First isolation of an antifungal lipid transfer peptide from seeds of a Brassica species

An antifungal peptide with a molecular mass of 9412 and an N-terminal sequence exhibiting notable homology to those of lipid transfer proteins was isolated from seeds of the vegetable Brassica campestris. The purification protocol entailed ion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on a Superdex peptide column. The antifungal peptide was adsorbed on Affi-gel blue gel and Mono S. It inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola with an IC(50) value of 8.3 microM and 4.5 microM, respectively. It exhibited dose-dependent binding to lyso-alpha-lauroyl phosphatidylcholine. The present findings constitute the first report on a non-specific lipid transfer protein from the seeds of a Brassica species.     

Effects of stilbene derivatives on arachidonate metabolism in leukocytes

The effects of various alpha-phenylcinnamic acid derivatives (i.e., alpha-(3,4-dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid, alpha-(3,4-dihydroxyphenyl)-4-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3, 4-dihydroxycinnamic acid) synthesized from 3,4-dihydroxyphenyl acetic acid and hydroxy-benzaldehyde, and 3,3',4-trihydroxystilbene obtained by decarboxylation of alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase activities were studied. 3,3',4-Trihydroxystilbene was found to inhibit the 5-lipoxygenase product, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HETE), and cyclooxygenase products, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2; its concentrations for 50% inhibition (IC50) were 0.885 +/- 0.016 microM for the leukocyte lipoxygenase product, 5-HETE, 7.70 +/- 0.104 microM for the formations of HHT and 7.96 +/- 0.143 microM for the formation of thromboxane B2. Alpha-(3,4-Dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3,4-dihydroxycinnamic acid also inhibited the formations of 5-HETE, HHT and thromboxane B2, although less strongly. Their IC50 values were, respectively, 91.3 +/- 3.62 microM, 947.5 +/- 28.7 microM, 453.3 +/- 229.3 microM and 148.8 +/- 50.6 microM for the formation of 5-HETE, 894.0 +/- 5.57 microM, 792.5 +/- 15.9 microM, greater than 1000 microM and 925.0 +/- 7.64 microM for the formation of HHT and 941.0 +/- 18.0 microM, 825 +/- 14.4 microM, greater than 1000 microM and 932.7 +/- 3.93 microM for the formation of thromboxane B2.     

Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)(2)SO(4) precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS-PAGE it was found to be a monomeric protein with molecular mass 72+/-2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 microM) and highest specificity constant (V(max)/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 microM and Ki=205 microM, respectively).     

Purification of allivin,a novel antifungal protein from bulbs of the round-cloved garlic

A novel antifungal protein, designated allivin, was isolated from bulbs of the round-cloved garlic Allium sativum var. round clove with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and FPLC-gel filtration on Superdex 75. Allivin possessed an N-terminal sequence demonstrating very little similarity to sequences of Allium sativum chitinases and ribosome inactivating proteins. Allivin exhibited a molecular weight of 13 kDa in gel filtration and SDS-polyacrylamide gel electrophoresis. It displayed antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola and Physalospora piricola. It inhibited translation in a cell-free rabbit reticulocyte system with an IC50 of 1.6 microM.     

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.     

Alpha-adrenergic inhibition of renal cortical adenylate cyclase

Adenylate cyclase in homogenates of rat renal cortex was inhibited by alpha-adrenergic agonists. Inhibition required sodium ion and GTP. A maximum inhibition of 17.8 +/- 1.4% (S.E.M.) was produced by l-epinephrine in the presence of 0.2 M NaCl, 10 microM GTP and 10 microM propranolol. Similar inhibition was produced by l-norepinephrine and alpha-methylnorepinephrine. The EC50 values for l-epinephrine, l-norepinephrine and alpha-methylnorepinephrine were respectively 1.9 +/- 0.7 microM, 2.3 +/- 1.6 microM and 5.1 +/- 1.8 microM. Clonidine was a partial agonist causing 50% as much inhibition as epinephrine. Phenylephrine and methoxamine did not inhibit at concentrations up to 100 microM. Micromolar concentrations of phentolamine and yohimbine prevented the inhibition of adenylate cyclase by epinephrine. However, prazosin was ineffective. Thus the adenylate cyclase coupled alpha-receptors have alpha-2 specificity. Inhibition of adenylate cyclase by alpha-adrenergic agonists was not observed in homogenates of renal medulla.     

Exchange of fluorinated glucose across the red-cell membrane measured by 19F-n.m.r. magnetization transfer.

The 19F n.m.r. spectrum of 3-fluoro-3-deoxy-D-glucose (3FG) in a red-cell suspension was observed to contain separate resonances from the intra- and extra-cellular populations of both the alpha- and beta-anomers. This phenomenon was used with an n.m.r. spin-transfer procedure to measure the rate of exchange of the anomers across the human red-cell membrane under equilibrium-exchange conditions at 37 degrees C. The beta-anomer crossed the membrane significantly more quickly than the alpha-anomer. At a total 3FG concentration of 9.3 mM; the first-order rate constants for the efflux of the alpha- and beta-anomers were 0.41 +/- 0.15 and 0.88 +/- 0.20 s-1 respectively. The measurable 3FG exchange was inhibited by 75 and 100% respectively by the glucose-transport inhibitors cytochalasin B and phloretin. Glucose inhibited the exchange of 3FG, and the results were consistent with glucose and 3FG binding to the hexose-transport protein with similar affinity.     

Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells

Concomitant Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activation occurs during stimulation of acid secretion in cultured rabbit parietal cells, possibly related to a necessity for volume regulation during the secretory process. We investigated whether cytoplasmic volume changes occur during secretagogue stimulation of cultured rabbit parietal cells. Cells were loaded with the fluorescent dye calcein, and the calcein concentration within a defined cytoplasmic volume was recorded by confocal microscopy. Forskolin at 10(-5) M, carbachol at 10(-4) M, and hyperosmolarity (400 mosmol) resulted in a rapid increase in the cytoplasmic dye concentration by 21 +/- 6, 9 +/- 4, and 23 +/- 5%, respectively, indicative of cell shrinkage, followed by recovery to baseline within several minutes, indicative of regulatory volume increase (RVI). Depolarization by 5 mM barium resulted in a decrease of the cytoplasmic dye concentration by 10 +/- 2%, indicative of cell swelling, with recovery within 15 min, and completely prevented forskolin- or carbachol-induced cytoplasmic shrinkage. Na(+)/H(+) exchange inhibitors slightly reduced the initial cell shrinkage and significantly slowed the RVI, whereas 100 microM bumetanide had no significant effect on either parameter. We conclude that acid secretagoguges induce a rapid loss of parietal cell cytoplasmic volume, followed by RVI, which is predominantly mediated by Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange.