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L Herbette

Publications and source records attributed to L Herbette.

17 recordsLinked to original sources
PubMed1992

Equilibrium and kinetic studies of the interactions of salmeterol with membrane bilayers.

The interaction of salmeterol with model membranes has been studied with regard to equilibrium and kinetic behavior, including determination of the membrane-based partition coefficient, the rate of dissociation of salmeterol from membranes, and the rate of association. These data were obtained in various membrane preparations and under various conditions (e.g., temperature, cholesterol content). The compound is very lipophilic, compared with other beta 2 agonists such as salbutamol, and has a rapid association rate and a moderate dissociation rate. The equilibrium data support the assertion that the salmeterol action measured in perfused tissue involves an exo-site for nonspecific binding that may be identified with or related to the lipid bilayer. The kinetic data in unilamellar and multilamellar liposomes of synthetic lipids further suggest that the approach to the exo-site and the active site may involve components in the native system other than the lipid bilayer in which the beta 2 receptor is located. These additional components may explain the slow onset and the extraordinarily long duration of action.

Albuterol↗
PubMed1985

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction.

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.

Animals↗
PubMed1985

Interaction of amphiphilic molecules with biological membranes. A model for nonspecific and specific drug effects with membranes.

The nonspecific interactions of propranolol, timolol, and ethanol with model and sarcoplasmic reticulum membranes were determined utilizing radioisotopic association differential scanning calorimetry, and neutron diffraction. Differential scanning calorimetry performed on mixtures of these amphiphilic compounds and model membrane bilayers composed of dimyristoyllecithin showed that propranolol was approximately 25 times more lipid-soluble than timolol and at least 100 times more lipid-soluble than ethanol. Neutron diffraction showed that the solvation of propranolol was within the fatty acyl chain region of the lipid bilayer. This solvation correlated with the effect of propranolol to inhibit ATP-dependent calcium transport in isolated rabbit skeletal muscle sarcoplasmic reticulum, a membrane that lacks beta-adrenergic receptors. In contrast, the major site of interaction of ethanol was within the aqueous compartment hydrating the sarcoplasmic reticulum membrane. A model for nonspecific drug interaction with the sarcoplasmic reticulum membrane based on the site of interaction of these amphiphiles and their relative potencies to inhibit calcium transport by these membranes is proposed. In principle, this model could be extended to specific drug interactions with membranes.

Animals↗
PubMed1984

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane.

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.

Animals↗
PubMed1984

Direct determination of the calcium profile structure for dipalmitoyllecithin multilayers using neutron diffraction.

The distribution of calcium in lamellar phases of dipalmitoyllecthin (DPPC) multilayers was directly determined by neutron diffraction and stable isotope substitution of 44Ca for 40Ca. A significant resonance effect on the intensities of the lamellar diffraction pattern was observed for millimolar concentrations of these calcium isotopes. The calcium difference profile indicated that calcium was localized in the phospholipid headgroup region, being excluded from the hydrocarbon core as was water separately determined from the water profile structure obtained by H2O/D2O exchange. A reciprocal space analysis of the difference structure factors indicated that calcium binds preferentially to within 1-2 A of the phosphate moiety of the phospholipid head groups of the DPPC bilayer.

Calcium↗
PubMed1983

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction.

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47-72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.

Animals↗
PubMed1983

Organization of calcium pump protein dimers in the isolated sarcoplasmic reticulum membrane.

The arrangement of the calcium pump protein in the isolated sarcoplasmic reticulum (SR) membrane was examined by optical diffraction of freeze-fracture electron micrographs. Several states of protein particle organization were observed: random, hexagonal and tetragonal packing, and a mixture of hexagonal and tetragonal packing. This suggests that the time-averaged positions of protein particles in the plane of the SR membrane are weakly defined. In addition, there appears to be a greater degree of local or short-range order compared to long-range order within the field of freeze-fracture particles. We utilized measurements from tetragonally or hexagonally packed arrays to determine a unit cell area occupied by each freeze-fracture particle and its associated lipid matrix. When these unit cell areas and the stereologically determined area per freeze-fracture particle were compared to the cross-sectional area occupied by a single calcium pump protein and its associated lipid, obtained by x-ray and neutron diffraction methods, we concluded that each freeze-fracture particle probably represents a dimer of pump protein molecules in the plane of the SR membrane.

Animals↗
PubMed1983

Comparisons of the interaction of propranolol and timolol with model and biological membrane systems.

The nonspecific interaction of the beta-adrenergic blocking drugs, propranolol and timolol, with model and biological membranes has been investigated. Radioisotope measurements of the association of these drugs with dimyristoyl lecithin (DMPC) bilayers showed that both propranolol and timolol had a significantly greater molar association (mole of drug per mole of lipid) with DMPC above its phase transition temperature than below. Timolol had a much lower molar association with DMPC as compared with propranolol both above and below the phase transition temperature. For the DMPC model membrane system, the molar association of propranolol as measured by radioisotope and inferred from calorimetric studies was similar. Neutron diffraction utilizing propranolol deuterated in the naphthalene moiety showed that the naphthalene moiety of propranolol partitions into the hydrocarbon core of the DMPC lipid bilayer, and that the charged amine side chain is most likely positioned in the aqueous phospholipid head group region. For timolol, the association as measured by radioisotope methods was apparently greater than the partitioning inferred from calorimetric studies using freezing point depression analysis, suggesting a more complex interaction of timolol as compared with propranolol with the DMPC lipid bilayer. The association of propranolol and timolol with sarcoplasmic reticulum vesicles (SR) was similar to that with highly purified protein-depleted SR lipids, and DMPC above its phase transition. The association of propranolol with the SR membrane (mole of propranolol per mole of SR phospholipid) correlated with its ability to inhibit calcium uptake, whereas only a fraction of the total association of timolol with the SR membrane appeared to lead to inhibition of calcium uptake. These results suggest that the major nonspecific interactions of propranolol and timolol are with the SR membrane lipids, and that the magnitude of their interactions depends on both the lipid solubility of the drug and the physical state of the fatty acyl chains of the membrane. Both propranolol and timolol appear to perturb the functional properties of the calcium pump protein in the SR membrane (inhibition of ATP-induced calcium uptake) indirectly by partitioning into the bulk lipid matrix of the SR lipid bilayer, although other sites of interaction cannot be excluded.

Animals↗
PubMed1982

Static and time-resolved structural studies of the Ca2+-ATPase of isolated sarcoplasmic reticulum.

X-ray and neutron diffraction studies of oriented multilayers of isolated light sarcoplasmic reticulum (SR) have provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10 A resolution. These studies utilized biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via exchange proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have indicated that significant changes occur in the membrane profile structure within a single turnover of the Ca2+-transport cycle. These studies utilized the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100- to 500-millisecond data collection times, and double-beam spectrophotometry to monitor Ca2+ transport in the oriented SR membrane multilayer.

Animals↗
PubMed1982

The interaction of drugs with the sarcoplasmic reticulum.

Efforts to determine the mechanisms by which drugs interact with membranes can be facilitated by studies of the SR, which has a relatively simple structure. The fact that the SR lacks most of the specific receptors found on other membranes, notably the sarcolemma, is an advantage, as well as a limitation, in that "nonspecific" drug effects can be evaluated readily in the SR. The presence in the SR of a well-characterized calcium pump protein allows the effects of a given drug on membrane function to be analyzed, and drug effects on the functional properties of the SR can now be correlated with their probable sites of action within the membrane. Equally important is the ability to acquire information regarding the relation between the structure of an amphiphilic drug and its mechanism of interaction with the SR membrane, because these interactions depend on the structural properties of both the membrane and the added agent. The potential sites of interaction of amphiphiles schematically portrayed in Figure 3 may be relevant to membranes other than the SR, as the overall structural properties of this membrane appear not to be unique. For this reason, the interaction of drugs with the sarcoplasmic reticulum membrane may provide structural models that are applicable to other biological membranes.

Adrenergic beta-Antagonists↗
PubMed1982

Ion channels in membranes.

The membranes that separate the myocardial cell interior from the extracellular space and delimit compartments within the myocardial cell represent ion-impermeable phospholipid barriers. Embedded in these phospholipid membranes are intrinsic membrane proteins, some of which serve as ion channels. The voltage-sensitive ion channels that control the sarcolemmal action potential appear to be highly regulated intrinsic membrane proteins that contain "gates" that respond to changing membrane potential by opening and closing an ion-selective "pore" that allows specific ions to cross the membrane. Pharmacologic blockade of the sarcolemmal ion channels is selective, not only for individual classes of ion channels, but also for specific states of a given type of channel. The basis for this selectivity remains unclear, but may derive from a preferential interaction between a given drug and a specific type of ion-channel protein, or a selective drug action on a structurally specific region of the membrane phospholipid that is in intimate contact with the ion-channel protein.

Animals↗
PubMed1981

31P-NMR studies of oriented multilayers formed from isolated sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum.

31P-NMR spectra were obtained from oriented multilayer preparations of normal sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum with lipid to protein ratios varying between 41 : 1 and 110 : 1. The dependence of the 31P-NMr spectra on the alignment of the membranes with respect to the magnetic field was used to draw two conclusions about the motion of the phospholipid molecules that contribute to the observed spectra. First, the phosphate group and the two adjacent methylene groups are able to rapidly rotate (i.e., tau R much less than 10-5 S) around the normal to the plane of the membrane. Second, the restricted internal motion of the phosphate group and the glycerol CH2OP group is very similar to that found in liposomes formed from sarcoplasmic reticulum phospholipids. Calibration experiments showed that all (100 +/- 7%) of the phospholipid molecules in the membrane can be accounted for in the observed spectra. Thus, essentially all the phospholipid molecules in the sarcoplasmic reticulum and the reconstituted sarcoplasmic reticulum membranes have the same motion in the polar headgroup region as found in model bilayer membranes. Since a large fraction of the phospholipid molecules (between one-quarter and one-half, depending on ;the lipid to protein ratio) are immediately surrounding the calcium-pump protein, we conclude that the calcium-pump protein does not perturb the motion of these 'boundary-layer' lipids.

Animals↗
PubMed1981

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio.

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.

Animals↗
PubMed1981

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes.

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.

Animals↗
PubMed1980

A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP.

2-Nitrobenzyl derivatives have been used for several years as photolabile protecting groups in synthetic organic chemistry. Recently, P3-1-(2-nitro phenylethyladenosine 5'-triphosphate "caged ATP" was synthesized and its photolysis was shown to generate ATP in situ. This and related reactions have great potential for structural and kinetic studies of both intact and soluble biological systems and it is thus important to define the kinetic characteristics of the photolytic reaction. Caged ATP (2.5 mM) was photolyzed at 347 nm by a single 30-nsec pulse from a frequency-doubled ruby laser of 25 mJ energy to generate 500 microM ATP. The kinetics of the overall reaction were determined by monitoring the kinetics of ATP-induced dissociation of actomyosin, a reaction of known kinetic characteristics. Release of 500 microM ATP was found to be controlled by a process having a rate constant of 2.2 X 10(9) [H+] sec-1 at 22 degrees C at pH 5.8-9.5, which corresponds to 220 sec-1 at pH 7. This process is believed to be the breakdown of an aci-nitro compound, which was identified on the basis of its spectral properties and the photochromicity of related 2-nitrobenzyl compounds.

Actomyosin↗
PubMed1977

A direct analysis of lamellar x-ray diffraction from hydrated oriented multilayers of fully functional sarcoplasmic reticulum.

The profile structure of functional sarcoplasmic reticulum (SR) membranes was investigated by X-ray diffraction methods to a resolution of 10 A. The lamellar diffraction data from hydrated oriented multilayers of SR vesicles showed monotonically increasing widths for higher order lamellar reflections, indicative of simple lattice disorder within the multilayer. A generalized Patterson function analysis, previously developed for treating lamellar diffraction from lattice-disordered multilayers, was used to identify the autocorrelation function of the unit cell electron density profile. Subsequent deconvolution of this autocorrelation function provided the most probable unit cell electron density profile of the SR vesicle membrane pair. The resulting single membrane profile possesses marked asymmetry, suggesting that a major portion of the Ca++ -ATPase resides on the exterior of the vesicle. The electron density profile also suggests that the Ca++-dependent ATPase penetrates into the lipid hydrocarbon core of the SR membrane. Under conditions suitable for X-ray analysis, SR vesicles prepared as partially dehydrated oriented multilayers are shown to conserve most of their ATP-induced Ca++ uptake functionality, as monitored spectrophotometrically with the Ca++ indicator arsenazo III. This has been verified both in resuspensions of SR after centrifugation and slow partial dehydration, and directly in SR multilayers in a partially dehydrated state (20-30 percent water). Therefore, the profile structure of the SR membrane that we have determined may closely resemble that found in vivo.

Adenosine Triphosphatases↗