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Retrogradation kinetics for a potato starch‐water system (10% w/w gel) was monitored by Fourier Transform Infrared spectroscopy and compared with waxy maize starch. The spectra showed the C‐C and C‐O stretching region (1300‐800 cm−1) to be sensitive to the retrogradation process. A multi‐stage process was observed during the retrogradation of potato starch and characterized as the formation of short‐ and long‐range order. The first stage was characterized as the formation of helices and the fast formation of crystalline amylose regions. The second stage was described as the induction time for amylopectin helix aggregation. Stage three was described as the helix‐helix aggregation and the crystallization of amylopectin. The overall‐first order calculated rate constant of potato starch was (9.6±1.4) 10− 3h−1. The calculated rate constant were in agreement with the known difference in retrogradation kinetics of waxy maize and potato starch. The effects were explained by the differences in retrogradation rate of amylopectin and amylose. Potato starch consists of amylose as well as amylopectin. Whereas amylose crystallization occurs within a few hours, amylopectin crystallization is slow and takes a few weeks.

Incorporation of the channel-forming antibiotic gramicidin into the membrane of human erythrocytes highly (up to 30-fold) enhances rates of reorientation (flip) of lysophosphatidylcholine and palmitoylcarnitine to the inner membrane layer after their primary incorporation into the outer layer. Despite the high increase of flip rates by gramicidin, the asymmetric orientation of the inner membrane layer phospholipids phosphatidylethanolamine and phosphatidylserine is stable as demonstrated by the lack of accessibility of these lipids toward cleavage by exogenous phospholipase A2. On the other hand, gramicidin enhances the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2, which indicates changes in the packing of phosphatidylcholine following gramicidin binding. The increase of flip becomes detectable when about 10(5) copies of gramicidin per cell have been bound (gramicidin to membrane phospholipid ratio of 1:2000). This is a 1000-fold higher concentration than that required for an increase of K+ permeability mediated by the gramicidin channel. Acceleration of flip is thus not simply correlated with channel formation. The enhancement of flip is markedly dependent on structural details of gramicidin. Formylation of its four tryptophan residues abolishes the effect. Even at high concentrations of formylated gramicidin at which the extents of binding of native and of formylated gramicidin to the membrane are comparable, no flip acceleration is produced. Enhancement of flip by gramicidin occurs after a temperature-dependent lag phase. At 37 degrees C, flip rates begin to increase within a few minutes and at 25 degrees C, only after 3 h. This lag phase is most likely not due to limitations by the rate of binding of gramicidin to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

It is shown by 3t P-NMR and small angle X-ray scattering that induction of an hexagonal H n phase in dioleoylphosphatidylcholine model membranes by external addition of gramicidin A' depends on the solvent which is used to solubilize the peptide. Addition of gramicidin from dimethylsulfoxide or trifluoroethanol solution leads to H n phase formation whereas addition of the peptide from ethanol does not. This solvent dependence is shown by circular dichroism to be correlated with the peptide conformation. The channel conformation appears to be responsible for H n phase formation by gramicidin.Gramicidin A is an effective promoter of hexagonal HII phases in model and biological membranes [1]. This effect is a consequence of the specific chemical structure of this pentadecapeptide, in particular with respect to the presence of the four tryptophans at the C-terminal part of the molecule [1][2][3]. It was proposed that these bulky residues would provide a pronounced cone shape to the molecule which, within the shape-structure concept of polymorphism [4], together with the peptide's tendency to self associate into cylindrical structures [5][6][7] would explain the Hll phase-inAbbreviations: CD, circular dichroism; DMSO, dimethylsulfoxide; DOPC, dioleoylphosphatidylcholine.

Gramicidin films at the air/water interface are shown to exhibit a phase transition at 225 A2/molecule which might be caused by either cluster formation, reorientation of molecules, conformational changes or multilayer formation. It is further shown that coupling of a charged group on either NH2- or COOH-terminus or elongation of the peptide by two amino acids, only slightly affects the surface area characteristics whereas modification of the tryptophans or even replacement of a single tryptophan by phenylalanine leads to drastic alterations in the surface-area characteristics and a (partial) loss of the phase transition demonstrating that the tryptophans play an important role in the interfacial behavior of gramicidin. The lack of a solvent history effect on the interfacial behavior indicates a rapid conformational interconversion of the peptide at the air/water interface. Gramicidin in mixtures with dioleoylphosphatidylcholine and lysopalmitoylphosphatidylcholine shows a condensing effect whereas gramicidin shows ideal mixing with dioleoylphosphatidylethanolamine. The condensing effect most likely is related to the aggregational state of the peptides which is different in phosphatidylcholines and phosphatidylethanolamines.

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The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.

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