Physarum actinin previously isolated [Hatano, S., & Owaribe, K. (1976) in Cell Motility (Goldman, R., Pollard, T., & Rosenbaum, J., Eds.) Vol. 3, Book B, p 499, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] was found to be a 1:1 complex of actin and fragmin which is a regulatory factor in the formation of actin filaments. Since fragmin did not contain a cysteine residue, it was purified from the complex by the selective cleavage of actin with 2-nitro-5-thiocyanobenzoic acid, followed by column chromatography. Fragmin had nearly the same molecular weight as actin, but had a quite different amino acid composition. When added to G-actin before polymerization, fragmin accelerated the initial viscosity increase of actin solutions induced by salts, but kept the final viscosity much lower than normal F-actin. When added to F-actin after polymerization, fragmin drastically reduced the viscosity of actin solutions. In both cases, the final products of reaction of fragmin with actin were short F-actin filaments. The number average length of the filaments decreased with the increasing molar ratio of fragmin to actin, and the length distribution was always exponential. Fragmin required for its activity a concentration of free Ca2+ higher than 10(-6) M. When the concentration of free Ca2+ was lower than 10(-7) M, fragmin affected neither actin polymerization nor F-actin. The regulation by Ca2+ was reversible.

Long-term iron depletion for CHC patients is a promising modality for lowering the risk of progression to HCC.

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A group of synthetic peptides having an amino acid sequence related to the N-terminal region of the influenza virus hemagglutinin HA-2 chain can induce phospholipid membrane fusion in a pH-dependent manner. These peptides bind to membranes to form alpha-helices even at pH's where no fusion activity is seen. We determined the orientation of these alpha-helical peptides in lipid multibilayers using attenuated total reflection infrared spectroscopy and found that the peptide alpha-helices took a preferential orientation, the helix axis being about 70 degrees from the normal of the membrane plane, or in other words rather parallel to the membrane plane. The orientation was almost independent of pH and a modification of the N-terminal amino group which reduced the fusion activity of the peptides. The determination was carried out for peptides in lipid multibilayers in dry or hydrated (membranes equilibrated with D2O vapor) conditions. Although a slight decrease in the helix orientation angle from the membrane normal was noticed for a hydrated system, the difference between the results for dry and hydrated conditions was small.

Fusion of small unilamellar vesicles of egg phosphatidylcholine can be triggered with synthetic 20-residue peptides. Taking the N-terminal amino acid sequence of HA-2 polypeptide of influenza virus as a guideline, we designed and synthesized several peptides having amphiphilic structures. Among the peptides so far studied, those active to induce membrane fusion took an alpha-helical conformation in the presence of phospholipid bilayers, while a peptide which was unable to induce membrane fusion was in a beta-structure. Mixing of a pair of positively and negatively charged peptides, which had a complementary arrangement of electric charges to each other, resulted in alpha-helix formation at neutral pH, the condition of forming a randomly coiled conformation for each peptide. We concluded that alpha-helix formation was one of the necessary conditions to trigger a process of membrane fusion, at least in the present set of peptides. Characteristic features of these amphiphilic peptides are also described.

A twenty amino acid hydrophobic peptide with the same sequence as that of the HA2 N-terminal segment of influenza virus hemagglutinin was synthesized and studied as to its fusion activity. The peptide caused rapid and efficient fusion of egg yolk phosphatidylcholine sonicated vesicles at acidic pH but not at neutral pH. The threshold pH was ca. 6.2 and the maximum fusion occurred at pH 4.8, the half-maximal pH for fusion being 5.6. The pH dependence was similar to that of the parent virus. The fusion efficiency was dependent on the ration of lipid to peptide, increasing with decreasing ratio. The fusion can be rapidly switched on and off by adjusting the pH, to the acidic side and neutral, respectively. The peptide with an acetylated or succinylated N-terminus also showed low pH-induced fusion activity but the pH range was shifted by ca. 1 unit to the acidic side. The results indicate that the HA2 hydrophobic segment in the virus fusion protein is directly involved in the fusion reaction and protonation of the acidic residues in the segment is required for the activity.

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