Biomedical subjects

J M Sturtevant

Publications and source records attributed to J M Sturtevant.

At least 91 records · Page 5Linked to original sources

PubMed1985

Thermotropic behavior of retinal rod membranes and dispersions of extracted phospholipids.

High sensitivity, differential scanning calorimetry studies of bovine retinal rod outer segment (ROS) disk membranes and aqueous dispersions of the extracted ROS phospholipids have been performed. ROS disk membranes were found to exhibit a broad peak of excess heat capacity with a maximum at less than about 3 degrees C, ascribable to a gel-to-liquid crystalline phase transition of a fraction of the phospholipids. A similar thermotropic transition was observed for aqueous dispersions of the total extracted and purified ROS phospholipids. Comparison of the results obtained for the dispersion of total ROS phospholipids to those of the purified head group fractions suggests that the thermotropic behavior reflects a gel-to-liquid crystalline transition, leading to lateral phase separation, involving those phosphatidylcholine (PC) molecules containing saturated fatty acyl chains, possibly together with the highest melting ROS phosphatidylethanolamine (PE) and phosphatidylserine (PS) components. The interpretation of the thermal behavior of the ROS disk membranes depends on whether the transition is assumed to derive from the ROS PC and/or PE/PS fractions, and whether the transbilayer arrangement of the ROS phospholipids is assumed to be symmetric or asymmetric. The calorimetric data can be simply explained in terms of an asymmetric distribution of the major ROS disk membrane phospholipids (G.P. Miljanich et al., J. Membrane Biol. 60:249-255, 1981). In this case, the transition would arise from the PE/PS fractions in the outer ROS disk membrane monolayer, and the anticipated transition from the PC in the inner monolayer would be broadened due to interaction with cholesterol. For the ROS membranes at higher temperatures, two additional, irreversible transitions are observed at 57 and 72 degrees C, corresponding to the thermal denaturation of opsin and rhodopsin, respectively.

Animals↗

PubMed1984

Effects of ganglioside GM1 on the thermotropic behavior of cholera toxin B subunit.

The B, or binding, subunit of cholera enterotoxin forms a pentameric ring structure in the intact toxin, and also when the subunit is isolated from the A subunit. The thermal denaturation of the B subunit ring was examined by differential scanning calorimetry in the presence and absence of ganglioside GM1, its natural 'receptor'. In the absence of ganglioside an irreversible endotherm was observed with maximal excess apparent heat capacity, Cmax, at 74.6 degrees C. When the ganglioside was added in increasing amounts, multiple transitions were observed at higher temperatures, the most prominent having a Cmax at 90.8 degrees C. At high ganglioside concentrations, the 74.6 degrees C transition was not observed. In addition to the thermodynamic results a model is proposed for the interaction of GM1 and B subunit pentamer. This model is derived independently of the calorimetric results (but is consistent with such data) and is based upon considerations of the geometry of the GM1 micelle B subunit pentamer.

Binding Sites↗

PubMed1984

The effects of various peptides on the thermotropic properties of phosphatidylcholine bilayers.

The effects of an amino acid derivative (N-benzoyl-L-argininamide), four small peptides (Phe-Gly-Phe-Gly, gastrin-related peptide (Trp-Met-Arg-Phe-NH2), tetragastrin (Trp-Met-Asp-Phe-NH2), pentagastrin (Boc-beta Ala-Trp-Met-Asp-Phe-NH2] and one medium-sized peptide, glucagon (29 residues), on the gel-to-liquid crystalline transition of a multilamellar suspension of dimyristoylphosphatidylcholine have been studied by means of high-sensitivity differential scanning calorimetry. At low concentrations of added solutes, the temperature at which the excess apparent specific heat in the gel-to-liquid crystalline phase transition of the lipid is maximal is lowered by an amount proportional to the total concentration of the peptide, with proportionality constants ranging from -0.018 K mM-1 for Phe-Gly-Phe-Gly to -3.1 K mM-1 for the gastrin-related peptide. The lipid mixtures involving the first two solutes listed above exhibited approximately symmetrical curves of excess apparent specific heat vs. temperature. The curves for the other solutes were asymmetric, and could be well represented as the sum of either two or three two-state curves. The asymmetry, which was especially pronounced in the cases of pentagastrin and glucagon, thus appeared to be due to the presence of components having lower and/or higher transition temperatures than that of the lipid. Pentagastrin and glucagon (R.M. Epand and J.M. Sturtevant, Biochemistry 20 (1981) 4603) have much smaller effects on the gel-to-liquid crystalline phase transition of dipalmitoylphosphatidylcholine than on that of the dimyristoyl analog.

Arginine↗

PubMed1984

Thermodynamics of phospholipid-sucrose interactions.

The effect of 0-1.0 M sucrose on the phase-transition properties of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (1,2-DPPC) was examined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. Increasing the concentration of sucrose caused a small, but experimentally significant, increase in the temperature (Tm) of maximal excess apparent specific heat (Cmax) and in delta T 1/2 (the transition width at 1/2 Cmax), a reduction in Cmax, and a small decrease (approximately 8-10% at 1.0 M sucrose compared with 0 M sucrose) in the calorimetric enthalpy (delta Hcal) of the gel-to-liquid crystalline transition. The calorimetric parameters of the pretransition of 1,2-DPPC were not significantly affected by sucrose in the concentration range examined, except there was a 1.0 degree C increase in the temperature (Tp) of maximal excess apparent specific heat in the presence of 1.0 M sucrose. The results are discussed in terms of the possible molecular mechanisms that could have caused the observed changes and are contrasted with the results obtained by C. -H. Chen et al. (1981, Biophys. J., 36:359-367).

Calorimetry↗

PubMed1984

Thermodynamic properties of purple membrane.

We measured the density, expansivity, specific heat at constant pressure, and sound velocity of suspensions of purple membrane from Halobacterium halobium and their constituent buffers. From these quantities we calculated the apparent values for the density, expansivity, adiabatic compressibility, isothermal compressibility, specific heat at constant pressure, and specific heat at constant volume for the purple membrane. These results are discussed with respect to previously reported measurements on globular proteins and lipids. Our data suggest a simple additive model in which the protein and lipid molecules expand and compress independently of each other. However, this simple model seems to fail to describe the specific heat data. Our compressibility data suggest that bacteriorhodopsin in native purple membrane binds less water than many globular proteins in neutral aqueous solution, a finding consistent with the lipid surround of bacteriorhodopsin in purple membrane.

Bacteriorhodopsins↗

PubMed1984

Effect of lanthanum ions on the phase transitions of lecithin bilayers.

Interaction of lanthanum ions (La3+) with 1,2 dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) causes an increase in Tc, the temperature of maximal excess heat capacity, and the width of the gel-to-liquid crystalline transition. At a mole ratio of La3+ to DPPC sufficient to remove the hydrocarbon chain tilt angle of DPPC, the changes in the thermodynamic parameters of the pretransition are minor, Tc and the width were unaltered and the enthalpy was reduced by only 10%. This suggests that the change in tilt angle is not a necessary concomitant of the pretransition.

Calorimetry↗

PubMed1984

Multicomponent phase transitions of diacylphosphatidylethanolamine dispersions.

The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PE's) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02-1.0 K min-1. At all scan rates nonhydrated PE's show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PE's, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02-0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PE's may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.

Biophysical Phenomena↗

PubMed1984

Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage lambda repressor.

The thermal stabilities of mutant phage lambda repressors that have single amino acid replacements in the NH2-terminal domain have been studied by means of circular dichroism and differential scanning calorimetry. The variations in stability determined by these physical methods correlate with the resistance to proteolysis at various temperatures and can be compared with the temperature-sensitive activity of the mutants in vivo. In general, mutant proteins bearing solvent-exposed substitutions have thermal stabilities identical to wild type, whereas buried substitutions reduce stability. In one case, a single amino acid replacement increases the thermal stability of the repressor.

Amino Acids↗

PubMed1984

The effects of water-soluble solutes on the phase transitions of phospholipids.

A previous treatment [Sturtevant, J.M. (1982) Proc. Natl. Acad. Sci. USA 79, 3963-3967], based on ideal solution theory, of the influence of small molecules on the main phase transitions of phospholipid bilayers is extended to include the effect of water solubility of the added solute. This effect is manifested primarily in a sharpening of the phase transition, resulting from a buffering by the aqueous phase of the concentration of the solute in the lipid phases.

Lipid Bilayers↗

PubMed1983

Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli.

The thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli have been studied by isothermal and scanning calorimetry. The binding reaction with arabinose is characterized by an enthalpy change of -15.3 +/- 0.5 kcal mol-1 at 25 degrees C, and a large decrease in apparent heat capacity, amounting to -0.44 +/- 0.05 kcal K-1 mol-1, which is constant over the temperature range 8 to 30 degrees C. Very similar results were obtained with D-galactose. These calorimetric results have been combined with binding constants determined by equilibrium dialysis (Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227-2233) to obtain free energy and entropy changes over the range 5 to 30 degrees C, and by extrapolation to 60 degrees C. The protein undergoes reversible unfolding on being heated with an increase in enthalpy at 53.5 degrees C of 151.8 +/- 1.1 kcal mol-1 (169.2 +/- 1.2 kcal mol-1 at 59.0 degrees C) and in apparent heat capacity of 3.16 +/- 0.07 kcal K-1 mol-1. In the presence of arabinose, the unfolding enthalpy is increased to 200.7 +/- 1.8 kcal mol-1 at 59.0 degrees C, the increase being due to the enthalpy of dissociation of the ligand which amounts to 31 kcal mol-1 at the unfolding temperature. The unfolding temperature is increased by the presence of excess arabinose or galactose, an effect which is due solely to displacement by the added ligand of the unfolding-dissociation equilibrium. The thermodynamic data are discussed in connection with the detailed structural information available for this system from x-ray crystallography (Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213-13217, and references cited therein).

Arabinose↗

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

Precision scanning calorimetry of bile salt-phosphatidylcholine micelles.

Precision scanning calorimetry has been used to examine the thermal behavior of mixed micelles formed between bile salts and dipalmitoylphosphatidylcholine (DPPC). Complex thermal transitions are observed which change dramatically with the mole ratio of bile salt to DPPC, dilution, and ionic strength. Comparison of the behavior of sodium taurocholate (TC) mixed micelles with sodium taurodeoxy-cholate (TDC) mixed micelles indicates similarity in the thermal transitions at high dilution or when the actual micellar composition is similar. It was found through equilibrium dialysis that considerably less TC than TDC is incorporated into mixed micelles with DPPC at a given bile salt concentration. Accounting for these concentration differences provides a means for more direct analysis of changes in the thermal transitions with mole ratio and dilution for the two bile salt components. Resolution of the thermal transitions into several component contributions is employed as an aid to interpretation of the differential scanning calorimetry curves. The curve resolutions lead to estimates of van't Hoff and calorimetric enthalpies of the individual contributions. The results of the curve resolutions, along with the behavior of the total enthalpies of the transitions, are consistent with a transformation in micellar structure occurring when the actual micellar composition is a mole ratio of bile salt to DPPC of about 1 to 1. The transformation region is near that found from X-ray evidence and is thought to correspond to a change from disk-shaped to spherical micelles.

Bile Acids and Salts↗

PubMed1982

Calorimetric evidence for phase transitions in spin-label lipid bilayers.

Dispersions of pure, spin-label phosphatidylcholines in aqueous buffer have been investigated with the Privalov high-sensitivity differential scanning calorimeter. The lipids studied are mixed-chain ones in which C-2 of glycerol bears a spin-label derivative of stearic acid and the fatty acid group at C-1 is palmitate. A well-defined phase transition is observed at 30.3-30.7 degrees C for the phosphatidylcholine labeled near the polar end of the stearate chain (label at C-5). A sharp transition (32-34 degrees C) is also observed for the lipid spin-labeled near the terminal methyl of stearate (label at C-16), but the thermodynamic parameters for this lipid depend strongly on the history of the sample. Calorimetric evidence for hysteresis in the phase transition of the C-16-labeled lipid is presented. In contrast to the above spin-label lipids, the lipid labeled at C-12 does not show a sharp transition in the region 5-35 degrees C. In general, therefore, the thermal behavior of the spin-label phosphatidylcholines resembles that of phosphatidylcholines bearing double bonds or branched methyl groups at similar locations on acyl chains. During synthesis of mixed-chain lipids, migration of acyl chains occurs. Methyl esterification procedures which are compatible with the acid-labile spin-label group are described. Gas chromatographic analysis of methyl esters shows that chain migration during synthesis gives 15-20% of the spin-label fatty acid at the glycerol C-1 position.

Calorimetry, Differential Scanning↗

PubMed1982

A scanning calorimetric study of small molecule-lipid bilayer mixtures.

The influence of small molecules on the main phase transition of dimyristoyl phosphatidylcholine was observed by high-sensitivity differential scanning calorimetry. The variation with temperature of the excess heat capacity is markedly affected by solid solution formation, which appears to be of frequent occurrence in solutions in lipid bilayers. The experimental results are in some cases in reasonable agreement with ideal solution theory.

Calorimetry, Differential Scanning↗

PubMed1981

Thermal denaturation of streptomyces subtilisin inhibitor, subtilisin BPN', and the inhibitor-subtilisin complex.

The thermal unfolding of the microbial proteinase inhibitor Streptomyces subtilisin inhibitor (SSI) [Sato, S., & Murao, S. (1973) Agric, Biol. Chem. 37, 1067-1074), the bacterial proteinase subtilisin BPN' (EC 3.4.21.14), and the complex formed by these two proteins has been studied by differential scanning calorimetry (DSC). The thermal denaturation of SSI at pH 7.00 is fully reversible while those of subtilisin BPN' and its complex with SSI are not. The DSC data show that dimeric SSI remains dimeric as the temperature is raised until it unfolds and that it then dissociates during the unfolding process. The apparent specific heat of denatured SSI decreases rapidly with increasing temperature, a behavior not previously observed for proteins. The shape of the DSC curves observed with the enzyme-inhibitor complex suggests that the two components of the complex undergo their unfolding transitions more or less independently. The enthalpies of unfolding of mixtures of enzyme and inhibitor in various molar ratios indicate a substantially large enthalph of interaction than that deduced from fluorescence titrations (Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202).

Bacterial Proteins↗

PubMed1981

A calorimetric study of peptide-phospholipid interactions: the glucagon-dimyristoylphosphatidylcholine complex.

The isothermal enthalpy of reaction glucagon with dimyristoylphosphatidylcholine is found to be large and markedly temperature dependent, changing from -150 kcal/mol of glucagon at 25 degrees C to + 80 kcal/mol of glucagon at 23 degrees C. The observed enthalpy is shown to arise mainly from the glucagon-induced alteration of the distribution of the phospholipid between gel and liquid crystalline phases. The reaction is accompanied by a large change in the apparent heat capacity around the phase transition temperature of the pure lipid which explains the small variation of the free energy of binding with temperature despite the large changes in enthalpy. The equilibrium binding constant calculated from the enthalpy as a function of lipid-peptide ratio is in good agreement with that previously found from fluorescence titrations, but the number of lipid molecules bound per glucagon molecule at 25 degrees C is increased from 20 to 50, suggesting that glucagon can bind to 50 lipid molecules but that only 20 of these are close enough to affect the fluorescence of the hormone. Differential scanning calorimetry of dimyristoylphosphatidylcholine in the presence of glucagon shows a transition curve composed of two components. The major component, corresponding to 80% of the enthalpy change, is centered at 26.1 degrees C and has a cooperative unit of 45 lipid molecules. The total enthalpy change for the transition in the presence of glucagon is only +3.2 kcal/mol, compared with +4.8 kcal/mol for the pure lipid.

Animals↗

PubMed1981

Thermodynamics of the binding of D-glucose to yeast hexokinase.

The binding of D-glucose to baker's yeast hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) was studied by isothermal and differential scanning calorimetry (DSC) and by fluorometric titration. The enthalpy and heat capacity changes associated with the binding of glucose were found to be nearly zero at both low and high ionic strengths over the temperature range from 7 to 29 degrees C. Thus, the free-energy change, amounting to -5.1 kcal mol(-1) at 25 degrees C and high ionic strength, is nearly independent of the temperature and is primarily of entropic origin. DSC study of the thermal unfolding of the free enzyme at low ionic strength gave an excess heat capacity curve with two maxima. This result appears to reflect a difference in thermal stability of the two domains in the hexokinase molecule which are indicated by X-ray crystallography [Bennett, W.S., & Steitz, T. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852]. In contrast, the unfolding of free enzyme at high ionic strength was fully cooperative. The excess heat capacity curve for the unfolding of the glucose-bound enzyme had only one peak at both low and high ionic strengths. This is consistent with the X-ray result that the binding of glucose induces a conformational change in the enzyme which brings the two lobes into close proximity. It is interesting that such a significant, molecule-wide conformational change is accompanied by only very small net changes in enthalpy and heat capacity.

Binding Sites↗

PubMed1981

Scanning calorimetric investigation of the polymerization of the coat protein of tobacco mosaic virus.

The endothermic polymerization of the coat protein of tobacco mosaic virus has been studied by high-sensitivity differential scanning calorimetry, with control experiments involving turbidimetry and sedimentation velocity measurements. The variation of the apparent extent of polymerization under conditions close to equilibrium as the temperature is raised follows a course which is difficult to duplicate on the basis of simple models for the process. The enthalpy of polymerization at low protein concentration varies from 12.5 kcal (mol of monomer)-1 (17500 daltons) under conditions where the product is largely a mixture of short helical rods to 6.0 kcal ol-1 for the formation of double disks containing 34 monomer units. In the former case, the polymerization is accompanied by a decrease in apparent heat capacity of 350 cal K-1 mol-1 while in the latter there is an increase of 150 cal K-1 mol-1. These results constitute evidence that these two types of polymerization involve intersubunit bonds of quite different chemical character.

Calorimetry, Differential Scanning↗