Magainins from Xenopus skin are antimicrobial peptides with broad spectra, and their action mechanisms are considered to be the permeabilization of bacterial membranes. To elucidate their molecular mechanisms, three analog peptides of magainin 2, each having a Trp residue substituted for Phe at the 5th, 12th, or 16th position, were synthesized, and their interactions with acidic phospholipid membranes were investigated by fluorescence. The Trp substitution did not significantly affect the properties of the parent peptide. The binding isotherms of these peptides to the membranes, which were obtained on the basis of fluorescence changes upon membrane binding of the peptides, were sigmoidal, suggesting the association of the bound peptide molecules. A quantitative analysis indicated that the formed aggregate is a dimer. The observation that the initial rate constant of magainin 2 induced leakage of calcein from liposomes was dependent on the fourth power of the peptide concentration demonstrates the formation of a tetrameric pore. A blue shift and intensity enhancement of Trp fluorescence in the presence of the membranes indicate that those Trp residues are buried in the hydrophobic region of the bilayers. Furthermore, the depths of the Trp residues, which were determined using the n-doxylphosphatidylcholine quenching technique, were about 10 A from the bilayer center irrespective of the peptide aggregational state. Thus, it was concluded that the orientation of the magainin 2 alpha-helix is parallel to the membrane surface. A model of the pore formation will be proposed on the basis of these observations.

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The internal pH (pHi) of Toxoplasma gondii was estimated by measuring the accumulation of the weak base 9-aminoacridine in buffers with various ionic compositions. The pHi of the metabolizing parasite increased when the extracellular K+ was elevated in alkaline medium or when the external pH (pHe) was substantially increased in medium employing high external K+ (90 mM). The parasite in mouse peritoneal fluid, or in potassium sulfate buffer (pH 8.2), where the pHi was demonstrated to be increased to 7.9, became motile when acidic buffer was substituted for the original suspension medium. This acid-induced independent movement subsided within 5 min but was repeatedly induced if the pHe was serially lowered to 6.0. Basic buffers, on the other hand, abolished motility when applied to the moving parasites. Nigericin, which is known to collapse pH gradients across the membrane, also abolished motility.

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Effects of Na+ on respiratory NADH oxidase were studied in membranes prepared from Gramnegative marine bacteria belonging to the genera Alcaligenes, Alteromonas, Flavobacterium and Vibrio. NADH oxidases of 9 out of the 10 strains examined were found to require Na+ for maximum activity. Without exception, the Na+-dependent site was located at the level of the NADH : quinone oxidoreductase region of the respiratory chain and was highly sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). NADH dehydrogenases in the Na+-dependent segment were sensitive to Ag+ and able to oxidize reduced nicotinamide hypoxanthine dinucleotide as a substrate. Since the NADH dehydrogenase was neither dependent on Na+ nor sensitive to HQNO, it was suggested that reduction of quinone by the NADH :quinone oxidoreductase involves the formation of an intermediate, presumably semiquinone, and that electron transfer from the intermediate requires Na+ and is sensitive to HQNO. These results showed a striking similarity to those obtained with the Na+-dependent NADH : quinone oxidoreductase, the Na+ pump, of Vibrio alginolyticus, indicating that this enzyme is widely distributed among Gram-negative marine bacteria and shares common properties.

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