Differential deuterium isotope shifts and one-bond 1H−13C scalar couplings in the conformational analysis of protein glycine residues

Summary The one-bond deuterium isotope shift effect for glycine C resonances exhibits a conformational dependence comparable to that of the corresponding ¹J_HC scalar coupling in both magnitude (11 Hz at 14.1 T) and dihedral angle dependence. The similarity in the conformational dependence of the ¹J_HC and deuterium isotope shift values suggests a common physical basis. Given the known distribution of (,) main-chain dihedral angles for glycine residues, the deuterium isotope shifts and the ¹J_HC scalar couplings can determine conformations in the left-and right-handed helical-to-bridge regions of the (,) plane to an accuracy of approximately 13°. In the absence of stereochemical assignments, the differential deuterium isotope shifts and the ¹J_HC scalar couplings can be combined with limited independent structural information (e.g., the sign of ) to determine the chirality of the deuterium substitution.     

Possible sites of release of neurosecretory granules in the sinus gland of the crayfish,Orconectes nais

Summary The sinus gland is a neurohemal organ located in the crayfish eyestalk and represents a storage site for neurohormones prior to their release into the circulation. The sinus gland contains three classes of electron dense, membrane-limited granules. Class 3 granules are the largest and most electron dense of the granules found in the sinus gland. Granules of class 1 are the smallest, while those of class 2 are the most abundant. Generally, all granules undergo similar changes during their release.Release of neurosecretory material may be initiated by a preliminary fragmentation of the parent granules into smaller granules. Following the formation of numerous smaller granules, these move to the plasma membrane and their limiting membrane apparently fuses with it thus releasing its contents into the external lamina which is applied to the sinusoidal surface of the axon terminals. Granule release does not appear to occur along the entire plasma membrane adjacent to the blood sinus but, instead, probably occurs only at specific active sites on the membrane. The active sites are characterized in part by an accumulation of small granules and clear vesicles against the cytoplasmic side of the plasma membrane. At the site of release of the neurohormone, there is often an accumulation of dense homogeneous material beneath the axolemma.Occasionally, axon endings filled with large, electron lucent vesicles are seen. These clear granules vary from 1150–1750 Å in diameter and often exhibit broken limiting membranes. Few small vesicles are seen near the plasma membrane of these endings; however, instances of invaginations of the plasma membrane occur. The significance of endings filled with clear granules is discussed.Supported by a grant from the National Research Council of Canada (No. A-4675).     

Deuterium stable isotope ratios as tracers of water resource use: an experimental test with rock doves

Naturally-occurring deuterium stable isotope ratios can potentially be used to trace water resource use by animals, but estimating the contribution of isotopically distinct water sources requires the accurate prediction of isotopic discrimination factors between water inputs and an animals body water pool. We examined the feasibility of using estimates of water fluxes between a bird and its environment with a mass-balance model for the deuterium stable isotope ratio of avian body water (D_body) to predict isotopic discrimination factors. Apparent fractionation and thus discrimination factors were predicted to vary with the proportion of an animals total water losses than could be attributed to evaporative processes. To test our ability to predict isotopic discrimination, we manipulated water intake and evaporative water loss in rock doves (Columba livia) by providing them with fresh water or 0.15 M NaCl solution in thermoneutral or hot environments. After we switched the birds from drinking water with D=–95 VSMOW (Vienna Standard Mean Ocean Water) to enriched drinking water with D=+52 VSMOW, steady-state D_body was approached asymptotically. The equilibrium D_body was enriched by 10–50 relative to water inputs. After isotopic equilibrium was reached, the degree of enrichment was positively related (r²=0.34) to the fraction of total water loss that occurred by evaporation supporting the major prediction of the model. The variation we observed in discrimination factors suggests that the apparent fractionation of deuterium will be difficult to predict accurately under natural conditions. Our results show that accurate estimates of the contribution of different water sources to a birds body water pool require large deuterium isotopic differences between the sources.     

Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment

Summary The ratio of deuterium to hydrogen (expressed as D) in hydrogen released as water during the combustion of dried plant material was examined. The D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C₃ plants show mean D values of-132 for shoots and -117 for roots; C₄ plants show mean D values of -91 for shoots and-77 for roots and CAM plants a D value of-75 for roots and shoots. The difference between the D value of shoot material from C₃ and C₄ plants was confirmed in species growing under a range of glasshouse conditions. This difference in D value between C₃ and C₄ species does not appear to be due to differences in the D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C₃ and C₄ plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in D values are discussed. In CAM plants grown in the laboratory or collected from the field D values range from-75 to +50 and are correlated with ¹³C values. When deprived of water, the D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different D value of available water in areas of increasing aridity. Whatever the origin of the variable D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.     

Sensitivity of Nostoc commune UTEX 584 (Cyanobacteria) to water stress

Cells of Nostoc commune UTEX 584 from liquid cultures expressed an upshift in nitrogenase activity when immobilised on inert supports and exposed to matric water potentials between -1.10 and -99.5 MPa. Cells incubated at 0.10 MPa (a_w=c 1.0) maintained increased activity for at least 48 h following immobilization. At water potentials below -23.1 MPa (a_w=0.85), the upshift was transitory. Nitrogenase activity decreased rapidly when immobilised cells were incubated at lower values of _m.Desiccated cells stored at -99.5 MPa (a_w=0.50) underwent an upshift in nitrogenase activity, and in the size of the intracellular ATP pool, when rewetted with either distilled water or liquid MB_o medium (_o =-0.18 MPa). The upshift in nitrogenase activity was chloramphenicol-sensitive and was preceeded by a lag. The duration of the lag depended on the time taken to equilibrate cells to-99.5 MPa, the time desiccated, and the conditions of storage and rewetting. Cells that had no, or very low, nitrogenase activity when rewetted in air, showed a marked stimulation of nitrogenase activity in the presence of 5% v/v CO₂ under both aerobic and anerobic conditions.When rewetted in the presence of 1% w/v glucose (_o =-0.14 MPa), vegetative cells remained intact, but heterocysts underwent autolysis and nitrogenase activity was not detected, even in the presence of 5% v/v CO₂.Abbreviations TTC 2,3,5-triphenyl-2-tetrazolium chloride - _m matric water potential - _o osmotic water potential - a_w water activity     

Critical moisture content for successful cryopreservation of embryonic axes of <Emphasis Type="Italic">Fortunella polyandra</Emphasis> determined by differential scanning calorimetry (DSC)

The relationships between water content of desiccated embryonic axes (using different methods of desiccation), the availability of water determined by differential scanning calorimetry (DSC) analysis and recovery percentage after liquid nitrogen (LN) exposure of Fortunella polyandra embryonic axes were investigated. The objectives were to understand thermal properties of desiccated embryonic axes during cryopreservation and to determine the critical moisture contents for successful cryopreservation of the embryonic axes. Excised embryonic axes were desiccated under laminar air flow (0, 10, 15, 30 and 45 min), over silica gel (0, 5, 15, 30 and 60 min), and ultra-rapidly (0, 5, 10, 20 and 25 min). Desiccation under laminar air flow resulted in an optimal water content of 0.150 gH₂O g^?1 dw and a survival of 50 % after cryopreservation, while the unfrozen water content (WC_u) was 0.126 gH₂O g^?1 dw. After drying over silica gel, the optimal water content was 0.190 gH₂O g^?1 dw, where the survival was 40 % after cryopreservation and the WC_u was determined as 0.177 gH₂O g^?1 dw. Using the flash-drying method, the optimal water content was found to be 0.145 gH₂O g^?1 dw, the survival was 50 % after cryopreservation and the WC_u was 0.133 gH₂O g^?1 dw. Embryonic axes of F. polyandra showed low-to-moderate tolerance to desiccation. The results of the freezing transitions for all the desiccation times and methods showed that the onset temperature and the peak of the mean enthalpy decreased in size with decreasing water content. DSC elucidated the critical moisture contents and the cooling and melt enthalpies for successful cryopreservation of F. polyandra embryonic axes.     

The mean depth of soil water uptake by two temperate grassland species over time subjected to mild soil water deficit and competitive association

Little is known concerning the soil water use dynamics of white clover (WC) and ryegrass (RG) grown in mixtures. A greenhouse study, on a deep soil, was conducted to determine the mean depth of soil water uptake of WC and RG plants grown in a competitive association and subjected to a moderate soil water deficit. Plant growth period simulated that experienced by newly sown grassland in temperate regions. Three irrigation solutions, each containing a different hydrogen isotope (deuterium) concentration, expressed as delta notation (), were provided at three different soil depths through specially constructed tubes and containers (0.50 m diameter, 1 m depth) in order to create a soil deuterium profile gradient. Young leaves and not the entire plant were harvested in order to preserve the competitive plant association over time. Patterns of leaf D value were constant for both WC and RG. Lower leaf D values in RG compared to WC was attributed to RG more efficient stomatal control. Increases in the mean depth of soil water uptake as soil water deficit increased was similar between plants. The mean depth of soil water uptake of WC was at all times greater than that of RG. After 3 months of competitive growth, WC roots obtained water from a soil depth 30% greater than that of RG. In our experimental conditions, the ability of WC to obtain water from substantially lower soil depths may give it a competitive advantage over RG during the period subsequent to pasture sowing if surface soil water deficits are experienced and deeper soil layers contain water.     

Ecophysiological studies on orchids of Madagascar: incidence and plasticity of crassulacean acid metabolism in species of the genus Angraecum Bory

The present study is an investigation on photosynthetic options in an orchid taxon and deals with the mainly epiphytes comprising genus Angraecum Bory. The incidence of crassulacean acid metabolism (CAM) in Angraecum species collected at various habitats in Madagascar was surveyed by analysis of stable carbon isotope composition (¹³C values). The values showed both inter- and intraspecific variability and suggest that in situ about 50% of the analysed species perform C3 photosynthesis, 20% moderate CAM (fixation of external CO₂ during day-and night-time) and 30% pronounced CAM (CO₂ uptake entirely during the night). The photosynthetic behaviour of the species indicated by the ¹³C values was clearly related to the habitat from where the samples derived. In A. eburneum, A. sororium and particularly in A. sesquipedale the stable carbon isotope analysis was complemented by measurements of CAM performance under controlled conditions. The experiments with A. sesquipedale revealed that drought and temperature are important factors modulating CAM, whereas variation of the leaf-to-air water vapor pressure difference was less effective. Altogether, the results of the study support the view that the high biological adaptability and thus the ecological success of the genus Angraecum is largely based on genotypic diversity and intraspecific plasticity of the photosynthetic behaviour.     

H/D isotope effects on protein hydration and interaction in solution

An approach has been suggested to study the H/D isotope effect on protein-water and protein-protein intermolecular interactions by determining the content of non-freezing water using low-temperature (1)H NMR in mixed (H2O/D2O) water solutions. Direct data are obtained on the amount of H2O adsorbed (absolute hydration) in presence of the heavy isotope (deuterium D), and isothermals of H2O/D2O fractionation at protein surface groups are presented for temperatures between -10 degrees C and -35 degrees C and solutions of varying composition. The fractionation factor, phi = [x/(1 - x)]/[x(0)/(1 - x(0))], where x and x(0) are the fractions of deuterons in hydration and bulk water, respectively, appeared to be extremely high: phi > 1 at 0.03 < x(0) < 0.10. The high values of phi indicate a decrease in apparent hydration of protein molecules. A probable reason of the effect can be an inter-protein molecular solvent-mediated interaction induced by D2O. The excess of phi over 1 appears to provide a quantitative estimate of the fraction of hydration water affected by such interaction.     

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D₂O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (R_i) of 3 the lamellar gel (L or L_c)-to-hexagonal type II (H_II) phase sequence is observed on increasing the temperature. For R_i3 the thermotropic sequence (L or L_c)-L-H_II is detected. On increasing hydration from R_i=3, the H_II phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, L and H_II phases are R_i 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the L to the H_II phase.Abbreviations NMR Nuclear magnetic resonance - DDPE 1,2-Didodecyl-rac-glycerol-3-phosphoethanol-amine - DHPE 1,2-Dihexadecyl-sn-glycerol-3-phosphoethanol-amine - DOPE 1,2-Dioleoyl-sn-glycerol-3-phosphoethanol-amine - POPE 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphoetha-nolamine - DAPE 1,2-Diarachinoyl-sn-glycerol-3-phosphoethanol-amine - DMPC 1,2-Dimyristol-sn-glycerol-3-phosphocholine - DPPC 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine - T_c lamellar gel-to-lamellar fluid transition temperature - T_h lamellar fluid-to-hexagonal transition temperature     

Effect of inhibitor complex formation on the hydration properties of alpha-chymotrypsin. Changes induced in protein hydration by toxylation of the native enzyme

The effect of enzyme-inhibitor complex formation on the hydration properties of the macromolecular moiety was investigated on the model system of α-chymotrypsin and its Ser-195 tosyl derivative. The primary (A-shell) hydration of the native and modified enzyme was compared by sorption measurements. The secondary (B-shell) hydration water was investigated by differential scanning calorimetry. Tosylation is known to induce pronounced conformational changes in the chymotrypsin molecule. These structural modifications have the following effects on the hydration of the native enzyme. The water binding capacity of the protein surface is significantly increased, as shown by both the calorimetric and the sorption results. The amount of unfreezable water of primary hydration is increased by 50 mol H₂O/mol chymotrypsin. The heats (ΔH ) and entropies (ΔS ) of the interaction of water with chymotrypsin are strongly reduced in the modified enzyme. This effect is interpretable by a reduction of the H bonding potential of the protein surface. Parallel to this decrease in δH , the heats of fusion of the secondary hydration water (Q_fus) are significantly increased by tosylation (Q_fus = 256.2 ± 7.8 and 294.2 ± 4.8 J g^?1 H₂O for the native and the tosylated enzyme, respectively). This increase in Q_fus reflects an increase in the extent of H bonding in the B-shell hydration sphere. These changes in the hydration of the native enzyme, associated with the reaction: native chymotrypsin → tosylchymotrypsin, are interpreted by cooperative phase transitions of water molecules in the primary and secondary hydration water. One of these transitions was found to exhibit a significant, linear enthalpy–entropy compensation effect. The compensation temperature \documentclass{article}\pagestyle{empty}\begin{document}$ \hat{\beta} $\end{document} is 290.7 ± 2.8°K. This \documentclass{article}\pagestyle{empty}\begin{document}$ \hat{\beta} $\end{document} value agrees well with compensation temperatures reported in the literature for a series of biochemical reactions in aqueous solution (250–320° K). This agreement in \documentclass{article}\pagestyle{empty}\begin{document}$ \hat{\beta} $\end{document} may point to a common source of both compensation phenomena.     

D2O-induced cell excitation

Summary The effects of deuterium oxide (D₂O) on giant internodal cells of the fresh water algaChara gymnophylla, were investigated. D₂O causes membrane excitation followed by potassium leakage. The primary effect consists of an almost instantaneous membrane depolarization resembling an action potential with incomplete repolarization. A hypothesis was proposed which deals with an osmotic stress effect of D₂O on membrane ion channels followed by the supression of the electrogenic pump activity. The initial changes (potential spike and rapid K⁺ efflux) may represent the previously undetected link between the D₂O-induced temporary arrest of protoplasmic streaming and the early events triggered at the plasma membrane level as the primary site of D₂O action.     

On the density of intracellular water

The density of individualArtemia cysts has been determined by sedimentation velocity measurements at unit gravity. Dried cyst (< 0.02 g H₂O/g dry weight) densities, _s were obtained by successive sedimentation in two nonpenetrating organic solvents. This removes geometric terms from the equation relating density to sedimentation velocity. Hydrated cysts ( 1.68 g H₂O/g dry weight) were sedimented in 0.0750 m NaCl to obtain their density ( _c). Values of _s, _c, and their ratios were found to be independent of cyst volume; therefore, the weight fraction of water in hydrated cysts is very nearly the same in cysts of greatly different size. It can be concluded that measurement of the water content of large populations of these cysts accurately reflects the water content of individual cysts, a point which has been assumed in previous work on this system. If _s does not change appreciably when dried cysts are fully hydrated then the density of their water, _w, can be calculated to be 1.022 g/cm³ (±0.0011 ). That value is significantly higher than the density of pure water and is very close to estimates of _w in skeletal muscle and amphibian oocytes obtained by others. However, the assumption that _s is independent of hydration is open to serious criticism, for all these studies. Consequently, conclusions and interpretations derived from such measurements must be considered to be tentative and uncertain.     

Hydration Properties of the Molecular Chaperone α-Crystallin in the Bovine Lens

Topographic studies of crystalline fractions from different morphological layers of the young adult bovine lens were conducted. Crystallin profiles were obtained for each lens layer, using thin-layer isoelectric focusing in polyacrylamide gel (IEF). Water soluble (WS) crystallins from the lens equator revealed a separation into HM (high molecular weight) _L-, _H-, _L-, _S-, and -crystallins. The nature of the water insoluble (WI) protein fraction in the separated lens layers reflected the aggregated state of _L-, _L-, _S-, and -crystallins in different regions of the lens, concealed in the central cavity of the -crystallin chaperone model. The IEF data demonstrate a possible chaperone-like function for -crystallin in the nucleus and inner cortex of the lens, but not in the outer cortex. The water binding properties of bovine lens -crystallin, calf skin collagen, and bovine serum albumin (BSA) were investigated with various techniques. The water adsorptive capacity was obtained in high vacuum desorption experiments volumetrically, and also gravimetrically in controlled atmosphere experiments. The NMR spin–-echo technique was used to study the hydration of protein samples and to determine the spin–-spin relaxation times (T₂) from the protons of water adsorbed on the proteins. Isolated bovine lenses were sectioned into 11-12 morphological layers (from anterior cortex through nucleus to posterior cortex). The water content in relation to dry weight of proteins was measured in individual morphological lens layers. During water vapor uptake at relative humidity P/P₀ = 0.75, -crystallin did not adsorb water suggesting that hydrophobic regions of the protein are exposed to the aqueous solvent. At relative humidity P/P₀ = 1.0, the adsorption of water by -crystallin was 17% with a single component decay character of spin echo (T₂ = 3 msec). Addition of water to -crystallin to about 50% of its weight/weight in the protein sample showed T₂ = 8 msec with only one single component decay of the spin–-echo signal. The single component decay character of the spin echo indicates water tightly bound by -crystallin. Under a relative humidity P/P₀ = 1.0, collagen and BSA adsorbed, correspondingly, 19.3 and 28% of water and showed a two-component decay curve with T₂ about 5 and 40 msec. The findings demonstrate the presence of two water fractions in collagen and BSA which are separated in space. The IEF data suggest a tight binding of water with -crystallin with similar distribution patterns in the lens layers. To conclude, it was found that -crystallin can immobilize water to a greater extent than other proteins such as collagen and BSA. These results shed new light on structural properties of -crystallin and its superhydration properties and have important implications for understanding the mechanism of the chaperone-like action of this protein in the lens and non-ocular tissues.     

Structural and dynamical examination of the low-temperature glass transition in serum albumin

The nuclear magnetic transverse decay and the proton second moment of bovine serum albumin samples dry and hydrated with different water isotope compositions show that at temperatures around 170 K, there is a dramatic change in the dynamics of the water associated with the protein interface. By comparison, observation of the protein protons when hydrated with deuterium oxide provides no evidence for significant dynamical changes near 170 K. The proton second moment of the hydrated protein shows that the protein structure becomes more open with increasing hydration from the lyophilized condition and that the side chains extend from the protein surface into the solvent in the hydrated but not the dry cases. The proton second moment of serum albumin hydrated with H(2)O increases dramatically with decreasing temperature near 170 K, demonstrating that the water forms a rigid solid around the protein which effectively fills the surface irregularities created by the protein fold. Solvation with dimethyl sulfoxide yields small effects compared with water.     

Pseudocyphellaria dissimilis: a desiccation-sensitive,highly shade-adapted lichen from New Zealand

Summary Pseudocyphellaria dissimilis, a foliose, cyanobacterial lichen, is shown not to fit into the normal ecological concept of lichens. This species is both extremely shade-tolerant and also more intolerant to drying than aquatic lichens previously thought to be the most desiccation-sensitive of lichens. Samples of P. dissimilis from a humid rain-forest site in New Zealand were transported in a moist state to Germany. Photosynthesis response curves were generated. The effect of desiccation was measured by comparing CO₂ exchange before and after a standard 20-h drying routine. Lichen thalli could be equilibrated at 15° C to relative humidities (RH) from 5% to almost 100%. Photosynthesis was saturated at a photosynthetically active radiation (PAR) level of 20 mol m^-2 s^-1 (350 bar CO₂) and PAR compensation was a very low 1 mol m^-2 s^-1. Photosynthesis did not saturate until 1500 bar CO₂. Net photosynthesis was relatively unaffected by temperature between 10° C and 30° C with upper compensation at over 40° C. Temporary depression of photosynthesis occurred after a drying period of 20 h with equilibration at 45–65% relative humidity (RH). Sustained damage occurred at 15–25% RH and many samples died after equilibration at 5–16% RH. Microclimate studies of the lichen habitat below the evergreen, broadleaf forest canopy revealed consistently low PAR (normally below 10–20 mol m^-2 s^-1) and high humidities (over 80% RH even during the day time). The species shows many features of an extremely deep shade-adapted plant including low PAR saturation and compensation, low photosynthetic and respiratory rates and low dry weight per unit area.     

Modelling endplate currents: dependence on quantum secretion probability and decay of miniature current

Quantification of the time course and amplitude of endplate currents (EPC) was made with respect to dispersion of quanta secretion and to changes in the exponential decay of miniature endplate currents (_mepc). The relationship between RPC amplitude and _mepc follows a double-exponential curve with ₁= 0.3 ms and ₂ = 6 ms. If the amplitude of fully synchronised EPC is taken as 100%, then the loss of EPC amplitude is already 42% with physiological parameters of dispersion (the half-rise and decay constant of distribution of secretion probability = 0.5 ms, _mepc =1 ms). This loss is even more substantial if secretion is more dispersed or miniature endplate currents decay faster. Correspondence to: F. Vyskocil     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

Spatio – Temporal Features of Protein Specific Motions. The Influence of Hydration

The angular dependencies of inelastic intensities of Rayleigh scatteringof Moessbauer radiation were measured for lysozyme and myoglobin (fordifferent degrees of hydration: from h = 0.05 till h = 0.7). The treating ofthe data at h > 0.05 approves the existence of segmental motions(-helices for myoglobin, -helices and -sheets forlysozyme) as well as of individual motions. Further hydration increase themean-square displacements for both types of intraglobular motions for theseproteins, while the motions of the globule as a whole remain nearlythe same as for h = 0.05. Results of the study of the radial distributionfunction deduced by Fourier – transform from the diffuse x-raymeasurements together with RSMR data allow to conclude that the waterduring hydration of proteins competes with the intramolecular hydrogenbonds, loosens the protein and increases the internal dynamics. At the sametime water arranges the ordering of macromolecule from `glassy' state ath 0.02 to the native state at h = 0.4–0.7. Differentarchitecture of proteins leads to the different structural dynamics as in thecase of lysozyme and myoglobin.     

Thermodynamic analysis of nonelectrolyte permeation across the toad urinary bladder

Summary Permeability coefficients (P's) and apparent activation energies (E _a s) for nonelectrolyte permeation across the toad urinary bladder have been analyzed in terms of the thermodynamics of partition between membrane lipids and water. Particular attention has been paid to the contributions made by –CH₂– and –OH groups: on the average, the addition of one –CH₂– group to a molecule increasesP fourfold, while the addition of one –OH group reducesP 500-fold. Using these changes inP, we have calculated the incremental free energies (F), enthalpies (H), and entropies (S) for partition, hydration, and solution in membrane lipids. The results for toad bladder have been compared and contrasted with those extracted from the literature for red blood cells, lecithin liposomes, and bulk phase lipid solvents. The partition of –CH₂– groups into toad bladder and red cell membranes is dominated by entropy effects, i.e., a decrease in entropy of the aqueous phase that pushes the group out of water, and an increase in entropy of the membrane lipid that pulls the group into the membrane. This process resembles that in frozen liposome membranes. In melted liposomes and bulk lipid solvents the free energy of solution in the lipid is controlled by enthalpy of solution. Partition of –OH groups in all systems is governed by hydrogen bonding between the –OH group and water. However, the solution of the –OH group in toad bladder membranes is complex, and processes such as dimer and tetramer formation in the lipid phase may be involved. The results presented in this and the previous paper are discussed in terms of the structure of phospholipid bilayer membranes. Attention is drawn to the possible role of structural defects in the quasi-crystalline structure of the lipid (so-called 2gl kinks) in the permeation of small molecules such as water, urea, methanol and acetamide.