Purification and characterization of a fatty acyl-ester hydrolase from post-germinated sunflower seeds.

Fatty acyl-ester hydrolase was not detectable in dry sunflower seeds using various p-nitrophenyl-acyl-esters, 1,2-O-didodecyl-rac-glycero-3-glutaric acid-resorufin ester or emulsified sunflower oil as substrate. After inhibition of the seeds, acyl-ester hydrolase activity slowly developed in cotyledon extracts and was maximal after 5 days. No activity was directly measurable on oil bodies. The enzyme was purified 615-fold to apparent homogeneity, as determined by performing SDS-PAGE electrophoresis, and biochemically characterized. With p-nitrophenyl-caprylate the optimum pH was around 8.0. The purification procedure involved an acetone powder from 5-day dark-germinated seedlings, chloroform-butanol extraction and three chromatography steps. We obtained 35 micrograms of pure enzyme from 250 g of fresh cotyledons with an activity yield of around 7%. It should be possible to subsequently improve this low recovery as we gave priority here, in the first instance, to purity at the expense of the yield. The enzyme consisted of one glycosylated polypeptide chain with a molecular mass of approx. 45 kDa and, as far as we could tell, it did not seem to require metal ions to be fully active, as it was not inhibited by EDTA or o-phenanthroline and not activated by various mono or bivalent metal ions. The amino acid composition showed the presence of four cysteine and four tryptophan residues. The enzyme was partially inhibited by dithiothreitol, DTNB and PCMB. The fact that high inhibition was observed in the presence of PMSF indicates that a serine residue may possibly be involved in the catalytic mechanism. The hydrophobicity index was about 53.6% which places this enzyme in the class of the soluble proteins in good agreement with the fact that it was mainly present in the soluble part of the crude extract. Partial characterization of glycan chains, using antiglycan antibodies, showed the presence of complex Asn-linked glycans. The enzyme was active on purified sunflower glycerol derivatives. It was also able to hydrolyze monooleyl and dioleyl glycerols, as well as phosphatidylcholine, but not cholesteryl esters. Using p-nitrophenyl-acyl-esters as substrate, the highest activity was observed with middle-chain derivatives (C6 and C8). Its maximum activity was about 0.015 units mg-1 with sunflower oil. It was not activated by an organic solvent such as isooctane. This enzyme probably is involved in acyl-ester hydrolysis which follows triacylglycerol mobilization by true lipases.

Phospholipase D from soybean (Glycine max L.) suspension-cultured cells: purification, structural and enzymatic properties.

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from soybean (Glycine max L.) suspension-cultured cell was purified around 1,200-fold to homogeneity by acetone precipitation, Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4B affinity chromatography. The purified enzyme released 1,600 mumol of choline per min per mg of protein. The enzyme is monomeric with a molecular mass of 92 kDa, as estimated by SDS-PAGE. One of the most interesting characteristics of the purified soybean phospholipase D was the dependence of the pH optimum on the Ca2+ ion concentration in the assay. With 10 mM, 20 mM and 40 mM Ca2+ ions, the optima were at pH 7.5, 6 and 5.5, respectively. The specific adsorption of phospholipase D onto octyl-Sepharose gel suggests that the molecule becomes more hydrophobic in the presence of Ca2+ ions. The amino acid sequence of the first 18 N-terminal residues of soybean phospholipase D revealed a high degree of homology with those previously published for cabbage leaf and castor bean endosperm enzymes. Western blots of the soybean phospholipase D showed an immunoreactivity with antibodies raised against a synthetic peptide corresponding to the 15 N-terminal amino acid residues of phospholipase D from cabbage leaves.

Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast.

The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol-transfer protein (Sec14p(sc)) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro. Genetic and biochemical studies conducted in S. cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so-called Kennedy pathway only. This inhibition is controlled by the binding of phospholipids to the Sec14p(sc) protein. Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14(ts) mutant of S. cerevisiae. This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14(ts) mutant at the restrictive temperature of 37 degrees C. Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion. In addition, the phosphatidylinositol-transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14(ts) mutant. The best sequence similarity at the amino acid level is found with the Sec14p protein of S. cerevisiae (36.5% similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product. Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded. This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis.

Crystallization and preliminary crystallographic study of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp).

The plant phospholipase D (PLD) is considered to be a key enzyme involved in various physiological processes such as signal transduction and membrane metabolism. Crystals of the PLD protein from Vigna unguiculata have been produced from the recombinant 768 amino-acid protein. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 157.7, b = 65.6, c = 90.2 A, beta = 111.5 degrees. There is one molecule in the asymmetric unit. Frozen crystals diffract to at least 1.94 A resolution using synchrotron radiation. A search for heavy-atom derivatives using ytterbium and tungstate is currently under way in order to solve the three-dimensional structure.

Use of the tape stripping technique for directly quantifying esterase activities in human stratum corneum.

The enzymes secreted in the intercellular spaces of stratum corneum (SC), the outermost layer of the epidermis, are thought to be involved in normal desquamation and skin barrier function. Their activity can barely be measured due to the difficulty in isolating enough biological material. Human SC layers were obtained from the forearm of healthy volunteers by the tape stripping technique. Assays for esterase activities were carried out in specially designed plates which contained the SC blotted on tape strips, using various fluorescent methylumbelliferone acyl esters as substrates. Triacylglycerol hydrolase activities were also studied by this method. By using radiolabeled triolein and fluorescent 4-methylumbelliferyl 7-oleate as substrates, true lipase activities could be detected and quantitated in SC at pH 5.5 and 7.5. These activities were shown to be strongly inhibited by tetrahydrolipstatin while this was not the case with 4-methylumbelliferyl 7-heptanoate. The method described here combines the painless tape stripping technique with a sensitive plate assay analysis. Since the whole process needs little manipulation, this method can permit rapid quantitation of multiple enzyme activities from a single strip. Therefore, it will permit the study of the involvement of enzyme activities in epidermis aging and skin pathologies.