Asymmetric antagonistic effects of an inhalation anesthetic and high pressure on the phase transition temperature of dipalmitoyl phosphatidic acid bilayers

The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.     

The effects of n-alcohols on sarcoplasmic reticulum vesicles

Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous $\text{n-}\text{alcohols}$ from methanol to $\text{n-}\text{heptanol}$ caused an inhibition of calcium uptake and an enhancement of ATPase activity. The $\text{n-}\text{alcohol}$ treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of $\text{n-}\text{alcohol-treated}$ vesicles was about twice that of control vesicles. With increasing number ($\text{n}$) of carbon atoms of the $\text{n-}\text{alcohols}$, the maximum increment of ATPase activity increased, and both the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$) required to inhibit calcium uptake by 50% and the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.

$\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}\text{=23.5·10}{}^{\mathrm{?0.593n}}\text{M}$

$\text{N}{}_{\mathrm{ATPase}}\text{=35.5·10}{}^{\mathrm{?0.593n}}\text{M}$

The ratio, $\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$ to $\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$, was constant for all $\text{n}$ values. The apparent free energy of binding of the methylene groups of $\text{n-}\text{alcohols}$ to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of $\text{n-}\text{alcohols}$ in octanol and water (?670 cal/mole). The effects of $\text{n-}\text{alcohols}$ on membrane vesicles are discussed on the basis of these data.     

The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

Comparison of the promoting activity of pristane and n-alkanes in skin carcinogenesis with their physical effects on micellar models of biological membranes

In 1976 (Horton, A.W., Butts, C.K. and Schuff, A.R. (1976) Colloid Interface Sci. 5, 159–168) we assayed pristance (2,6,10,14-tetramethylpentadecane) in a model interfacial system that has given excellent correlation with cocarcinogenic activity among $\text{n-}\text{alkanes}$, as tested in cycloalkane diluents. It was predicted that this branched-chain derivative of the diterpenes would have higher activity than $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$, one of the most cocarcinogenic of the $\text{n-}\text{alkanes}$ in such diluents. Pristane was compared with $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$ and two other $\text{n-}\text{alkanes}$ for their promoting activities in cyclohexane for C3H male mice after a single application of 7,12-dimethylbenz$\text{[a]}\text{anthracene}$. The branched-chain alkane proved to be more active. 20% $\text{n-}\text{tetracosane}$ in cyclohexane was inactive, which correlated with its effects in this diluent in the physical assay system. The promoting activity of 75% $\text{n-}\text{octane}$ in cyclohexane, predicted by the physical assay, was confirmed by tests on mice. The combined by-products of the synthesis of tetracosane, including C₁₂ alkanes and alkenes, C₁₉ and C₂₀ alkylbenzenes, and C₂₄ alkenes, proved to be a very active promoter. However, a mixture in cyclohexane of purified tetracosane with this composite of potential impurities was inactive. From the alkanes behavior in physical systems involving vatious membrane phospholipids and steroids, it is hypothesized that the primary step in their biological activity is a chain-chain interaction with membrane lipids that alters the properties of liquid-crystalline phases at aqueous interfaces. Resulting changes in the microfluidity of the lipid phase and the lateral mobility of critical hormone receptors and enzyme systems, such as the nucleotidyl cyclases, would perturb control systems that maintain the normal behavior of the initiated cell. Thus, its progression to a proliferating neoplasm may be favored.     

Electrophoretic measurements about the relation between transition voltage and ζ-potential of biological membranes

The effects of inorganic cations, $\text{n-}\text{hexanol}$, saccharose and ²H₂O on the electrophoretic mobility and ζ-potential of membrane vesicles from nerve myelin were measured and the results compared with the corresponding effects of the same reagents on the transition voltage, $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, of the nerve axon membrane. Different cation concentrations and ²H₂O affect both potentials, the ζ-potential and $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, in a parallel way. Saccharose and $\text{n-}\text{hexanol}$, however, shift $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$ but leave the electrophoretic mobility of the myelin vesicles unchanged. These results suggest that $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$ shifts are not necessarily linked to changes in the membrane surface charge density but may also be caused by an interaction between the reagent and non-polar groups of the membrane interior.     

The influence of cholesterol on head group mobility in phospholipid membranes

The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0–70°C for various mixtures of $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency $\text{f}{}_2$ observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42°C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42°C no sudden changes in $\text{f}{}_2$ are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42°C, however, a decrease in $\text{f}{}_2$ at cholesterol contents up to 20–30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.     

Infinite cis influx of cyclic AMP into human erythrocyte ghosts

Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The $\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>c</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}$ is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ at 30°C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments.Lowering the temperature reduces $\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>c</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}$ from 0.69 mM at 30°C to 0.478 mM at 20°C, 0.108 mM at 10°C and 0.072 mM at 4°C ($\text{Q}{}_{10}\text{= 2.4}$). The $\text{Q}{}_{10}$ for activation of influx permeability of 10^?5 M cyclic AMP is 1.55.     

Studies on glutathione transport utilizing inside-out vesicle prepared from human erythrocytes

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Line-weaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity. Most active at acidic pH and at 25°C, this transport activity was easily lost during the storage of vesicles at 4°C. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 0.63 mM; guanosine triphosphate (GTP) substituted for ATP gave a 340% stimulation of transport activity. Neither dithiothreitol nor thiol reagents affected this transport process. The other phase had a low affinity for GSSG and a high transport velocity. Most active at pH 7.2 and 37°C, this transport activity was stable during storage of vesicles at 4°C for several days. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg-ATP was 1.25 mM; GTP substituted with no change in activity. Dithiothreitol increased the V but did not alter the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, and thiol reagents inhibited the transport. These findings suggest that there are two independent transfer processes for GSSG in human erythrocytes.     

Serum principal iodothyronines and the influence of cold exposure associated with shearing in the sheep

The effect of cold exposure caused by shearing on serum thyroid hormone (TH) concentrations in sheep kept at an ambient temperature of 8.5°C was studied. While the deep body temperature fell to the lowest level 4 h after shearing the concentration of triiodothyronine (T₃) increased to a peak value at that time. Thyroxine (T₄) and metabolically inactive reverse triiodothyronine (rT₃) levels reached their peak value after 24 h. The $\text{T}{}_3\text{T}{}_4$ ratio reached a maximum at about 4 h and $\text{rT}{}_3\text{T}{}_4$ and $\text{rT}{}_3\text{T}{}_3$ ratios rose to maximum values about 24 h after shearing. This sequence of events suggest a biphasic response to cold—an immediate secretion of TH from the thyroid gland, followed by adaptive alteration in T₃ and rT₃ generation in the extrathyroidal tissues.     

Ruthenium(II) complexes derived from 2-(methylamino)pyridine

2-(Methylamino)pyridine reacts with RuCl₂(CO)₃ to give a carbamoyl complex, [$\text{Ru(C(O)N(CH}{}_3\text{)(C}{}_5\text{H}{}_4\text{N}$)Cl(CO)₂], which yields with pyridine (py) and acetylacetone (Hacac), respectively, [$\text{Ru(C(O)N(CH}{}_3\text{)C}{}_5\text{H}{}_4\text{N}$)Cl(CO)₂(py)] and [$\text{Ru(C(O)N(CH}{}_3\text{)C}{}_5\text{H}{}_4\text{N}$)(CO)₂(acac)]. These complexes are characterized spectroscopically. The amino group of the ligand is carbonylated and the resulted carbamoyl ligand is chelating through a pyridine ring-N and a carbamoyl-C atom. 2-Aminopyridine and 2-aminopyrimidine react similarly with RuCl₂(CO)₃ to give the corresponding carbamoyl complexes.     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ($\text{K′}{}_{\mathrm{<mtext>s</mtext>}}$) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome $\text{P-450}$ is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome $\text{P-450}$ binding reaction and in the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$.     

Influence of duration of incubation on zooplankton respiration and excretion results

Respiration (O), ammonium (NH₄), phosphate (PO₄), total nitrogen (N_T) and phosphorus (P_T) excretions were measured on mixed zooplankton during 3-, 6-, 9-, 12-, 21-, and 24-h incubation periods at 20–23 C. The excretion rates of PO₄, N_T. and P_T decrease during a 21-h period, while rates of respiration and excretion of NH{IN4} are constant. The percentage of inorganic nitrogen excreted increases regularly from 3 h (30–40% of total nitrogen) to 21 h (70–80%) and it could be either due to a bacterial activity which was measured or to a decrease with time of organic nitrogen excreted because of starvation. $\text{O}\text{N}{}_{\mathrm{T}}$, $\text{O}\text{PO}{}_4$, $\text{O}\text{P}{}_{\mathrm{T}}$, and $\text{NH}{}_4\text{PO}{}_4$ ratios increase during the first 9 h of incubation; the percentage of inorganic phosphorus excreted is higher at the very beginning and then remains constant from 6 to 24 h. $\text{O}\text{NH}{}_4$ and $\text{N}{}_{\mathrm{T}}\text{P}{}_{\mathrm{T}}$ ratios are constant during a 24-h term, which makes them useful metabolic indexes.     

Phase transitions in phospholipid vesicles Fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol

We have studied the solid to liquid-crystalline phase transition of sonicated vesicles of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine. The transition was studied by both fluorescence polarization of perylene embedded in the vesicles, and by the efflux rate of trapped ²²Na⁺.Fluorescence polarization generally decreases with temperature, showing an inflection in the region 32–42°C with a mid-point of approximately 37.5 °C. On the other hand, the perylene fluorescence intensity increases abruptly in this region. To explain this result, we have proposed that, for $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ where $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ is the transition temperature, perylene is excluded from the hydrocarbon interior of the membranes, whereas, $\text{T < T}{}_{\mathrm{<mtext>c</mtext>}}$ this probe may be accommodated in the membrane interior to a large extent.The self-diffusion rates of ²²Na⁺ through dipalmitoylphosphatidylglycerol vesicles exhibit a complex dependence on temperature. There is an initial large increase in diffusion rates (approximately 100-fold) between 30 and 38 °C, followed by a decrease (approximately 4-fold) between 38 and 48 °C. A monotonic increase is then observed at temperatures higher than 48 °C. The local maximum of ²²Na⁺ self-diffusion rates at approximately 38 °C coincides with the mid-point of phase transition as detected by changes in fluorescence polarization of perylene with the same vesicles. Vesicles composed of dipalmitoylphosphatidylcholine show the same general behavior in terms of ²²Na⁺ self-diffusion rates at different temperatures, except that the local maximum occurs at approximately 42 °C.The temperature dependence of the permeability and the appearance of a local maximum at the phase transition region could be explained in terms of a domain structure within the plane of the membranes. This explanation is based on the possibility that boundary regions between liquid and solid domains would exhibit relatively high permeability to ²²Na⁺.Mixed vesicles composed of equimolar amounts of dipalmitoyl phospholipids and cholesterol show no abrupt changes in the temperature dependence of either perylene fluorescence polarization or ²²Na⁺ diffusion rate measurements. This is taken to indicate the absence of agross phase transition in the presence of cholesterol.     

Initial membrane reaction in peptidoglycan synthesis. Perturbation of lipid-phospho-N-acetylmuramyl-pentapeptide translocase interactions by n-Butanol

$\text{Phospho}\text{-N-}\text{acetylmuramyl-pentapeptide translocase}$, the initial membrane enzyme in the biosynthesis of peptidoglycan, requires a lipid microenvironment for function. $\text{n-}\text{Butanol}$ was reversibly intercalated into membranes to perturb the hydrophobic interactions in this microenvironment in order to define further the role of lipid. In the concentration range for maximal stimulation of enzymic activity (0.12–0.18 M), $\text{n-}\text{butanol}$ causes a 40% decrease in the fluorescence emission of the dansylated product, undecaprenyl $\text{diphosphate}\text{-(N}{}^{\mathrm{?}}\text{-}\text{dansyl)pentapeptide}$. Since no change in emission maximum occurs below 22°C in the presence of 0.12 M $\text{n-}\text{butanol}$, it is concluded that intercalation of this alkanol causes an increase in fluidity. Above 22°C this concentration of $\text{n-}\text{butanol}$ causes both a decrease in the fluorescence emission and a red shift in the emission maximum. It is concluded that a polarity change as well as fluidity change occurs above 22°C. $\text{n-}\text{Butanol}$ also causes a significant change in the phase transition experienced by the dansylated lipid product. Thus, it is possible with $\text{n-}\text{alkanols}$, e.g. $\text{n-}\text{butanol}$, to perturb lipid-translocase interactions resulting in an increase in fluidity in the microenvironment of the enzyme. This change in fluidity correlates with a stimulation of enzymic activity.     

Cytochrome b5/cytochrome b5 reductase interaction in microsomes: kinetics at subzero temperature.

The cytochrome $\text{b}{}_5\text{b}{}_5$ reductase system solubilized from microsomes exhibits monophasic reduction kinetics over the temperature range 15 ° to ?25 °C in aqueous/ethylene glycol co-solvent, whereas in intact microsomes, the process becomes increasingly heterogeneous below 0 °C, reflecting heterogeneities in membrane structure observable as distributions in reaction rates and activation energies.     

The effect of n-alkanols on the stationary current voltage behavior and action potential of myelinated nerve

Stationary current voltage characteristics and the action potential of single myelinated nerve fibres were measured to examine the effect of $\text{n-}\text{alkanols}$ (methanol to octanol) on the electrophysiological function of the axon membrane. K⁺-depolarized membranes show alkanol-dependent shifts of $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, the membrane transition voltage, whereas in veratridine-depolarized membranes such $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}\text{-}\text{shifts}$ are not observed. In the latter case, $\text{n-}\text{alkanols}$ reduce both the stationary Na⁺ current and the conductivity step between the high- and low-ohmic conductivity state of the membrane. Action potential amplitude, however, is less affected by the alkanols as is the stationary Na⁺ current. The results are compared with the alkanol-dependent changes of the thermotropic phase transition in phospholipid bilayers.     

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin.

Half $\text{met-N}{}_3{}^{\mathrm{?}}$ hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half $\text{met-N}{}_3{}^{\mathrm{?}}$, combined with the presence of a low energy $\text{N}{}_3{}^{\mathrm{?}}\text{→ Cu(II)}$ charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half $\text{met-N}{}_3{}^{\mathrm{?}}$ is found to be capable of coordination of a second $\text{N}{}_3{}^{\mathrm{?}}$ at the copper(II) site.