Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 19 de 19
Filter
Add more filters










Publication year range
1.
Biochemistry ; 33(40): 12276-82, 1994 Oct 11.
Article in English | MEDLINE | ID: mdl-7918448

ABSTRACT

The peptide hormone human calcitonin (hCT) has a marked tendency to aggregate in aqueous solutions, resulting in viscous and turbid dispersions consisting of long fibrils approximately 80 A in diameter. Both transmission (T-FTIR) and attenuated total reflection Fourier transform infrared (ATR-FTIR) experiments were applied on hCT adsorption and aggregation kinetics. By means of the surface sensitive ATR-FTIR spectroscopy at a hydrophobic/hydrophilic interface, early adsorption and aggregation steps of hCT could be followed in situ under real time conditions. Since the aggregation of hCT is associated with conformational changes, the secondary structure sensitive amide I'-band (D2O) could be used as a diagnostic marker. ATR-FTIR spectra recorded during the aggregation kinetics of hCT showed an increase of the amide I'-band intensity by a factor of 3.4, interpreted as pronounced adsorption of hCT molecules from bulk solution to the germanium plate. Furthermore, variations in the line shape of the amide I'-band were interpreted. At the beginning, hCT adopted a random coil conformation followed by distinct formations of alpha-helical and intermolecular parallel beta-sheet structures. Finally, the random coil content declined to 63%, whereas alpha and beta contents rose to 8% and 29%, respectively. From our kinetics results the alpha-structures were formed faster than the beta-structures. This was interpreted as an initial induction of amphiphilic helices during the adsorption process of hCT monomers. ATR-FTIR spectroscopy provides a sensitive analytical tool suggested to monitor interfacial adsorption and aggregation phenomena also of other peptides and proteins.


Subject(s)
Calcitonin/chemistry , Protein Structure, Secondary , Adsorption , Humans , Normal Distribution , Spectroscopy, Fourier Transform Infrared
2.
Biochim Biophys Acta ; 984(3): 301-12, 1989 Sep 18.
Article in English | MEDLINE | ID: mdl-2550077

ABSTRACT

Na+/K+-ATPase can be isolated from the outer medulla of mammalian kidney in the form of flat membrane fragments containing the enzyme in a density of 10(3)-10(4) protein molecules per microm2 (Deguchi et al. (1977) J. Cell. Biol. 75, 619-634). In this paper we show that these membrane fragments can be bound to a germanium plate coated with a phospholipid bilayer. With this system infrared spectroscopic studies of the enzyme have been carried out using the technique of attenuated total reflection (ATR). At a coverage of the lipid surface corresponding to 30-40% of a monolayer of membrane fragments, characteristic infrared bands of the protein such as the amide I and II bands can be resolved. About 24% of the NH-groups of the peptide backbone are found to be resistant to proton/deuterium exchange within a time period of several days. Evidence for orientation of the protein with respect to the supporting lipid layer is obtained from experiments with polarized light, the largest polarization effects being associated with the -COO- band at 1400 cm-1. Experiments with aqueous media of different ionic composition indicate that the average orientation of transition moments changes when K+ in the medium is replaced by Tris+ or Na+.


Subject(s)
Sodium-Potassium-Exchanging ATPase , Animals , Buffers , Cations, Monovalent , Cell Membrane/ultrastructure , In Vitro Techniques , Kidney Medulla/enzymology , Membrane Lipids , Membrane Proteins , Protein Conformation , Rabbits , Spectrophotometry, Infrared/methods
3.
Proc Natl Acad Sci U S A ; 83(5): 1315-9, 1986 Mar.
Article in English | MEDLINE | ID: mdl-3081894

ABSTRACT

Surfacing and membrane-penetrating ability of vinculin and bovine serum albumin have been studied on a macroscopic level by means of a Langmuir film balance and on a molecular level by means of infrared attenuated total reflection spectroscopy. It is suggested that the driving force of the nonspontaneous process of membrane penetration by native vinculin is the spontaneous formation of rigid vinculin monolayers in the membrane. Lateral adhesion of vinculin molecules results from the formation of intermolecular pleated-sheet structures. Vinculin surface activity was found to result from an alpha-helical segment oriented approximately perpendicular to plane of the membrane. There is a conformational equilibrium between this helix and random structure. High ionic strength (110 mM) favors helix formation that leads to the greater than 100-fold enhancement of surfacing velocity relative to the velocity observed at a lower ionic strength (10 mM). Vinculin has a second helical segment oriented parallel to the plane of the membrane that is in a conformational equilibrium with the pleated-sheet structure.


Subject(s)
Muscle Proteins , Amides , Animals , Chemical Phenomena , Chemistry, Physical , Chickens , Gizzard, Avian , Hydrogen-Ion Concentration , Membrane Lipids , Osmolar Concentration , Phosphatidylcholines , Spectrophotometry, Infrared , Structure-Activity Relationship , Surface Properties , Vinculin
4.
Biochemistry ; 23(12): 2730-4, 1984 Jun 05.
Article in English | MEDLINE | ID: mdl-6466611

ABSTRACT

The activation of acetylcholinesterase [EC 3.1.1.7 (AChE)] by monovalent and divalent metal ions has been investigated by kinetic experiments under steady-state conditions (pH-stat method). It has been shown that at low ionic strength the enhancement of the activity by both monovalent and divalent metal ions can be explained as an electrostatic effect. Thereby, enhancement of the concentration of monovalent metal ions (Na+ and K+) acts by reducing the penetration depth of the electric field of carboxylate groups located at the active center whereby divalent metal ions (Ca2+ and Mg2+) act by a complex formation with these charged groups.


Subject(s)
Acetylcholinesterase/metabolism , Calcium/pharmacology , Electric Organ/enzymology , Magnesium/pharmacology , Potassium/pharmacology , Sodium/pharmacology , Animals , Cations, Divalent , Cations, Monovalent , Enzyme Activation , Kinetics , Mathematics , Torpedo
5.
Biochemistry ; 23(8): 1808-10, 1984 Apr 10.
Article in English | MEDLINE | ID: mdl-6326811

ABSTRACT

Infrared attenuated total reflection spectroscopy (IR-ATR) revealed that the hydrophilic adrenocorticotropin-(11-24)-tetradecapeptide ( ACTH11 -24, net charge 6+) assumed an irregular secondary structure when incorporated into the aqueous layers between equilibrated multibilayers of planar membranes prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine ( POPC ). This structure was characterized by a perpendicular orientation of the peptide bonds on the bilayer surfaces, as observed earlier for the corresponding segment of adrenocorticotropin-(1-24)-tetracosapeptide (ACTH1-24, 6+). Once incorporated, ACTH11 -24 was not removed by washing, in agreement with its strong positive charge. In contrast to ACTH1-24, ACTH11 -24 was not measurably adsorbed to the neutral membranes from 0.1 mM aqueous solutions. The more hydrophobic adrenocorticotropin-(1-10)-decapeptide is also not adsorbed. We therefore concluded that adsorption of ACTH1-24 to neutral membranes was dependent on its amphiphilic primary (amphipathic primary) structure that resulted from the covalent combination of the hydrophobic ACTH1-10 segment with the hydrophilic ACTH11 -24 segment. This conclusion was consistent with the results obtained by vesicle-mediated hydrophobic photolabeling and equilibrium dialysis.


Subject(s)
Adrenocorticotropic Hormone , Cosyntropin , Liposomes , Peptide Fragments , Phosphatidylcholines , Circular Dichroism , Protein Conformation , Spectrophotometry, Infrared
6.
Biochemistry ; 22(18): 4257-64, 1983 Aug 30.
Article in English | MEDLINE | ID: mdl-6313037

ABSTRACT

Infrared attenuated total reflection (IR-ATR) spectroscopy was used to study conformational and topological aspects of the interaction between two adrenocorticotropin fragments and dioleoylphosphatidylcholine membranes. Corticotropin-(1-10)-decapeptide, ACTH1-10, was found to exist as a rigid antiparallel pleated sheet structure in dry membranes. In aqueous environment, it completely escaped from the lipid. This dominant preference for the aqueous phase is a possible explanation for the very low biological potency of ACTH1-10 in some assays. On the other hand, the very potent corticotropin-(1-24)-tetracosapeptide, ACTH1-24, was firmly incorporated into dry and wet membranes. Aqueous environment even promoted the peptide-lipid interaction. Under these latter conditions, part of the molecule entered the bilayer and adopted a helical structure with the axis oriented perpendicularly to the bilayer plane. Contact of a 0.1 mM solution of ACTH1-24 in liquid deuterium oxide with the pure lipid membrane system resulted in measurable adsorption of the peptide to the membrane with the same conformational and topological characteristics as described above (perpendicularly oriented helix entering the bilayer). The helical part of the ACTH1-24 molecule entering the bilayer was the quite hydrophobic N-terminal decapeptide unit ("message" segment). The adjacent hydrophilic C-terminal tetradecapeptide unit ("address" segment) remained on the membrane surface. As the message region is essential for triggering corticotropin receptors, its intrusion into the membrane and its adoption of an oriented, helical conformation may facilitate receptor stimulation.


Subject(s)
Adrenocorticotropic Hormone , Cosyntropin , Liposomes , Peptide Fragments , Phosphatidylcholines , Adrenocorticotropic Hormone/analogs & derivatives , Amino Acid Sequence , Humans , Lipid Bilayers , Models, Molecular , Molecular Conformation , Protein Conformation
7.
Biophys J ; 34(2): 173-87, 1981 May.
Article in English | MEDLINE | ID: mdl-6894555

ABSTRACT

A new phase transition of L-alpha-dipalmitoyl phosphatidylcholine (DPPC) monohydrate from the "biaxial" phase to a crystalline phase (C phase) has been found at 71 degrees C by means of infrared attenuated total reflection (IR-ATR) spectroscopy. The transition is characterized by drastic conformational changes in the glycerophosphorylcholine moiety, which led on the one hand to an alignment of the turn near the ester group in the hydrocarbon chain at glycerol C(2) position. On the other hand a uniform conformation of the glycerophosphorylcholine moiety is found to be typical for the C phase, in contrast to nonuniform head group conformations of DPPC in other regions of the DPPC/water phase diagram investigated so far.


Subject(s)
Pulmonary Surfactants , Crystallization , Molecular Conformation , Spectrophotometry, Infrared
8.
Biophys Struct Mech ; 8(1-2): 45-59, 1981.
Article in English | MEDLINE | ID: mdl-7326355

ABSTRACT

Three mechanisms have been suggested to describe the inhibition of acetylcholinesterase (EC. 3.1.1.7) by an excess of acetylcholine. (i) Substrate inhibition occurs through the reaction of acetylcholine with acetylated enzyme. The deacetylation of this ternary complex is supposed to be completely inhibited. (ii) A ternary complex is formed as in (i). However, the deacetylation is not completely inhibited. (iii) A two-site-mechanism is discussed. Acetylcholine binds either to the active site or to the modifier site. Binding to the latter changes the activity of the active site. Steady state treatment was applied to (i)-(iii). A least squares fit led to catalytic parameters. It is demonstrated that mechanism (ii) is the most simple one which can describe satisfactorily the experimental data. Limits for a set rate constants are derived from the catalytic parameters. A numerical integration shows that the steady state approximation may be used even when the mechanisms are rather complex.


Subject(s)
Acetylcholinesterase/metabolism , Acetylcholine/metabolism , Animals , Cholinesterase Inhibitors , Fishes , Kinetics
10.
J Membr Biol ; 54(3): 203-12, 1980 Jun 15.
Article in English | MEDLINE | ID: mdl-6893058

ABSTRACT

Attenuated total reflection infrared spectroscopy has been used to determine the equilibrium distribution of the peptide antibiotic alamethicin RF30 between dipalmitoyl phosphatidylcholine bilayers and the aqueous environment. The distribution coefficient K = cWeq/cMeq turned out to be concentration dependent, pointing to alamethicin association in the membrane with increasing concentration in the aqueous phase (cWeq). This concentration was varied within 28 and 310 nM, i.e., in a range typical for black film experiments. Furthermore. diffusion coefficients of alamethicin in the hydrophobic phase of the membrane (DM) and across the membrane/water interface (DI) have been estimated from the time course of the equilibration process. It was found that the diffusion rate of the uncharged analogue RF50 is about 10 times higher than that of the RF30 component, exhibiting one negative charge at the C-terminus. The time constants for transmembrane diffusion of alamethicin RF30 varied between 2.2hr at low concentration and 3.2 hr at higher concentration. The corresponding low concentration value of the RF50 component was found to be 0.25 hr.


Subject(s)
Alamethicin , Anti-Bacterial Agents , Lipid Bilayers , Pulmonary Surfactants , Diffusion , Kinetics , Mathematics , Models, Biological , Spectrophotometry, Infrared , Water
11.
Neurochem Int ; 2C: 185-92, 1980.
Article in English | MEDLINE | ID: mdl-20487782

ABSTRACT

Acetylcholinesterase from Torpedo marmorata Risso was adsorbed as a monolayer on to a germanium internal reflection plate. The behaviour of the enzyme in strong electric fields was investigated by means of infrared attenuated total reflection (ATR) spectroscopy. A positive potential on the germanium plate was found to result in an electric-field induced dissociation of the -COOH groups of Asp and Glu residues. Independently, a field-induced conformational change of the protein was observed. Since a negative charge on the enzyme facilitates the trapping of positively charged acetylcholine, and this negative charge can be controlled via the potential of the support to which it is adsorbed, a new electrostatic regulation model for acetylcholinesterase activity in excitable membranes is proposed.

12.
Biophys Struct Mech ; 6(1): 67-80, 1979 Dec.
Article in English | MEDLINE | ID: mdl-540124

ABSTRACT

Infrared attenuated total reflection (ATR) spectroscopy may be used to investigate biological material (e.g., membranes, proteins, erythrocytes etc.) under biological conditions provided that adhesion of the sample can be achieved in aqueous environment. Uncharged lipid multilayer model membranes can be attached by hydrophobic interaction when hydrophobic internal reflection plates (e.g., ZnSe, CdTe) are used. However, if an electric field is applied across the membrane, germanium reflection elements would be preferred because of their low electric resistance (approximately 50 omega cm). This material can also be used if cells or proteins are linked chemically to the ATR plate because of the hydrophilic surface which is similar to that of glass and, thus, enables chemical modification by silanization. It has turned out that good adhesion of uncharged and negatively charged model membranes to germanium plates is achieved when they are coated with a monomolecular layer of aminopropylsilane. There is some evidence that erythrocytes remain more stable when adsorbed to a polymerized aminosilane coating (organic silanization) rather than to the corresponding monolayer (aqueous silanization). Negatively charged germanium surfaces have been obtained by succinylation of the aminosilane coating. Furthermore it has been demonstrated that proteins can be bound to the aminosilane coating by means of carbodiimide. Immobilized acetylcholinesterase was still enzymatically active.


Subject(s)
Membranes/ultrastructure , Proteins , Acetylcholinesterase , Animals , Erythrocytes/ultrastructure , Germanium , Humans , Membranes, Artificial , Phosphatidylcholines , Silanes , Spectrophotometry, Infrared/methods
13.
Proc Natl Acad Sci U S A ; 76(8): 3852-6, 1979 Aug.
Article in English | MEDLINE | ID: mdl-291045

ABSTRACT

The conformation of the linear peptide antibiotic alamethicin in dipalmitoyl phosphatidylcholine multilayers was investigated in the absence of an electric field by means of infrared attenuated total reflection spectroscopy. Alamethicin was found to be incorporated into the lipid membrane not only in the dry state but also in an aqueous environment. Its molecular conformation, however, changed from a helix when dry to an extended chain when aqueous. The extended chain aggregated to di- and multimers spanning the lipid bilayer. The equilibrium concentration of alamethicin in the surrounding water was 90 nM, which is in the range of concentrations used in black film experiments. The corresponding molar ratio of lipid to peptide was 80:1. Concerning the molecular mechanism of electric field-induced pore formation, one has to conclude that the dipole model proposed by several authors is very unlikely because it is based on the assumption that the major part of alamethicin is adsorbed on the membrane surface, from which small amounts flip into the membrane under the influence of an electric field. An alternative mechanism is proposed, based on a field-induced conformational change of the peptide from the extended state to a helix. This transition is favored by the resulting dipole moment of the alamethicin helix.


Subject(s)
Alamethicin , Anti-Bacterial Agents , Ion Channels , Phosphatidylcholines , Ionophores , Lipid Bilayers , Membrane Lipids , Motion , Protein Conformation
14.
Z Naturforsch C Biosci ; 32(1-2): 20-45, 1977.
Article in English | MEDLINE | ID: mdl-139776

ABSTRACT

Polarized infrared ATR spectra of dry oriented multilayers of dipalmitoylphosphatidylethanolamine, sheep brain phosphatidylethanolamine, dipalmitoylphosphatidyl-N-methylethanolamine, dipalmitoylphosphatidyl-N-N-dimethylethanolamine, dipalmitoylphosphatidylcholine and egg phosphatidylcholine are reported. Structural features of hydrocarbon chains and polar headgroups are discussed. The average deviation fo hydrocarbon chains from the normal to the plane of the bilayer was found to be 20-30 degrees. However it was not possible to decide whether the chains are oriented parallel to each other. The fatty acid ester groups in beta- and gamma-position have different conformations. The phosphate group of dipalmitoylphosphatidylethanolamine exists probably in the protonated (formula: see text) and not in the ionized (greater than PO2-) state. However, the latter state is expected for all other phospholipids of this series. The deviation of the bisector of (formula: see text) of the greater than PO2- group from the normal to the bilayer is less than 45 degrees and the mean orientation of all polar head groups is rather parallel than perpendicular to the plane of the bilayer. The polar headgroup of phosphatidylcholine assumes at least two different conformations of the O-C-C-N moiety, i.e. gauche and trans. A variety of conformers has to be expected also for the polar head groups of most of the other phospholipids investigated in this work.


Subject(s)
Phosphatidylcholines , Phosphatidylethanolamines , Animals , Chickens , Crystallography , Ethanolamines , Fatty Acids , Membranes, Artificial , Molecular Conformation , Optical Rotation , Phosphates , Pulmonary Surfactants , Sheep , Spectrophotometry, Infrared/methods
16.
Biochim Biophys Acta ; 450(1): 101-6, 1976 Oct 21.
Article in English | MEDLINE | ID: mdl-987805

ABSTRACT

Conventional and modulated excitation infrared spectra of egg phosphatidylcholine are measured in the spectral range of 4000-900 cm-1 by modulation of relative humidity with an amplitude of +/- less than 5% at a mean value of 75% (T=30 degrees C). From the modulated-excitation spectrum, hydration sites are found to be the greater than PO2 group, the greater than C=O group and the choline group.


Subject(s)
Phosphatidylcholines , Binding Sites , Egg Yolk , Female , Molecular Conformation , Spectrophotometry, Infrared , Water
17.
Biophys J ; 16(7): 791-810, 1976 Jul.
Article in English | MEDLINE | ID: mdl-938719

ABSTRACT

Structural deteriorations in biomembranes, as inevitably induced while structural information is gathered by electron optical methods, were evaluated by infrared spectroscopy. Tripalmitin model membranes were irradiated with 100 keV-electrons in an electron microscope. The intensity decay of group vibrations over the dose reveals the sequence of damage in the polar and nonpolar part of the molecule. The C-C backbone, being the most important structural feature, shows a significant latency effect up to 0.6 e-/A2 and is completely disordered by 3 e-/A2, corresponding to about three inelastic processes per molecule.


Subject(s)
Membranes, Artificial , Microscopy, Electron , Palmitates/radiation effects , Palmitic Acids/radiation effects , Radiation Effects , Electrons , Spectrophotometry, Infrared
18.
Biophys Struct Mech ; 1(2): 75-96, 1975 Feb 19.
Article in English | MEDLINE | ID: mdl-1234025

ABSTRACT

Results of an investigation of the stability of n-layers of barium stearate, cadmium arachidate and tripalmitin by means of electron microscopy and attenuated total reflection infrared spectroscopy are reported. Odd and even numbered barium starate n-layers with n - 1,2,3,4,5 are found to rearrange spontaneously from a regular film into ultrastructures of irregular, flat islands of varying thickness. The kinetics of the phase transformation of the first layer depends on the substrate, that of n-layers appears to be dependent on n, the temperature, and the surrounding medium. The kinetic behaviour of odd and even numbered layers is distinctly different. Similar studies on cadmium arachidate layers reveal much slower kinetics of the rearrangement process. In the case of tripalmitin n-layers it is shown that electron microscopy and infrared spectroscopy yield valuable complementary information about ultrastructure and molecular structure of the layers in correlation with the rearrangement process, which also occurs with this system. Consequences of the results of this paper for work published in various fields are briefly discussed.


Subject(s)
Eicosanoic Acids , Membranes, Artificial , Palmitates , Palmitic Acids , Stearates , Stearic Acids , Barium , Cadmium , Kinetics , Microscopy, Electron , Spectrophotometry, Infrared , Triglycerides
19.
Appl Opt ; 10(4): 819-24, 1971 Apr 01.
Article in English | MEDLINE | ID: mdl-20094545

ABSTRACT

The design and construction of a modulated excitation infrared spectrophotometer that allows detection of photoinduced transients in the spectral range 3000-4000 em(-1) is described. The lifetime range of photoproduced species is 10(-1)-10(-5) sec. Information about kinetics may be obtained by amplitude and phase measurements. The sensitivity achievable by single and double modulation is discussed in detail. Performance of the instrument is illustrated by detection of four transient photoproducts of pyrocatechol, distinguishable by phase measurements. Estimated lifetimes are within 1 msec and 40 msec.

SELECTION OF CITATIONS
SEARCH DETAIL
...