Mechanism of Magainin 2a Induced Permeabilization of Phospholipid Vesicles

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)hduced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages-an initial rapid phase, with t1/2 ≈ 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptidelipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a

Mechanism of Magainin 2a Induced Permeabilization of Phospholipid Vesicles

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)hduced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages-an initial rapid phase, with t1/2 ≈ 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly equilibrate between vesicles; this was demonstrated by determining a binding isotherm for the peptidelipid interaction, and by showing that addition of unloaded vesicles rapidly quenches peptide-induced leakage from loaded vesicles. Transient dye release in the presence of an equilibrating peptide can be explained in two ways: (1) the peptide exists only transiently in an active form; (2) the vesicles are only transiently leaky. Preincubation of mgn2a at assay concentrations in buffer alone or with unloaded vesicles did not inactivate the peptide. Therefore, rapid leakage is probably due to transient destabilization of the vesicle upon addition of mgn2a

Raman Spectroscopy of Synthetic Antimicrobial Frog Peptides Magainin 2a and PGLa

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus lueuis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 Å upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the gel-liquid-crystalline phase transition. Mgn2a appears to bind to the surface of liposomes made of negatively charged lipids without spontaneously penetrating the bilayer. Finally, mgn2a and PGLa interact together with liposomes in a synergistic way that enhances the helix content of one or both of the peptides and allows the peptides to more easily penetrate the bilayer. PGLa mixed with a small nonperturbing amount of magainin 2 amide is 25-43 times as potent as PGLa alone at inducing the release of carboxyfluorescein from liposomes. The results suggest that the mechanism of antimicrobial activity does not involve a channel formed by transmembrane helical peptides

Effect of the general anesthetic halothane on the activity of the transverse tubule Ca\u3csup\u3e2+\u3c/sup\u3e-activated K\u3csup\u3e+\u3c/sup\u3e channel

The effect of the general anesthetic halothane on the activity of the rat skeletal muscle Ca2+-activated K+ channel in planar lipid bilayers was investigated. Halothane concentrations in the clinical range (1.0-0.2 mM) alter the regulation of the channel by both Ca2+ and membrane potential. At Ca2+ concentrations between 10 and 250 µM and membrane potentials between 0 and -30 mV, halothane significantly decreases the open state probability without changing the channel conductance. The results demonstrate that halothane can act directly on the Ca2+-activated K+ channel or its lipid environment to alter the channel Bating kinetics

Regulation of Cellular Ca\u3csup\u3e2+\u3c/sup\u3e by Yeast Vacuoles

The role of vacuolar Ca2+ transport systems in regulating cellular Ca2+ was investigated by measuring the vacuolar Ca2+ transport rate, the free energy available to drive vacuolar Ca2+ transport, the ability of the vacuole to buffer lumenal Ca2+, and the vacuolar Ca2+ efflux rate. The magnitude of the Ca2+ gradient generated by the vacuolar H+gradient best supports a 1 Ca2+:2 H+ coupling ratio for the vacuolar Ca2+/H+ exchanger. This coupling ratio along with a cytosolic Ca2+ concentration of 125 nM would give a vacuolar free Ca2+ concentration of ~30 μM. The total vacuolar Ca2+ concentration is ~2 mM due to Ca2+ binding to vacuolar polyphosphate. The Ca2+ efflux rate form the vacuole is less than the growth rate indicating that the steady-state Ca2+ loading level of the vacuole is dependent mainly on the Ca2+ transport rate and the rate that vacuolar Ca2+ is diluted by growth. Based on the kinetic parameters of vacuolar Ca2+ accumulation in vitro, the maximum rate of Ca2+accumulation in vivo is expected to be ~0.2 nmol of Ca2+ min-1 mg protein-1, a rate that is similar to the cellular Ca2+ accumulation rate. The cytosolic Ca2+ concentration increases from 0.1 μM to 1-2 μM as the extracellular Ca2+ concentrarion is raised from 0.3 mM to 50 mM. The rise in cytosolic Ca2+ concentration increases cellular Ca2+ from 10 to 300 nmol Ca2+/mg by increasing the rate of vacuolar Ca2+ accumulation but does not significantly alter the cellular growth rate

Hydroxylation of \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e Ceramides Requires Sur2p and Scs7p

The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p- dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca2+-sensitive phenotype of csg2Δ mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation. Characterization of scs7 and sur2 mutants is expected to provide insight into the function of ceramide hydroxylation

A Novel Protein, CSG2p, Is Required for Ca\u3csup\u3e2+\u3c/sup\u3e Regulation in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

Nineteen mutants that lost the ability to grow in 100 mM Ca2+ (but remained insensitive to 50 mM Sr2+) were identified in a screen of approximately 60,000 mutagenized yeast colonies. Cells carrying mutations in the CSG2 gene grow normally in low Ca2+ medium but have decreased growth rates when the Ca2+ concentration is above 10 mM. The csg2 mutant cells accumulate much higher levels of Ca2+ in a compartment that is exchangeable with extracellular Ca2+ but the nonexchangeable Ca2+ pool which predominates in wild-type cells is not influenced. Sr2+ influx is not increased in the csg2 mutant cells. Mg2+ decreases the amount of Ca2+ in the nonexchangeable pool without influencing the csg2-induced exchangeable Ca2+ pool. The data indicate that the csg2 mutation causes a selective increase in Ca2+ accumulation into a pool which is distinct from the vacuolar pool. The CSG2 protein consists of 410 amino acids, contains nine putative transmembrane segments, four potential sites for N-linked glycosylation, and a sequence with homology to the EF-hand Ca2+-binding site

The \u3ci\u3eSaccharomyces cerevisiae TSC10/YBR265w\u3c/i\u3e Gene Encoding 3-Ketosphinganine Reductase Is Identified in a Screen for Temperature-sensitive Suppressors of the Ca\u3csup\u3e2+\u3c/sup\u3e-sensitive csg2Δ Mutant

Saccharomyces cerevisiae csg2Δ mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity of csg2Δ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2Δ suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3-ketosphinganine and microsomal membranes isolated from tsc10 mutants have low 3-ketosphinganine reductase activity. His6-tagged Tsc10p was expressed in Escherichia coli and isolated by nickelnitrilotriacetic acid column chromatography. The recombinant protein catalyzes the NADPH-dependent reduction of 3-ketosphinganine. These data indicate that Tsc10p is necessary and sufficient for catalyzing the NADPH-dependent reduction of 3-ketosphinganine to dihydrosphingosine