Cholesterol has a large effect on the binding of glucagon to dimyristoylphosphatidylcholine (DMPC... more Cholesterol has a large effect on the binding of glucagon to dimyristoylphosphatidylcholine (DMPC) and to dipalmitoylphosphatidylcholine (DPPC). At cholesterol concentrations of 20 mol% or greater there is virtually no interaction of glucagon with phospholipid. Glucagon can, however, solubilize DMPC or DPPC with 10% cholesterol. In the case of DPPC the solubilized lipid is depleted of cholesterol relative to the starting material. These results suggest that glucagon excludes cholesterol from its surroundings.It is shown from solubility, light scattering, and glucagon fluorescence measurements that the glucagon–DMPC complex containing 10% cholesterol is stable only in the region of the phase transition temperature. Scatchard analysis of lipid binding to glucagon indicates a decrease in the amount of bound lipid below 22 °C or above 24 °C. The effect of the phase transition on the interaction of glucagon with lipids is compared with that found for other membrane proteins. Several aspe...
... (3) C. C. McDonald, W. D. Phillips, and S. Penman, Science, 144, (4) V. Luzzati, A. Mathis, F... more ... (3) C. C. McDonald, W. D. Phillips, and S. Penman, Science, 144, (4) V. Luzzati, A. Mathis, F. Masson, and J. Witz, J. Mol. Bid, 10, (5) J. Witz and V. Luzzati, ibid., 11, 620 (1965). (6) D. N. Holcomb and I. Tinoco, Jr., Biopolymers, 3, 121 (1965). ...
Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at ... more Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at temperatures below the phase transition leading to a shift in the fluorescence emission maximum of tryptophan from 350 to 338 nm and a 3-fold enhancement of fluorescence intensity as well as a change in the polarization of fluorescence. The circular dichroism properties of the lipid-associated glucagon indicates that it has an increased content of alpha-helix. The phase transition temperature of the lipid as monitored by pyrene excimer fluorescence is not altered by interaction with glucagon although at higher glucagon/lipid ratios a decrease in excimer formation is noted at low temperature. Above the phase transition temperature, the addition of lipid has no effect on the fluorescence emission or circular dichroism of glucagon. Thus this hormone can interact with dimyristoyl glycerophosphocholine and this interaction is stronger below the phase transition temperature than above it.
Association of calcium-dependent isotypes of protein kinase C (PKC) with a phospholipid bilayer i... more Association of calcium-dependent isotypes of protein kinase C (PKC) with a phospholipid bilayer is regulated by a single Ca(2+)-binding site. The dependence of PKC association with phosphatidylserine-containing membranes on the concentration of Ca2+ is linear in the submicro- to submillimolar range. The Ca(2+)-regulated association of PKC with the membrane is sensitive to the factors that alter the diffuse double-layer potential produced by anionic lipids such as phosphatidylserine (PS). This indicates that the Ca(2+)-binding site on the membrane-bound enzyme senses a higher concentration of Ca2+ than is present in bulk solution. This is a consequence of the accumulation of Ca2+ in the layer adjacent to the plane of the membrane by the double-layer potential. Calculations based on the Gouy-Chapman-Stern theory of the diffuse double layer yielded a unique value of the Ca2+ dissociation constant for the Ca(2+)-PKC-bilayer complex equal to approximately 700 nM. The soluble form of the ...
The enthalpy, entropy and free energy of activation was measured for the transfer of the tryptoph... more The enthalpy, entropy and free energy of activation was measured for the transfer of the tryptophan residues of apolipoprotein A-I from a more hydrophobic environment of a lipoprotein particle containing dimyristoylphosphatidylcholine (with or without 12% cholesterol) to an aqueous solvent in the presence of varying concentrations of guanidinium chloride. The free energy of activation was approximately 25 kcal/mol at 50 degrees C for all the conditions studied. The enthalpy of activation was greatest under conditions where a large degree of unfolding occurs when the protein dissociated from lipid. However, under these conditions the unfavourable activation enthalpy was compensated for by a favourable activation entropy resulting in the insensitivity of the free energy of activation to the condition of measurement. Apolipoprotein A-I has an apparent affinity for gel-state lipid which results from the very slow rate of dissociation of the lipoprotein particle below 40 degrees C. It is...
Glucagon was acylated at position 12 using conditions favoring reaction with the epsilon-amino gr... more Glucagon was acylated at position 12 using conditions favoring reaction with the epsilon-amino group of lysine. The N epsilon-acetyl, N epsilon-hexanoyl, and N epsilon-decanoyl derivatives were prepared and purified. Secondary structure as measured by circular dichroism was lower in all derivatives than in glucagon, both in 95% methanol and in 25 mM sodium dodecyl sulfate at pH 2 and pH 12. N epsilon-Acetyl glucagon was less active than the native hormone in a radioreceptor assay and higher concentrations of this derivative were required to stimulate the adenylate cyclase activity of rat liver plasma membranes. The maximal extent of cyclase activation by this derivative was less than that found with the native hormone. N epsilon-Hexanoyl glucagon and N epsilon-decanoyl glucagon had greater activity than N epsilon-acetyl glucagon in receptor binding as well as in adenylate cyclase activation, although these two derivatives were not as active as the native hormone. N epsilon-hexanoyl ...
The ability of several chemically modified forms of glucagon to activate adenylate cyclase have b... more The ability of several chemically modified forms of glucagon to activate adenylate cyclase have been compared with their ability to displace 125I-glucagon from specific membrane binding sites. The results demonstrate that both NH2-terminal and COOH-terminal portions of the peptide, as well as the central region of the glucagon molecule, are all involved in receptor binding and subsequent activation of adenylate cyclase. Receptor binding was very sensitive to chemical modification of the polar residues of glucagon. For example, conversion of the sole lysine residue of glucagon to homoarginine resulted in over a 2-fold loss in receptor-binding affinity. Loss in ability to activate adenylate cyclase was at least as great as loss in receptor binding for all of the derivatives. In the case of derivatives modified at the COOH terminus, the loss in ability to activate adenylate cyclase correlated well with loss in receptor binding. In general, however, the loss of the ability to activate a...
We have studied the interaction of b-17, a potent synthetic antimicrobial b-peptide, with phospho... more We have studied the interaction of b-17, a potent synthetic antimicrobial b-peptide, with phospholipids. We find that unlike other antimicrobial peptides such as magainin II, b-17 facilitates the formation of nonbilayer phases, indicating that the peptide promotes negative curvature. Studies of liposomal leakage also indicate a different mode of mem- brane interaction relative to magainin II, but both leakage and
Cholesterol has a large effect on the binding of glucagon to dimyristoylphosphatidylcholine (DMPC... more Cholesterol has a large effect on the binding of glucagon to dimyristoylphosphatidylcholine (DMPC) and to dipalmitoylphosphatidylcholine (DPPC). At cholesterol concentrations of 20 mol% or greater there is virtually no interaction of glucagon with phospholipid. Glucagon can, however, solubilize DMPC or DPPC with 10% cholesterol. In the case of DPPC the solubilized lipid is depleted of cholesterol relative to the starting material. These results suggest that glucagon excludes cholesterol from its surroundings.It is shown from solubility, light scattering, and glucagon fluorescence measurements that the glucagon–DMPC complex containing 10% cholesterol is stable only in the region of the phase transition temperature. Scatchard analysis of lipid binding to glucagon indicates a decrease in the amount of bound lipid below 22 °C or above 24 °C. The effect of the phase transition on the interaction of glucagon with lipids is compared with that found for other membrane proteins. Several aspe...
... (3) C. C. McDonald, W. D. Phillips, and S. Penman, Science, 144, (4) V. Luzzati, A. Mathis, F... more ... (3) C. C. McDonald, W. D. Phillips, and S. Penman, Science, 144, (4) V. Luzzati, A. Mathis, F. Masson, and J. Witz, J. Mol. Bid, 10, (5) J. Witz and V. Luzzati, ibid., 11, 620 (1965). (6) D. N. Holcomb and I. Tinoco, Jr., Biopolymers, 3, 121 (1965). ...
Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at ... more Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at temperatures below the phase transition leading to a shift in the fluorescence emission maximum of tryptophan from 350 to 338 nm and a 3-fold enhancement of fluorescence intensity as well as a change in the polarization of fluorescence. The circular dichroism properties of the lipid-associated glucagon indicates that it has an increased content of alpha-helix. The phase transition temperature of the lipid as monitored by pyrene excimer fluorescence is not altered by interaction with glucagon although at higher glucagon/lipid ratios a decrease in excimer formation is noted at low temperature. Above the phase transition temperature, the addition of lipid has no effect on the fluorescence emission or circular dichroism of glucagon. Thus this hormone can interact with dimyristoyl glycerophosphocholine and this interaction is stronger below the phase transition temperature than above it.
Association of calcium-dependent isotypes of protein kinase C (PKC) with a phospholipid bilayer i... more Association of calcium-dependent isotypes of protein kinase C (PKC) with a phospholipid bilayer is regulated by a single Ca(2+)-binding site. The dependence of PKC association with phosphatidylserine-containing membranes on the concentration of Ca2+ is linear in the submicro- to submillimolar range. The Ca(2+)-regulated association of PKC with the membrane is sensitive to the factors that alter the diffuse double-layer potential produced by anionic lipids such as phosphatidylserine (PS). This indicates that the Ca(2+)-binding site on the membrane-bound enzyme senses a higher concentration of Ca2+ than is present in bulk solution. This is a consequence of the accumulation of Ca2+ in the layer adjacent to the plane of the membrane by the double-layer potential. Calculations based on the Gouy-Chapman-Stern theory of the diffuse double layer yielded a unique value of the Ca2+ dissociation constant for the Ca(2+)-PKC-bilayer complex equal to approximately 700 nM. The soluble form of the ...
The enthalpy, entropy and free energy of activation was measured for the transfer of the tryptoph... more The enthalpy, entropy and free energy of activation was measured for the transfer of the tryptophan residues of apolipoprotein A-I from a more hydrophobic environment of a lipoprotein particle containing dimyristoylphosphatidylcholine (with or without 12% cholesterol) to an aqueous solvent in the presence of varying concentrations of guanidinium chloride. The free energy of activation was approximately 25 kcal/mol at 50 degrees C for all the conditions studied. The enthalpy of activation was greatest under conditions where a large degree of unfolding occurs when the protein dissociated from lipid. However, under these conditions the unfavourable activation enthalpy was compensated for by a favourable activation entropy resulting in the insensitivity of the free energy of activation to the condition of measurement. Apolipoprotein A-I has an apparent affinity for gel-state lipid which results from the very slow rate of dissociation of the lipoprotein particle below 40 degrees C. It is...
Glucagon was acylated at position 12 using conditions favoring reaction with the epsilon-amino gr... more Glucagon was acylated at position 12 using conditions favoring reaction with the epsilon-amino group of lysine. The N epsilon-acetyl, N epsilon-hexanoyl, and N epsilon-decanoyl derivatives were prepared and purified. Secondary structure as measured by circular dichroism was lower in all derivatives than in glucagon, both in 95% methanol and in 25 mM sodium dodecyl sulfate at pH 2 and pH 12. N epsilon-Acetyl glucagon was less active than the native hormone in a radioreceptor assay and higher concentrations of this derivative were required to stimulate the adenylate cyclase activity of rat liver plasma membranes. The maximal extent of cyclase activation by this derivative was less than that found with the native hormone. N epsilon-Hexanoyl glucagon and N epsilon-decanoyl glucagon had greater activity than N epsilon-acetyl glucagon in receptor binding as well as in adenylate cyclase activation, although these two derivatives were not as active as the native hormone. N epsilon-hexanoyl ...
The ability of several chemically modified forms of glucagon to activate adenylate cyclase have b... more The ability of several chemically modified forms of glucagon to activate adenylate cyclase have been compared with their ability to displace 125I-glucagon from specific membrane binding sites. The results demonstrate that both NH2-terminal and COOH-terminal portions of the peptide, as well as the central region of the glucagon molecule, are all involved in receptor binding and subsequent activation of adenylate cyclase. Receptor binding was very sensitive to chemical modification of the polar residues of glucagon. For example, conversion of the sole lysine residue of glucagon to homoarginine resulted in over a 2-fold loss in receptor-binding affinity. Loss in ability to activate adenylate cyclase was at least as great as loss in receptor binding for all of the derivatives. In the case of derivatives modified at the COOH terminus, the loss in ability to activate adenylate cyclase correlated well with loss in receptor binding. In general, however, the loss of the ability to activate a...
We have studied the interaction of b-17, a potent synthetic antimicrobial b-peptide, with phospho... more We have studied the interaction of b-17, a potent synthetic antimicrobial b-peptide, with phospholipids. We find that unlike other antimicrobial peptides such as magainin II, b-17 facilitates the formation of nonbilayer phases, indicating that the peptide promotes negative curvature. Studies of liposomal leakage also indicate a different mode of mem- brane interaction relative to magainin II, but both leakage and
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