+). Multiple amino acid sequence alignments suggest that PLDalpha and PLDbeta both contain a Ca(2+)-dependent phospholipid-binding C2 domain near their N termini. In the present study, we expressed and characterized the putative C2 domains of PLDalpha and PLDbeta, designated PLDalpha C2 and PLDbeta C2, by CD spectroscopy, isothermal titration calorimetry, and phospholipid binding assay. Both PLD C2 domains displayed CD spectra consistent with anticipated major beta-sheet structures but underwent spectral changes upon binding Ca(2+); the magnitude was larger for PLDbeta C2. These conformational changes, not shown by any of the previously characterized C2 domains of animal origin, occurred at micromolar Ca(2+) concentrations for PLDbeta C2 but at millimolar levels of the cation for PLDalpha C2. PLDbeta C2 exhibited three Ca(2+)-binding sites: one with a dissociation constant (K(d)) of 0.8 microm and the other two with a K(d) of 24 micrometer. In contrast, isothermal titration calorimetry data of PLDalpha C2 were consistent with 1-3 low affinity Ca(2+)-binding sites with K(d) in the range of 590-470 micrometer. The thermodynamics of Ca(2+) binding markedly differed for the two C2 domains. Likewise, PLDbeta C2 bound phosphatidylcholine (PC), the substrate of PLD, in the presence of submillimolar Ca(2+) concentrations, whereas PLDalpha C2 did so only in the presence of millimolar levels of the metal ion. Both C2 domains bound phosphatidylinoistol 4,5-bisphosphate, a regulator of PC hydrolysis by PLD. However, added Ca(2+) displaced the bound phosphatidylinoistol 4,5-bisphosphate. Ca(2+) and PC binding properties of PLDalpha C2 and PLDbeta C2 follow a trend similar to the Ca(2+) requirements of the whole enzymes, PLDalpha and PLDbeta, for PC hydrolysis. Taken together, the results suggest that the C2 domains of PLDalpha and PLDbeta have novel structural features and serve as handles by which Ca(2+) differentially regulates the activities of the isoforms.     

Isolation of phospholipase D from cotton seeds]

A method of isolating homogenous phospholipase D from cotton seeds is described. The homogeneity of the enzyme preparation is demonstrated by means of polyacrylamide disc electrophoresis, gel filtration through Sephadex G-200 and determination of N-terminal amino acid residue, which turned to be glutamic acid. Molecular weight of the enzyme is found to be 71000 +/- 3000. Several equilibrium forms of the enzyme are observed.     

1,3-Diacylglycero-2-phosphocholines--synthesis, aggregation behaviour and properties as inhibitors of phospholipase D

A series of 1,3-diacylglycero-2-phosphocholines (1,3-PCs) with acyl chain lengths of C8-C18 were synthesised by chemical introduction of the phosphocholine moiety into the regioisomerically pure 1,3-diacylglycerols, which were obtained from glycerol and the vinyl esters of fatty acid by means of lipase from Rhizomucor mihei. The 1,3-PCs being regioisomers of the natural glycerophospholipids were studied with respect to their aggregation behaviour in the absence and in the presence of sodium dodecylsulfate (SDS) as well as their properties as substrates and inhibitors of phospholipase D (PLD) from cabbage. While the main structures of the pure 1,3-PCs were micelles (C8), liposomes (C10, C12) or planar bilayers (C14, C16, C18), the addition of SDS resulted in the formation of mixed micelles (C8, C10) and mixed liposomes (C12, C14, C16, C18). None of the 1,3-PCs was found to be hydrolysed by PLD, whereas all of them showed inhibitory properties in the standard assay for PLD. The inhibitory power was strongest with 1,3-didecanoylglycero-2-phosphocholine (IC50 = 43 microM).     

Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions

Multiple Phospholipase D (PLD) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of the genetic and biochemical heterogeneity has provided important clues as to the regulation and function of this family of enzymes. Polyphosphoinositides, Ca(2+), and G-proteins are possible cellular regulators for PLD activation. PLD-mediated hydrolysis of membrane lipids increases in response to various stresses. Recent studies suggest that PLD plays a role in the signaling and production of hormones involved in plant stress responses.     

Multiple forms of secretory phospholipase A2 in plants

Multiple secretory phospholipase A2 (sPLA2) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of this genetic and biochemical heterogeneity has provided important clues to the regulation and function of the individual enzymes. An increasing body of evidence shows that their lipid products, lysophospholipids and free fatty acids, mediate a variety of cellular responses, including plant growth, development, and responses to stress and defense. This review discusses the newly-acquired information on plant sPLA2s including the molecular and biochemical characteristics, and signaling functions of each isoform.     

Substrate specificity in phospholipid transformations by plant phospholipase D isoenzymes

Phospholipase D (PLD) catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond. In many plants, several isoforms of PLD have been identified without knowing their functional differences. In this paper, the specificities of two PLD isoenzymes from white cabbage (Brassica oleracea var. capitata) and two ones from opium poppy (Papaver somniferum L.), which were recombinantly produced in Escherichia coli, were compared in the hydrolysis of phospholipids with different head groups and in the transphosphatidylation of phosphatiylcholine with several acceptor alcohols. In a biphasic reaction system, consisting of buffer and diethyl ether, the highly homologous isoenzymes are able to hydrolyze phosphatidylcholine, -glycerol, -ethanolamine, -inositol and - with one exception - also phosphatidylserine but with different individual reaction rates. In transphosphatidylation of phosphatidylcholine, they show significant differences in the rates of head group exchange but with the same trend in the preference of acceptor alcohols (ethanolamine > glycerol ? l-serine). For l- and d-serine a stereoselectivity of PLD was observed. The results suggest a physiological relevance of the different hydrolytic and transphosphatidylation activities in plant PLD isoenzymes.     

Identification of a new phospholipase D in Carica papaya latex

Phospholipase D (PLD) is a lipolytic enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. It catalyzes the hydrolysis and transphosphatidylation of glycerophospholipids at the terminal phosphodiester bond. The presence of a PLD in the latex of Carica papaya (CpPLD1) was demonstrated by transphosphatidylation of phosphatidylcholine (PtdCho) in the presence of 2% ethanol. Although the protein could not be purified to homogeneity due to its presence in high molecular mass aggregates, a protein band was separated by SDS-PAGE after SDS/chloroform-methanol/TCA-acetone extraction of the latex insoluble fraction. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by micro-LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (723 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 2424 bp encoding a protein of 808 amino acid residues, with a theoretical molecular mass of 92.05 kDa. From sequence analysis, CpPLD1 was identified as a PLD belonging to the plant phosphatidylcholine phosphatidohydrolase family.