STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells.

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

Purification, primary structure, and properties of Euphorbia characias latex purple acid phosphatase.

A purple acid phosphatase was purified to homogeneity from Euphorbia characias latex. The native protein has a molecular mass of 130 ± 10 kDa and is formed by two apparently identical subunits, each containing one Fe(III) and one Zn(II) ion. The two subunits are connected by a disulfide bridge. The enzyme has an absorbance maximum at 540 nm, conferring a characteristic purple color due to a charge-transfer transition caused by a tyrosine residue (Tyr172) coordinated to the ferric ion. The cDNA nucleotide sequence contains an open reading frame of 1392 bp, and the deduced sequence of 463 amino acids shares a very high degree of identity (92-99%) to other purple acid phosphatases isolated from several higher plants. The enzyme hydrolyzes well p-nitrophenyl phosphate, a typical artificial substrate, and a broad range of natural phosphorylated substrates, such as ATP, ADP, glucose-6-phosphate, and phosphoenolpyruvate. The enzyme displays a pH optimum of 5.75 and is inhibited by molybdate, vanadate, and Zn2+, which are typical acid phosphatase inhibitors.

A compact STED microscope providing 3D nanoscale resolution.

The advent of supercontinuum laser sources has enabled the implementation of compact and tunable stimulated emission depletion fluorescence microscopes for imaging far below the diffraction barrier. Here we report on an enhanced version of this approach displaying an all-physics based resolution down to (19 +/- 3) nm in the focal plane. Alternatively, this single objective lens system can be configured for 3D imaging with resolution down to 45 x 45 x 108 nm in a cell. The obtained results can be further improved by mathematical restoration algorithms. The far-field optical nanoscale resolution is attained in a variety of biological samples featuring strong variations in the local density of features.

Activity and structural changes of Euphorbia characias peroxidase in the presence of trifluoroethanol.

Activity assays, conformational changes and transitional switches between secondary structures of a peroxidase from Euphorbia characias were studied in the presence of trifluoroethanol and in the presence or absence of calcium ions. The addition of trifluoroethanol up to 10-20% first induced a drastic decrease of alpha-helix content followed by an increase of tryptophan fluorescence emission intensity, a progressive re-induction of the formation of alpha-helical elements concomitant with loss of enzyme activity. In the presence of calcium ions, the fluorescence of the enzyme almost remained unchanged in the trifluoroethanol concentration range 5-20%. Further increase in trifluoroethanol concentration led to a protein structure characterized by a progressive re-induction of alpha-helical elements, a remarkable increase of the tryptophan fluorescence and a loss of enzyme activity. These results indicate that calcium ions in Euphorbia peroxidase play an essential role in maintaining the hydrophobic interactions on the protein structure preserving enzymatic activity.

CuI-semiquinone radical species in plant copper-amine oxidases.

The intermediate CuI-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

The physiopathological significance of ceruloplasmin. A possible therapeutic approach.

This article reviews and comments on the physiological roles of ceruloplasmin (Cp). We show that, in addition to its ascertained involvement in iron homeostasis, the protein, by virtue of its unique structure among multicopper oxidases, is likely involved in other processes of both an enzymatic and a nonenzymatic nature. In particular, based on the analysis of the kinetic parameters, on the one hand, and of the side-products of the oxidation, on the other, we propose that the long-recognized ability of Cp to interact with and oxidize non-iron substrates may be of physiological relevance. The striking example of 6-hydroxydopamine oxidation is presented, where we show that the catalytic action is carried out readily under physiological conditions, without release of potentially toxic oxygen intermediates.

Modulation of 6-hydroxydopamine oxidation by various proteins.

The spontaneous autoxidation of the neurotoxin 6-hydroxydopamine proceeds by a free radical chain reaction involving the superoxide anion radical and produces the corresponding chromogen 6-hydroxydopamine quinone and hydrogen peroxide. The rate of this reaction is increased in the presence of ceruloplasmin and peroxidase, and reduced by superoxide dismutase, catalase, and DT-diaphorase. We report some explanations of why these proteins may increase or reduce the rate of autoxidation of 6-hydroxydopamine.

On the use of 2,4,5-trihydroxyphenethylamine as a rapid method for laccase quantification.

The sensitivity of the autoxidation of 2,4,5-trihydroxyphenethylamine (6-hydroxydopamine) to increase by laccase was studied. The autoxidation of 6-hydroxydopamine proceeds by a free radical chain reaction involving O2- and produces the corresponding chromogen 6-hydroxydopaminequinone and hydrogen peroxide. The ability of laccase to increase the autoxidation of 6-hydroxydopamine at pH 5.5 has been used as sensitive assay for this enzyme. The results are tabulated and discussed.

Peroxidase from Astragalus maritimus: purification and properties.

A peroxidase has been purified to homogeneity from Astragalus maritimus seeds using ammonium sulfate precipitation and chromatography on DEAE-cellulose and hydroxylapatite. The purification obtained was 255 fold. The enzyme preparations were homogenous by the criteria of SDS-PAGE and analytical gel electrofocusing. The protein contained 0.11% of iron that corresponds to a minimum molecular size of 50,700. Determinations of molecular size by SDS-PAGE gave values of 48,000 +/- 1,000 while the one obtained by Sephadex gel filtration was 49,000. The pH optimum of the enzyme was 6.0. The activation energy was estimated to be 6 Kcal/mol. The prosthetic group was shown to be ferriprotoporphyrin IX. The presence of 13% neutral sugars was found. The spectrophotometric analysis showed the presence, in the visible region, of absorption maxima at 403, 490 and 633 nm. The Rz value (A403/A275) was 2.7.

Catalase and antiquitin from Euphorbia characias: two proteins involved in plant defense?

Here we report the cDNA nucleotide sequences of a calmodulin-binding catalase and an antiquitin from the latex of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent additional nodes in the Euphorbia defense systems, and a multi-enzymatic interaction contributing to plant's protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers.

Horse plasma ceruloplasmin molecular weight and subunit analysis.

Ceruloplasmin is a blue copper-containing serum glycoprotein with oxidase activity. It as been proposed that the physiological function of ceruloplasmin involves the oxidation of ferrous iron and its incorporation into apotransferrin. There are several reports demonstrating that ceruloplasmin is made up of multiple chains. Ryden has questioned the multichain structure of ceruloplasmin from human, pig, horse and rabbit sera, arguing that the dissociation observed by previous workers could be attributed to cleavage of labile bands in the protein by enzymatic contaminants present in commercial preparations of the protein. By introducing epsilon-aminocaproic acid, a general protease inhibitor, at the beginning of the enzyme preparation, Ryden proposed a single-chain structure for ceruloplasmin. On the contrary the results presented by Freeman and Daniel showed that human ceruloplasmin is a multichain protein. In this paper we report a new purification method for horse ceruloplasmin which furnishes a homogeneous protein preparation in high yield and with good reproducibility. This procedure allowed to determine with greater accuracy the molecular mass of the protein, of 120,000 daltons by gel chromatography and 115,000 daltons by SDS gel electrophoresis. The protein is composed of one unit only and contains 6 copper atoms. Horse ceruloplasmin is a glycoprotein containing about 20% carbohydrate by weight.

Superoxide Dismutase from Lens esculenta: Purification and Properties.

Superoxide dismutase has been purified to homogeneity from Lens esculenta cotyledons and shoots. The two forms appeared to be identical. The purified enzyme contained two electrophoretically distinct bands. It contained two ions of Cu and two ions of Zn. Gel filtration experiments indicate a molecular weight of about 33,000. The spectrum of ultraviolet and visible regions and electron paramagnetic resonance were similar to those of Cu-Zn mammalian superoxide dismutase.

Lentil seedling amine oxidase: interaction with carbonyl reagents.

Carbonyl reagents containing a primary amino group were covalently bound by lentil seedling amine oxidase to resolve conflicting data for both the number of functional active sites in the dimeric enzyme. Active site titration of highly purified enzyme samples with all the carbonyl reagents extrapolates to 1 mol of inhibitor/mol of enzyme subunit indicating the presence of a cofactor at each enzyme subunit. This result is at variance with numerous previous reports of only one functional cofactor for enzyme dimer in copper amine oxidase.

Evidence for alpha-proton abstraction and carbanion formation involving a functional histidine residue in lentil seedling amine oxidase.

Lentil seedling amine oxidase catalyzes the oxidation of putrescine and in the presence of tetranitromethane gives rise to the formation of nitroform anion. The initial rate of substrate and enzyme-dependent nitroform production is linearly related to the functional active site content and is proportional to the tetranitromethane concentration. Diethylpyrocarbonate modifies two histidyl residues on the lentil amine oxidase. Incubation of the enzyme with diethylpyrocarbonate at 25 degrees C and pH 7.0 irreversibly inhibits enzyme activity by a pseudo first-order kinetics process. The data obtained are consistent with the enzyme-dependent abstraction of an alpha-proton from the substrate to form an intermediate enzyme bound carbanion and indicate a functional role for histidine in lentil amine oxidase catalysis consistent with that of a general base in proton abstraction.

Amine oxidase from Lathyrus cicera and Phaseolus vulgaris: purification and properties.

Cu-Amine oxidases (amine oxygen oxidoreductase deaminating, copper containing E.C. 1.4.3.6.) are found in all forms of life (1). They catalyze the following general reaction: R-CH2-NH2 + O2 + H2O----R-CHO + NH3 + H2O2. Cu-amine oxidases (Cu-AOs) have been extracted from different leguminosae: Pisum sativum (2-3), Lathyrus sativus (4), Lens esculenta (5), Vicia faba (6), Cicer arietinum (7), Glycine max (8) but not from Phaseolus vulgaris. Palavan and Galston (9), in a study of polyamine biosynthesis during developmental stages of Phaseolus vulgaris, did not detect diamine or polyamine oxidase activity in Phaseolus. The present paper describes the purification of Phaseolus vulgaris seedlings amine oxidase (PhSAO) and also compares the properties of this enzyme to the Lathyrus cicera enzyme (LcSAO), obtained with the same method of purification.

Tryptamine as substrate and inhibitor of lentil seedling copper amine oxidase.

Copper amine oxidase from lentil seedlings was shown to be able to catalyze the oxidative deamination of the indoleamines tryptamine, 5-hydroxytryptamine, and 5-methoxytryptamine. These compounds showed saturation kinetics with Km values as normal substrates, but their oxidation led to irreversible loss of enzyme activity suggesting a covalent interaction with the enzyme, most probably through its cofactor 6-hydroxydopa (2,4,5-trihydroxyphenylalanine). These indoleamines acted as irreversible inhibitors of the enzyme only in the absence of oxygen but they brought about changes in the electronic spectra of the enzyme both in aerobiosis and in anaerobiosis. This study reports on the mechanism by which these compounds inhibit lentil amine oxidase which involves first the oxidation of indoleamines bound to 6-hydroxydopa followed by the formation of an irreversible covalent derivative. The same inhibitory mechanism could possibly lead to inactivation of mammalian amine oxidases involved in serotonin neurotransmitter metabolism in conditions of ischemia or hypoxia.

Inhibition of copper amine oxidase by haloamines: a killer product mechanism.

The observation that the alkylamines 2-Br-ethylamine and 2-C1-ethylamine and 1,2-diaminoethane, the shortest diamine, are irreversible inhibitors of several copper amine oxidases led to the investigation of the mechanism by which these compounds react with the highly active amine oxidase from lentil seedlings. 1,2-Diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine were found to be both poor substrates and irreversible inhibitors of lentil amine oxidase; inactivation took place in both the presence and absence of oxygen. All three compounds strongly affected the spectrum of the enzyme, leading to the formation of a stable band at 336 nm both in anaerobiosis and in aerobiosis, consistent with an interaction with the enzyme cofactor 6-hydroxydopa. On the contrary, the corresponding propylamine compounds 1,3-diaminopropane, 3-Br-propylamine, and 3-C1-propylamine were reversible inhibitors of lentil amine oxidase. Inhibition was shown to be due to the aldehyde oxidation products rather than the short chain amines themselves; a reaction mechanism is presented which involves attack of the aldehyde on the 6-hydroxydopa-derived free radical catalytic intermediate. With 1,2-diaminoethane, 2-Br-ethylamine, and 2-C1-ethylamine, the complex produced will form a stable 6-membered ring, causing irreversible inhibition of the enzyme.

Lentil seedlings amine oxidase: preparation and properties of the copper-free enzyme.

The reaction of copper-free lentil seedlings amine oxidase with substrates has been studied. While devoid of catalytic activity, this enzyme preparation is still able to oxidize two moles of substrate and to release two moles of aldehyde and two moles of ammonia per mole of dimeric protein. The same stoichiometry has been determined on the native enzyme in the absence of oxygen. Although copper is essential for the reoxidation of the reduced enzyme, a binding of oxygen to the copper-free protein has been demonstrated.

Substrate specificity of lentil seedling amine oxidase.

Copper diamine oxidase from lentil (Lens culinaris) seedlings was shown to be able to catalyze the oxidative deamination of a wide range of aliphatic and aromatic monoamine compounds, including some amino acids. Although the catalytic efficiencies were only 1-3% of that measured with the diamine substrate putrescine, they were still comparable to those of specialized monoamine oxidases. In particular, the lentil enzyme oxidized benzylamine and histamine with K(m) and Vmax values similar to those found for the mammalian enzymes benzylamine oxidase and histaminase. Cysteamine was found to be a substrate of the enzyme, whereas hypotaurine and taurine were found to be neither substrates nor inhibitors of the enzyme. Quite unexpectedly the amino acids L-ornithine and L-lysine were oxidized by lentil enzyme, and beta-alanine and gamma-aminobutyric acid were oxidized only at high concentrations of enzyme. These results suggest that enzymes normally classified as diamine oxidases could in fact have a more diversified role in metabolism than recognized so far.