Shun-ichi Ohnishi

Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes

Although myxoviruses share many ~~~~~~erni~~~ and morphological features with ~a~myxo~ruses, their infection mechanism is not completely elucidated It is established that paramywviruses penetrate the plasma membrane via envelope fusion [ 11, eventually i~d~~~g lysis and cell fusion, These ~~ti~ties have not been reported for influenza v&s, In t2] rnor~~oIo~i~a1 evidence appeared for e-nvelope fusion of influenza virus with &e cell rne~~~~~~e, but in [3] an ~~f~~ti~~ me~han~m via Tiropexis was suggested. fn [4,5 ] infectivity #f influensla virus harvested from tissue culture cells was greatly e~I~~~~ed when i~~~e~~a virus HA protein was split into two fragments by trypsin. In [6] the amino~termlnal region formed by proteolysis was found to be similar to the hydrophobic sequence of the F protein of hemagglue ti~~t~~

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis

virus of Japan (HVJ) (also known as Sendai arums), The F protein is involved in ,the envelope fu&n, hemolytic, and fusion actions of HVJ f7], These activities may be caused by direct i~te~a~tio~ of the. am~o-te~~al hydrophobic s~q~e~~~ with ~~d~~~~obi~ regions of the target cell ~emb~ne. The HA ~~~~ei~ of influenza virus may f~~t~o~ as the F protein of HVJ does in certain conditions, causing hemolysis and cell fusion. Here, we found so&at& virus inlcubated with cells in acidic medium of pH 5.2 caused high hemolytic and fusion activities, (I+3 &&i Co,, T-A-4201) with 2 setting of I mA. Bu%rs contained 085% (w/v) NaCl in ~~d~t~o~~ to 20 mM sodium acetate for pH 4.5-X8, IO n&l sodium phosph&te far pH 5.0-8.0 and 20 mM: gly tine-NaOH for pH 9.0-10.0. Protein concentration was determined by Lowry’s method [9] and hema~luti~~ti~~ assay was carried out using

Ca2+-induced lateral phase separations in phosphatidic acid-phosphatidylcholine membranes

alk”s pattern method [la]. Hema~utination acti~ty~m~ virus protein was 1.3 X IO5 for AePR8,3.0 X 10’ for WSN and 6.3 X 103 for HYJ(z). Hemolysis was assr~ed ~e~~~~~~t~~~etri~~l~ at 540 nm_ The s~n~I~~e~ assay method for phosph~l~~d transfer was describes in [S].

Studies on Tetrahymena membranes:: Temperature-induced alterations in fatty acid composition of various membrane fractions in Tetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 degrees C, while that for phosphatidylserine spin label had only one transition at 30 degrees C. When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogenous fluidity. Mg2+ or Mg2+ + ATP prevented the hemolysis-induced spectral changed. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or N-ethylmaleimide added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 degrees C and vanishing at 40 degrees C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.

Clustering of lecithin molecules in phosphatidylserine membranes induced by calcium ion binding to phosphatidylserine

Abstract The effects of Ca 2+ on phosphatidic acid-phosphatidylcholine membranes have been studied using phospholipid spin labels. ESR spectra of spin-labeled phosphatidic acid-phosphatidylcholine membranes and phosphatidic acid-spin-labeled phosphatidylcholine membranes are exchange-broadened immediately upon addition of CaCl 2 . These changes directly and conclusively indicate Ca 2+ -induced clustering of spin-labeled phosphatidylcholine and aggregation of spin-labeled phosphatidic acid bridged by Ca 2+ -chelation in the binary phopholipid membranes. In the Ca 2+ -chelated aggregates, the motions of the alkyl chains of phosphatidic acid are greatly reduced and the lipid molecules are more closely packed. The clusters and aggregates are formed in patches and the sizes are dependent on the fractions. Ba 2+ and Sr 2+ induce the lateral phase separations to the same extent as Ca 2+ . Mg 2+ is also effective but to a lesser extent. In acid solutions (pH 5.5), the Ca 2+ -induced lateral phase separations are of slightly lesser extent than in alkaline solution (pH 7.9). These results are compared with those for phosphatidylserine-phosphatidylcholine membranes reported previously and necessary conditions for the lateral phase separations are discussed.

Membrane fusion. Transfer of phospholipid molecules between phospholipid bilayer membranes

The fatty acid distribution pattern of lipids extracted from different subcellular components of Tetrahymena pyriformis was found to be significantly different from one type of membrane to another. The growth-temperature shift caused alterations in fatty acid composition. The ratio of palmitoleic to palmitic acid, especially, showed a sharp linear decline with increase of temperature in all of the membrane fractions. The spin labels were rapidly incorporated into Tetrahymena membranes. The order parameter of 5-nitroxide stearate spin label incorporated into various membrane fractions was found to be different for the different membrane fractions, suggesting the following order of the fluidity; microsomes > pellicles > cilia. The fluidity of the surface membranes, cilia and pellicles isolated from Tetrahymena cells grown at 15°C was noticeably higher than that of the membranes from cells grown at 34°C but was not so different with microsomal fractions. The motion of the spin label in the pellicular membrane was more restricted than in its extracted lipids, thus indicating the assumption that in Tetrahymena membranes the proteins influence the fluidity. It was also suggested that a sterol-like triterpenoid compound, tetrahymanol, which is principally localized in the surface membranes, would be involved in the membrane fluidity.

A spin-label study of phosphatidylcholine exchange protein. Regulation of the activity by phosphatidylserine and calcium ion.

Abstract The effects of calcium ion on phosphatidyl L-serine (PS) have been studied with PS membranes containing a lecithin spin label (L∗). The calcium ion makes the ESR spectra of the L∗ in PS membranes broadened owing to the intermolecular spin-spin exchange interactions. The results indicate that the calcium ion binds to PS molecules to form rapidly rigid calcium ion-bound PS aggregates, the lecithin molecules being thereby separated from the host PS bilayers to form clusters. The magnesium ion is ineffective for the aggregation and exerts a quite different effect only at much higher concentrations.

Ca2+-induced phase separation in phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine mixed membranes

Abstract Fusion of phosphatidylcholine (PC) vesicles and of PC-phosphatidylserine (PS) vesicles has been studied using spin-labeled PC and PS. Analysis of ESR spectra indicated transfer of phospholipid molecules between phospholipid vesicles at the instant of membrane contact by vesicular collision. The transfer rate of PC was not greatly affected by the presence of the anionic lipid in the membranes. The rate of PC transfer between PS-PC vesicles was nearly the same as that of PS transfer. Calcium ion greatly enhanced the transfer of phospholipid molecules between PS-PC vesicles. The enhancement of PS transfer occurred instantaneously. The phospholipid transfer is related to the fusion of vesicles.

Outer membrane of Salmonella typhimurium. Electron spin resonance studies

Exchange of phosphatidylcholine catalyzed by exchange protein has been studied by a new technique using spin-labeled phosphatidylcholine (PC∗). The exchange activity was assayed by the change in electron spin resonance (ESR) spectrum when PC∗ vesicles were incubated with unlabeled phospholipid vesicles. The method utilized decrease in the spin-spin exchange interaction and does not require separation of the donor and acceptor vesicles. 1. (1) The transfer rate, υ, from PC∗ to phosphatidylcholine vesicles described by υ = k[PC∗][protein](1 + K[PC]) for large excess of a acceptor vesicles where k is the rate constant for association of the protein with PC∗ vesicles and K the binding constant of the protein with phosphatidylcholine vesicles. The transfer rate at 23°C was 3.2 μmol · min−1 · ( protein)−1 · (M PC∗)−1 at [PC] = 14.7 mM, k = 136 mM−1 · min−1, and K = 79 M−1. 2. (2) The exchange activity was inhibited by addition of phosphatidylserine vesicles and also by mixed phosphatidylserine-phosphatidylcholine vesicles. The inhibition was abolished by Ca2+ and Mg2+. 3. (3) The ESR spectrum of PC∗ complexed with exchange protein showed strong immobilization of the lipid alkyl chain, suggesting van der Waals-type binding of the acyl chain to the protein interior. The endogeneous PC∗ molecule was readily exchanged with phosphatidylcholine but practically inexchangeable with phosphatidylserine in the vesicle membranes. 4. (4) A gel chromatographic analysis indicated weak interaction of the exchange protein with phosphatidylcholine vesicles but strong binding to phosphatidylserine vesicles. The inhibitory effect of the anionic lipid vesicles can be explained by their binding to the protein and incapability of exchange of the endogenous phosphatidylcholine with phosphatidylserine in the anionic membranes. The restoration by Ca2+ and Mg2+ may be due to binding of these cations to the anionic lipids, making the protein free from the vesicles. The divalent cations thus act regulatorily for the exchange reaction.

Rotational motion of rhodopsin in the visual receptor membrane as studied by saturation transfer spectroscopy

Ca2+-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidylethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94--100% at 23 degrees C and 84--88% at 40 degrees C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74--85% at 23 degrees C and 61--79% at 40 degrees C. Ca2+ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was 1.4 . 10(-7) M for phosphatidylserine-phosphatidylethanolamine and 1.2 . 10(-6) M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca2+ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17 degrees C) and phosphatidylcholine (-15 degrees C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca2+-chelated solid-phase phosphatidylserine domain.