Botulinum ADP-ribosyltransferase activity as affected by detergents and phospholipids

GTP-binding proteins with Mr values of 22,000 and 25,000 in bovine brain cytosol were ADP-ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3-catalyzed ADP-ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3-catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP-ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADP-ribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other.     

Phosphorus NMR analysis of phospholipids in detergents.

Various detergents can be used to dissolve phospholipids, resulting in very narrow 31PNMR resonances. The resonances are well resolved, allowing identification and quantitative analysis of phospholipids in a mixture. The chemical shift depends strongly on pH, reflecting changes in the state of ionization of the phospholipid headgroup moieties. Samples of phospholipids dissolved in aqueous detergents are conveniently prepared and give narrower 31P resonances than do phospholipids dissolved in organic solvents.     

Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents

Treatment of isolated human erythrocyte membranes with Triton X-100 at ionic strength ?0.04 preferentially released all the glycerolipid and glycoprotein species. At low ionic strength, certain nonglycosylated polypeptides were also selectively solubilized. The liberated polypeptides were free of lipids, but some behaved as if associated into specific oligomeric complexes. Each detergent-insoluble ghost residue appeared by electron microscopy to be a filamentous reticulum with adherent lipoid sheets and vesicles. The residues contained most of the membrane sphingolipids and the nonglycosylated proteins. The polypeptide elution profile obtained with nonionic detergents is therefore nearly reciprocal to that previously seen with a variety of agents which perturb proteins. These data afford further evidence that the externally-oriented glycoproteins penetrate the membrane core where they are anchored hydrophobically, whereas the nonglycosylated polypeptides are, in general, bound by polar associations at the inner membrane surface. The filamentous meshwork of inner surface polypeptides may constitute a discrete, self-associated continuum which provides rather than derives structural support from the membrance.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Reactivation of phospholipase A-inactivated enzyme by phospholipids and detergents

Specific degradation of the phospholipid membrane of guinea-pig liver microsomal fraction with phospholipase A inactivated glucuronyltransferase. The inactivation was reversed by phosphatidylcholine and mixed microsomal phospholipid micelles at concentrations similar to those present in intact microsomal preparations. The other commonly occurring phospholipids did not reactivate phospholipase A-treated enzyme. Since the mixed microsomal phospholipids consisted mainly of phosphatidylcholine, it is concluded that the reactivation by phospholipids is phosphatidylcholine-specific. Reactivation was also achieved by low concentrations of the cationic detergents cetylpyridinium chloride and cetyltrimethylammonium bromide. Higher concentrations of these detergents inactivated the glucuronyltransferase activity of intact and phospholipase A-treated microsomal fractions. Anionic detergents were potent inactivators of the glucuronyltransferase activity of untreated and phospholipase A-treated microsomal fractions, whereas non-ionic detergents had little effect on the activity of either preparation. Measurements of the zeta-potentials of the micellar species used in this study showed that no obvious relationship existed between the zeta-potentials and the ability to reactivate glucuronyltransferase. However, high positive or negative zeta-potentials were correlated with the ability of the amphipathic compound to inactivate glucuronyltransferase.     

Oxidized phospholipids as potential novel drug targets

The interactions of three therapeutic agents, viz. the antipsychotics HPD and CPZ, and the antineoplastic anthracycline DOX, with oxidatively modified phospholipids were studied by monitoring the quenching of fluorescence of an incorporated pyrene-labeled lipid derivative. All three drugs bound avidly to the two oxidized PCs bearing either an aldehyde or carboxylic function at the end of the sn-2 nonanoyl chain, with the highest affinity measured between CPZ and the latter oxidized lipid. Subsequent dissociation of the above drugs from the oxidized lipids by DNA, acidic phospholipids, and NaCl revealed the binding of these drugs with the aldehyde lipid to be driven by hydrophobicity similarly to their binding to lysophosphatidylcholine, whereas a significant contribution of electrostatics was evident for the lipid with the carboxylic moiety. These results connect to previous experimental data, demonstrating the induction by these drugs of oxidative stress and binding to membrane phospholipids. These issues are elaborated with reference to their clinical use and side effects.     

Anionic detergents as divalent cation ionophores across black lipid membranes

Summary Three ionic detergents commonly used in membrane-bound protein isolation and reconstitution experiments, SDS, cholate, and DOC, are shown to act as divalent cation ionophores when incorporated into black lipid membranes made from either oxidized cholesterol or a mixture of phosphatidylcholine and cholesterol (PC/cholesterol=51 mg). At a concentration greater than or equal to 1 m, SDS shows large selectivity differences between cations and anions and among the different cations tested (Ba²⁺, Ca²⁺, Sr²⁺, Mg²⁺, and Mn²⁺). Deoxycholate and cholate at concentrations greater than 4×10^–4 m and 10^–3 m, respectively, also act as divalent cation ionophores. The selectivity sequence measured for these two detergents is evidence for a strong ionic interaction between the divalent cation, and the anionic charged groups on the detergent. In the case of cholate, the conductance depends on the third or fourth power of the cholate concentration and shows a linear dependence on CaCl₂ concentration. The conductance for deoxycholate depends on the sixth or seventh power of the DOC concentration and is also linearly dependent on the CaCl₂ concentration. In an oxidized cholesterol black lipid membrane in the presence of 5mm CaCl₂, small concentrations of LaCl₃ (<1 m) inhibit the ionophoric activity of each of the detergents tested. Evidence is presented to show that this inhibitory effect is a nonspecific effect on oxidized cholesterol BLM's, and is not due to a direct effect of La³⁺ on detergent-mediated transport.     

Oxidized phospholipids as modulators of inflammation in atherosclerosis

PURPOSE OF REVIEW: This review will summarize recent evidence demonstrating that biologically active phospholipid oxidation products modulate inflammatory reactions. RECENT FINDINGS: Structural identification of new biologically active oxidized phospholipids and the finding that they can also be formed at inflammatory sites other than the atherosclerotic lesion have expanded the potential role of these compounds in inflammation beyond atherogenesis. Various signaling pathways are induced by oxidized phospholipids, leading to the expression of inflammatory genes by mechanisms that differ from those mediated by the classic inflammatory agonists tumor necrosis factor or lipopolysaccharide. Furthermore, oxidized phospholipids can bind to pattern recognition molecules and thus potently influence inflammation and immune responses during host defense. SUMMARY: During inflammatory processes biologically active lipid oxidation products accumulate that modulate the inflammatory process and may determine the fate and outcome of the body's reaction in acute inflammation during host defense. Oxidized phospholipids may induce and propagate chronic inflammatory processes; however, evidence is accumulating that cells and tissues respond towards these oxidatively formed stress signals also by activation of anti-inflammatory, cytoprotective reactions.     

Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators

In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specificin vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADHferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADHferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group or groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system.     

Oxidized phospholipids as potential molecular targets for antimicrobial peptides

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.     

Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.     

Properties of purified detergent-resistant phospholipase A of Escherichia coli K-12. Inactivation, and protection with detergents and phospholipids.

A crude preparation of membrane-bound phospholipase A (detergent-resistant) in Escherichia coli K-12 cells was found to be quite stable or even apparently activated on incubation at 100 degrees C, but became strikingly thermolabile when it was highly purified and Triton X-100 was removed from the purified enzyme preparation. The rate of inactivation showed a biphasic temperature dependence: inactivation was rapid at 37 degrees C and also above 70 degrees C. Inactivation above 70 degrees C changed the mobility of the enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, but inactivation at 37 degrees C did not affect the electrophoretic mobility. Triton X-100 effectively protected the enzyme against inactivation at 37 degrees C. The concentration required for the protection of the enzyme was more than its critical micelle concentration. Phospholipids, such as phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylcholine, also protected the enzyme against inactivation at 37 degrees C. These results suggest that the binding of hydrophobic compounds stabilizes the enzyme.     

The use of buttermilk as a source of phospholipids

A method for obtaining phospholipids from butter-milk, a by-product of butter making is described. The method consists in coagulating the fat globule membranes with mineral or organic acid, separating the resulted precipitate by centrifugation or filtering, lipid extraction by organic solvents mixture and isolation of phospholipids from the extract by acetone treatment or by column chromatography. Butter-milk, a new source of biological membranes, is characterized by a low cost and accessibility.     

Short-chain dehydrogenases/reductases (SDRs).

Short-chain dehydrogenases/reductases (SDRs) are enzymes of great functional diversity. Even at sequence identities of typically only 15-30%, specific sequence motifs are detectable, reflecting common folding patterns. We have developed a functional assignment scheme based on these motifs and we find five families. Two of these families were known previously and are called 'classical' and 'extended' families, but they are now distinguished at a further level based on coenzyme specificities. This analysis gives seven subfamilies of classical SDRs and three subfamilies of extended SDRs. We find that NADP(H) is the preferred coenzyme among most classical SDRs, while NAD(H) is that preferred among most extended SDRs. Three families are novel entities, denoted 'intermediate', 'divergent' and 'complex', encompassing short-chain alcohol dehydrogenases, enoyl reductases and multifunctional enzymes, respectively. The assignment scheme was applied to the genomes of human, mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae. In the animal genomes, the extended SDRs amount to around one quarter or less of the total number of SDRs, while in the A. thaliana and S. cerevisiae genomes, the extended members constitute about 40% of the SDR forms. The numbers of NAD(H)-dependent and NADP(H)-dependent SDRs are similar in human, mouse and plant, while the proportions of NAD(H)-dependent enzymes are much lower in fruit fly, worm and yeast. We show that, in spite of the great diversity of the SDR superfamily, the primary structure alone can be used for functional assignments and for predictions of coenzyme preference.