Kohsuke Fukushima

The transition of α-helix to β-structure of poly(d-lysine) induced by phosphatidic acid vesicles and its kinetics at alkaline pH

Abstract Static and dynamic circular dichroism (CD) measurements were carried out for poly( l -lysine) in suspensions of dilauroylphosphatidic acid (DLPA) vesicles at alkaline pH (8–11.5). The static experiments demonstrated that the α-helix of poly( l -lysine) induced by deprotonation in alkaline solutions is transformed to β-structure by the addition of DLPA vesicles. Stopped-flow CD measurements for such order-to-order transition revealed that the rate determining step is the unfolding process of α-helix to random coil. Previously, we have reported the conformational change of poly( l -lysine) induced by DLPA vesicles at neutral pH, where the β-structure transition from random coil was observed. Thus two types of transition of poly( l -lysine) are observed depending on bulk pH, i.e., from random coil to β-structure and from α-helix to β-structure. So far the phospholipid-induced conformations of poly( l -lysine) were interpreted in terms of counterbalance between the positively charged terminals of the lysyl chains and the negative headgroups of the phospholipid in vesicle. However, present work indicates the direct interaction other than electrostatic interaction between the lysyl chain and phosphate groups of the lipid.

Effect of sodium octanoate and sodium perfluorooctanoate on gel-to-liquid-crystalline phase transition of dipalmitoylphosphatidylcholine vesicle membrane

The gel-to-liquid-crystalline phase transition of dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was measured in the presence of sodium octanoate (SO) (pH 3 and 10) and sodium perfluorooctanoate (SPFO) (pH uncontrolled) by monitoring the scattered light intensity of the vesicle suspension. The phase transition temperature, Tm, decreased linearly with the concentration of added SO within the measured concentration range; the uncharged form of SO (pH 3) was much more effective for the depression of Tm than the charged form (pH 10). On the other hand, with increasing SPFO concentration, levelling off of Tm was observed after depression at an initial stage. From the depression of Tm, the partition coefficients, K, of these surfactants between bulk solution and DPPC vesicle membrane were estimated and compared with those obtained previously for other surfactant systems. The value of K for charged SO fell on the straight line of log K vs. Nc plot for anionic surfactants, where Nc is the carbon number of the hydrocarbon chain of surfactants, whereas K for uncharged SO showed a large positive deviation from the straight line of the plot for non-ionic surfactants. The latter suggested that some specific interaction, presumably hydrogen bond formation, may act between the protonated carboxyl group of SO and the lipid head group. The K value estimated for SPFO was much larger than that for charged SO. This difference in the affinity for the lipid bilayer between fluorocarbon surfactant and hydrocarbon surfactant may be attributed to the difference in their hydrophobicity.

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.

Surfactant partition between bulk water and DPPC vesicle membrane : solid-gel vs. liquid-crystalline membrane

The main phase transition temperature, Tm, of dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was measured in the presence of the cationic surfactants tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide. Variation of the perturbing effect of these surfactants on Tm with the lipid concentration was analyzed according to the theory recently proposed by Kaminoh et al. (Y. Kaminoh, C. Tashiro, H. Kamaya and I. Ueda (1988) Biochim. Biophys. Acta 946, 215-220), and the partition coefficients of the surfactant into solid-gel and liquid-crystalline membranes were estimated.

Kinetics of pressure-induced inactivation of bovine liver glutamate dehydrogenase

Kinetics of pressure-induced denaturation of bovine liver glutamate dehydrogenase (EC 1.4.1.3) were investigated in the pressure range 1.8-2.8 kbar by observing the residual activity after the pressure-release and the scattered light intensity during the incubation at high pressure. The residual activity decreased exponentially with the incubation time, whereas the scattered light intensity showed a bimodal profile indicating parallel aggregation and dissociation reactions. The latter suggested that two kinds of aggregates were formed during the incubation under pressure. The observed first-order rate constant for the inactivation, k obs, showed a minimum around 30 degrees C. These experimental results were interpreted in terms of the following reaction scheme; (formula; see text) where N represents the enzyme entity with native structure, D1 the partially denatured intermediate, D2 the irreversibly denatured state, and A1 and A2 the two kinds of aggregates, one of which (A1) is reversibly formed at an early stage of the incubation under high pressure. The apparent activation volume for the inactivation reaction was estimated to be delta V*app = -113 +/- 5 cm3 X mol-1 from the pressure dependence of k obs. The effect of coenzyme, NAD+, on the pressure-induced inactivation was also studied. The inactivation was retarded by the presence of the coenzyme, whereas the apparent activation volume for the holoenzyme (delta V*app = -104 +/- 2 cm3 X mol-1) did not differ significantly from that for the apoenzyme.

Response of bilayer phase transition temperature of acidic phospholipids to cationic surfactants

Abstract The effect of cationic surfactants on the bilayer phase transition of phospholipid vesicles was studied. Two types of acidic phospholipids, phosphatidic acid and phosphatidylglycerol, with different acyl chain length were examined. Cationic surfactants were alkyltrimethylammonium bromides with different alkyl chain length. The cationic surfactants with short chain length induced a monotonous decrease in the transition temperature, Tm, with increasing concentration, while those with long chain length exhibited a biphasic effect on Tm, i.e., increase in Tm at low concentrations and decrease in Tm at high concentrations. The surfactant chain length at which the biphasic effect on Tm appeared depended on the lipid acyl chain length; the longer lipid acyl chain needed the longer surfactant chain to bring about the biphasic effect. This behavior of the bilayer phase transition was common to acidic phospholipids regardless of the type of the lipid head groups. The complicated response of Tm to additives was explained by introducing interactions between the lipid and the surfactant in the membrane phase. According to this model, the behavior of Tm with respect to the additive concentration is determined by the combination of differences in the lipid-additive interaction energy and the free energy of additives between solid-gel and liquid-crystalline membranes.

Conformational change of poly(l-lysine) induced by lipid vesicles of dilauroylphosphatidic acid

The effect of negatively charged dilauroylphosphatidic acid (DLPA) vesicles on the conformation of poly(L-lysine) was investigated by circular dichroism measurements. DLPA vesicles induced a conformational change of poly(L-lysine) from the random coil to beta-structure in 5 mM Tes, pH 7.0. The fraction of induced beta-structure (F beta) was determined via a procedure of curve fitting of the observed spectra to the reference spectra. F beta increased linearly with the molar ratio, r, of DLPA to lysine residues up to r congruent to 0.7, and reached a saturation value of 1 at r greater than 1. Within the range 0.7 less than or equal to r less than or equal to 1, precipitation occurred. The effect of dilution of the negative charge on vesicle membranes was examined by mixing DLPA with dilauroylphosphatidylcholine (DLPC). Although the beta-structure of poly(L-lysine) was also induced by mixed vesicles, the saturation value of F beta decreased with decreasing DLPA content in mixed vesicles. The variation in saturation value of F beta with the composition of mixed vesicles was interpreted in terms of the change in average distance between DLPA head groups in mixed vesicles.

Light-scattering study on subunit association-dissociation equilibria of bovine liver glutamate dehydrogenase

The subunit dissociation of bovine liver glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) induced by guanidine hydrochloride ( GdnHCl ) in 0.2 M phosphate buffer (pH 7.3) was investigated by light-scattering molecular-weight measurements. With increasing GdnHCl concentration, two-step transition was observed in the molecular weight change. The dissociation behavior was well described by assuming the dissociation-association equilibria expressed as HK1 in equilibrium 2T K2 in equilibrium 6M where H, T, and M represent the hexameric, trimeric and monomeric forms of the enzyme, respectively. GdnHCl concentration dependence of the two equilibrium constants was interpreted in terms of the binding of GdnHCl on the protein. According to this treatment, the numbers of amino acid residues present at the trimer-trimer contact area within hexamer, N3, and at the monomer-monomer contact area within trimer, N1, were estimated to be as follows; N3 = 21 +/- 2 and N1 = 27 +/- 5. These values seem to be reasonable considering the physical model proposed for this enzyme.