Effect of formaldehyde on the level of cytochrome c in methylotrophic yeasts

The effect of formaldehyde on the cytochrome system of the methylotrophic yeasts Candida tropicalis, Candida boidinii, Candida methylica, Hansenula capsulata, Hansenula polymorpha, Pichia pastoris and nonmethylotrophic yeasts Saccharomyces cerevisiae was studied. In the investigated concentration range of 0.58 – 2.5 mol · 1^?1, formaldehyde decreases the level of the reduced cytochrome c in the above-mentioned microorganisms. The kinetics of the change in the reduced cytochrome c content has two phases: a phase of slow decrease and a phase of complete disappearance of the reduced cytochrome c in the cells. The duration of the first phase shortens with increasing concentrations of formaldehyde.     

Studies on the Critical Micellar Concentration and Phase Transitions of Stearoylcarnitine

The critical micellar concentration (CMC) of stearoylcarnitine was determined at different pH values at room temperature by fluorescence spectroscopy, monitoring the spectral changes of 8-anilinonaphthalene-1-sulfonate (ANS). The CMC was found to vary with pH, increasing from about 10 μM at pH 3.0 to ca. 25 μM at pH 7.0, but decreasing slightly with further increase in pH to approximately 19 μM at pH 10.0. Differential scanning calorimetry (DSC) shows that stearoylcarnitine dispersed in water at low concentration undergoes a broad thermotropic phase transition at 44.5°C, with a transition enthalpy of 15.0 kcal/mol. The transition temperature (T _t) shifts to ca. 50.5°C in the presence of 1 mM EDTA or when the concentration is increased significantly. The turbidity of aqueous dispersions of stearoylcarnitine was found to be considerably high at low temperatures, which decreases quite abruptly over a short temperature range, indicating that a transition occurs from a phase of large aggregates to one of much smaller aggregates, most likely micelles. The phase transition temperature was determined as 29.1°C at pH 3.0, which increased with increasing pH up to a value of 55.3°C at pH 8.6 and remains nearly constant thereafter up to pH 11.2. The pH dependence of CMC and T _t suggest that the pK _a of the carboxyl group of long chain acylcarnitines shifts to higher temperatures upon aggregation (micelles or bilayer membranes).     

Low-Moisture Food: A Physicochemical Approach to Investigate the Origin of Their Physical Instability versus Water or Sucrose

Low-moisture biopolymer-based systems are commonly encountered in food. Obviously, understanding the physical basis of their quality [texture, or performance over time or as a function of their composition (water or other added solutes)] is of primary importance. A polymer science approach using physical chemistry concepts based on physical state, phase transitions and molecular mobility can be applied to investigate the performances of food in particular versus moisture. Based on the example of starch-based samples and their texture property changes versus composition, the role of water and sucrose is considered through different aspects. The relations existing between the observed changes and physical state are investigated. While the motions associated with the glass transition were observed at high temperature, secondary relaxations are observed below Tg (at T _β): T _β decreased with water content and increased with increasing sucrose content. These local motions are suggested to contribute to the observed texture modifications versus water. Moreover, the stability of the glassy state was investigated by differential scanning calorimetry through the study of enthalpy relaxation (physical ageing). The amplitude of enthalpy relaxation decreased with both increasing sucrose and water content. All in all, this study strengthened the hypotheses that sub-Tg mobility could contribute to texture instability versus moisture or sugar content.     

Etude spectrale à basse temperature chez Euglena gracilis Z en culture synchrone sur milieu lactate: Variation de teneur en cytochrome 556

Summary Low temperature spectra are described for whole Euglena cells. Euglena growing in synchronous culture with lactate medium show a cyclic variation of cytochrome 556 content during each cellular generation. The greatest quantity of cytochrome 556 seems to coincide with the non-dividing phase of the cells, the phase in which the mitochondrial network is observed. On the other hand after treatment of the Euglena with antimycin A, a correlation exists between the formation of giant mitochondria and an increase in the quantity of cytochrome 556.These results demonstrate the existence of a cyclic variation of cytochrome 556 synthesis in Euglena during synchronous growth on lactate medium.     

Structural Polymorphisms of Rough Mutant Lipopolysaccharides Rd to Ra from Salmonella minnesota

The structural polymorphisms of rough mutant lipopolysaccharides (LPS) Rd, Rc, Rb, and Ra from Salmonella minnesota (strains R4, R7, Rz, R5, R345, and R60, respectively) were investigated as a function of temperature, water content, and Mg²⁺ concentration. The gel to liquid crystalline (B↔α) phase transition temperature (T_c) and the state of order within each phase were measured by Fourier transform infrared spectroscopy. The amount of bound water was determined by differential scanning calorimetry and the three-dimensional structures in each phase state were characterized by synchrotron radiation X-ray diffraction. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The B↔α acyl chain melting temperatures at high water contents (95-97%), T_c = 31 to 32°C for LPS Rd, 33 to 35°C for LPS Rc to Rb, and 36°C for LPS Ra, increase with decreasing water content and in the presence of Mg²⁺ cations with a concomitant broadening of the transition range. Below 30 to 50% water content, no distinct phase transitions can be observed. These effects are most pronounced for LPS with the shortest sugar chains. Below 50% water content, only lamellur structures can be observed in the temperature range 5 to 80°C, independent of the Mg²⁺ concentration. Above 50% water concentration, for large [LPS]:[Mg²⁺] molar ratios the predominant structure above T_c is nonlamellar; for smaller [LPS]:[Mg²⁺] molar ratios a superposition of lamellar and nonlamellar structures is found. For all LPS Rd to Rb at low Mg²⁺ concentrations, the phase transition is connected with a change in the three-dimensional structure from lamellar or mixed lamellar/nonlamellar to a pure nonlamellar, probably cubic structure. The tendency to form nonlamellar structures decreases with the length of the core oligosaccharide. At an equimolar ratio of [LPS] and [Mg²⁺] a multibilayered organization is observed. Some of the nonlamellar structures are cubic phases with periodicities between 12 and 16 nm. The molecular dimensions of the single endotoxin molecules in the absence and the presence of external water are estimated from the different lamellar periodicities, including those of free lipid A and deep rough mutant LPS Re. These observations are discussed with respect to the biological importance of LPS as a potent inducer of biological effects in mammals.     

Dimyristoylphosphatidic acid/cholesterol bilayers

Lipid bilayers and monolayers composed of dimyristoylphosphatidic acid (DMPA) and cholesterol were characterized by differential scanning calorimetry and film balance measurements. Increasing cholesterol content decreases the bilayer phase transition temperature and enthalpy in a manner similar to that observed before for other lipid/cholesterol systems. In monomolecular films at the air-water interface cholesterol exhibits the well known condensing effect in the liquid-expanded phase, while the liquid-condensed phase is less affected. As with the bilayer phase transition, the transition temperature and change in area at the liquid-condensed to liquid-expanded phase transition, as measured from isobars at 25 dynes/cm, decreases with increasing cholesterol content. The kinetics of the phase transition of DMPA/cholesterol bilayers were measured using the pressure jump relaxation technique with optical detection. Three relaxation times were observed. The relaxation times and amplitudes pass through maximum values at the transition midpoint. With increasing cholesterol content the maximum values of the relaxation times decrease but not in a linear fashion. The time constants display an intermediate maximum at ca. 10% to 12 mol% cholesterol. This observation is discussed in terms of a possible change in the nature of the phase transition from first-order with phase separation to a continuous second-order transition. The dependence of the relaxation amplitudes on cholesterol content gave evidence for nucleation being the rate limiting step for the transition in this particular system.Abbreviations DMPA dimyristoylphosphatidic acid - DMPC dimyristoylphosphatidylcholine - DMPE dimyristoylphosphatidylethanolamine - DPPC dipalmitoylphosphatidylcholine - DSC differential scanning calorimetry Part of this research has been presented at the VIII. Discussion Group Meeting Fast Reactions in Solution of the Royal Society of Chemistry and the Max-Planck-Gesellschaft, Berlin, 26th–29th August 1984     

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide

We have reported here on the structural polymorphism of lipid A, the “endotoxic principle” of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg²⁺] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70°C. These data were correlated with measurements of the β→α phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges—in the absence of Mg²⁺—from 45°C at high to 56°C at low water content, and—at an equimolar content of Mg²⁺—from 52°C at high to 59°C at low water concentrations. In the gel phase—in which the lipid A acyl chains are more disordered than those from saturated phospholipids—cubic phases are adopted at high water content (>60%) and at high [lipid A):[Mg²⁺] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal H_II, state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg²⁺ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.     

Separation of proteins and viruses using two-phase aqueous micellar systems

We discuss the utilization of a novel two-phase aqueous nonionic micellar system for the purification and concentration of biomolecules, such as proteins and viruses, by liquid–liquid extraction. The nonionic surfactant n-decyl tetra(ethylene oxide), C₁₀E₄, phase separates in water into two coexisting aqueous micellar phases by increasing temperature. The mild interactions of the C₁₀E₄ nonionic surfactant with biomolecules, combined with the high water content of the two coexisting micellar phases, suggest the potential utility of two-phase aqueous C₁₀E₄ micellar systems for the purification and concentration of biomolecules. In this paper, we review our recent experimental and theoretical studies involving the partitioning of several water-soluble proteins, including cytochrome c, soybean trypsin inhibitor, ovalbumin, bovine serum albumin, and catalase, in the two-phase aqueous C₁₀E₄ micellar system. In addition, we present results of our preliminary experimental investigation on the partitioning of bacteriophages, including φX174, P22, and T4.     

Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

Effect of carbon source on the accumulation of cytochrome P-450 and cytochrome c in the yeast Rhodosporidium toruloides

The appearance of cytochrome content in the yeast Rhodosporidium toruloides depended on the substrate supporting growth. The cells of R. toruloides growing on benzoate were found to contain cytochrome P-450. The concentration of cytochrome P-450 was maximal at the beginning of the exponential phase and then remained at a relatively constant level, but rapidly decreased at the beginning of the stationary phase. When benzoate was exhausted from the medium, the cytochrome P-450 level decreased to zero. On the other hand, cytochrome P-450 was not detected when R. toruloides grew on glucose. However, cytochrome P-450 was detected, when R. toruloides was grown on benzoate together with glucose.
The maximal content of cytochrome c in the cells was observed at the beginning of the exponential phase of growth on both substrates and decreased most rapidly during late stationary phase of growth. The content of cytochrome c in R. toruloides was 2–3 times lower during the growth on glucose as compared to the growth on benzoate.     

Conformational transitions of schizophyllan in aqueous alkaline solution

The conformational transitions of schizophyllan were studied in aqueous alkaline solutions by high-sensitivity differential scanning calorimetry (DSC) and optical rotation measurements. The temperature of half completion for reversible intramolecular conformational transition determined by DSC, centered at 7.4°C in water, increases to 37.2°C at 0.01M KOH with increasing alkaline concentration. The transition enthalpy per mole of the polysaccharide repeating unit is 2.62 ± 0.23 kJ mol⁻¹ independent of the alkaline concentration. The cooperative unit size for the transition decreases with increasing alkaline concentration. Optical rotation was measured as a function of pH at 25 and 60°C. A sharp decrease in optical rotation was observed at pH = 13, which is ascribed to the triple helix-coil transition. From data obtained by DSC and optical rotation measurements, in combination with results reported previously, a phase diagram for the conformation of schizophyllan as a function of temperature and pH is proposed. The irreversibility of the triple helix to single coil transition, induced by strong alkali, was investigated as a function of polymer concentration by gel permeation chromatography and electron microscopy. The renatured samples at polymer concentrations < 1.0 mg/mL, which are prepared by dissolution in 0.25M KOH followed by neutralization with HCl, are observed as a mixture of globular, linear, and circular structures, and larger aggregates with less-defined morphology by electron microscopy. Higher concentrations lead to increased proportions of multichain clusters (aggregates). Subsequent annealing of the renatured samples at 115–120°C increases the proportion of circular species. The change in molecular weight distribution of samples that accompanies the renaturation and annealing mentioned above can be well interpreted in terms of the proportion of species having different morphology as observed by electron microscopy. © 1996 John Wiley & Sons, Inc.     

Respiratory activity in the marine cyanobacterium Spirulina subsalsa and its role in salt tolerance

Intracellular ion concentration and respiratory activity in the marine cyanobacterium Spirulina subsalsa was analyzed during cell transition from saline to hypersaline medium. During salt upshock, an early phase of Na⁺ and Cl^- influx was observed, followed by an adaptation phase where both Na⁺ and Cl^- were excluded from the cell. Respiration in intact cells was enhanced during salt upshock. S. subsalsa spheroplasts exhibited a high rate of O₂ uptake, which was further enhanced in cells grown in hypersaline medium, upon addition of NaCl to the assay mixture. This effect was found to be specific to sodium ions. Plasma membrane fractions from cells grown in hypersaline medium exhibited a high rate of cytochrome oxidase activity, which was further stimulated by NaCl, and was sensitive to DCCD. Immunoblot analysis of Spirulina plasma membrane polypeptides with anti-cytochrome oxidase serum demonstrated high content of 53.4 kDa polypeptide of cytochrome oxidase, which was enriched in membranes obtained from hypersaline Spirulina cells. The enhanced respiration, and more specifically the enrichment of cytochrome oxidase activity in salt-adapted cells in situ, as well as its stimulation by NaCl in vitro and inhibition by DCCD, suggest that cytochrome oxidase is involved in the extrusion of sodium ions from cells of the salt-tolerant Spirulina subsalsa.Abbreviations DCCD dicyclohexylcarbodiimide - CCCP carbonylcyanide m-chlorophenyl hydrazone - TMPD N, N, N, N, tetramethyl p-phenylenediamine dichloride     

Stability of Lycopene in Oil‐in‐Water Emulsions

Lycopene can be dissolved within the oil phase of oil‐in‐water emulsions to increase bioavailability in water‐dispersible systems. It is sensitive to oxidative conditions and easily undergoes isomerization at high temperatures. Degradation kinetics and isomerization of lycopene in oil‐in‐water‐emulsions were investigated as a function of thermal treatment and oxygen content. Lycopene degradation was found to follow a first‐order kinetics and rate constants were determined. Higher temperatures are directly correlated with increasing lycopene losses. Moreover, thermal treatment leads to a significant decrease of the concentrations of all‐trans and 13‐cis isomer, while the concentration of the 9‐cis isomer increased. Oxygen‐free conditions reduce lycopene losses significantly.     

Phase diagram of deep rough mutant lipopolysaccharide from Salmonella minnesota R595.

The structural polymorphism of deep rough mutant lipopolysaccharide--in many biological systems the most active endotoxin--from Salmonella minnesota strain R595 was investigated as function of temperature, water content, and Mg2+ concentration. Differential scanning calorimetry was used to determine the amount of bound water and the enthalpy change at the beta<==>alpha gel to liquid crystalline acyl chain melting. The onset, midtemperature Tc, and completion of the beta<==>alpha phase transition were studied with Fourier-transform infrared spectroscopy. Synchrotron radiation X-ray diffraction was used to characterize the supramolecular three-dimensional structures in each phase state. The results indicate an extremely complex dependence of the structural behavior of LPS on ambient conditions. The beta<==>alpha acyl chain melting temperature Tc lying at 30 degrees C at high water content (95%) increases with decreasing water content reaching a value of 50 degrees C at 30% water content. Concomitantly, a broadening of the transition range takes place. At still lower water content, no distinct phase transition can be observed. This behavior is even more clearly expressed in the presence of Mg2+. In the lower water concentration range (< 50%) at temperatures below 70 degrees C, only lamellar structures can be observed independent of the Mg2+ concentration. This correlates with the absence of free water. Above 50% water concentration, the supramolecular structure below 70 degrees C strongly depends on the [LPS]:[Mg2+] ratio. For large [LPS]:[Mg2+] ratios, the predominant structure is nonlamellar, for smaller [LPS]:[Mg2+] ratios there is a superposition of lamellar and nonlamellar structures. At an equimolar ratio of LPS and Mg2+ a multibilayered organization is observed. The nonlamellar structures can be assigned in various cases to structures with cubic symmetry with periodicities between 12 and 16 nm. Under all investigated conditions, a transition into the hexagonal II structure takes place between 70 and 80 degrees C. These observations are discussed in relation to the biological importance of LPS as constituent of the outer membrane of gram-negative bacteria and as potent inducer of biological effects in mammals.     

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     

Zinc ions as cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase inhibitors: two sites of action

Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50–90%,depending on preparation) with an effective K _i of 10 µM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes,and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme:full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 µM. The rate of zinc inhibitory action in the slow phase is proportional to Zn²⁺ concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F O step corresponding to transfer of the 4th electron in the catalytic cycle).Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 160–170.Original Russian Text Copyright © 2005 by Kuznetsova, Azarkina, Vygodina, Siletsky, Konstantinov.This revised version was published online in April 2005 with corrections to the post codes.     

Application of the Entropy-Enthalpy Relationship in Water Transport through Plant Roots

Water transport through plant roots is determined by a single layer of cells, so that water passes through a plasmamembrane-cytoplasm-plasmamembrane system. The water transport shows an exponential relationship with temperature in two phases with an abrupt transition. The Arrhenius activation parameters log A and E are calculated for the two phases of water transport below and above the transition temperature. Between log A and E two linear and parallel relationships are observed, one for each phase of water transport. The difference of log A between these two relationships is a measure for a change in entropy in cell water structure at the transition temperature. The change in entropy was small (13.4 J · mol^?1· K^?1) in comparison to the difference in activation energy E for water transport above and below the transition temperature. The role of the plasmamembrane and cytoplasm in determining the cell water structure is discussed.     

Temperature and emergence effects on the net photosynthesis of two Zostera noltii Hornem. morphotypes

Apparent photosynthetic rates (APS) of two Zostera noltii Hornem. morphotypes were measured in air and in water at different temperatures with a closed infra-red gas analysis system (IRGA).Hyperbolic functions accurately described the photosynthesis-CO₂ relationships when the leaves were exposed to air. The photosynthetic behaviour in water, on the contrary, could not be described by Michaelis type kinetics, due to the existence of a rapid transition from the initial slope to the saturation phase. Both morphotypes (narrow-leaved, NLM and large-leaved, LLM) showed higher APS rates in water than in air, although the highest APS rates, in air as well in water, were recorded for the NLM.Temperature had a significant influence on the photosynthetic parameters: APS_max (maximum photosynthetic rate) decreased (in air and in water) with increased temperature in both morphytypes; compensation points (CP) in air increased at high temperature, especially in the LLM. NLM specimens showed enhanced affinity (lower Km) with increasing temperature in air. On the contrary, Km values in water were not significantly affected by temperature.The results suggest that NLM specimens are better adapted than the LLM to occur exposed to air. The distributional pattern of the two morphotypes in the Palmones Estuary is discussed on the basis of their photosynthetic behaviour.     

Studies on protein-lipid interactions in cytochrome c oxidase by differential scanning calorimetry

The interaction between cytochrome c oxidase and phospholipids was studied by differential scanning calorimetry. The active, lipid-sufficient cytochrome c oxidase undergoes thermodenaturation at 336 K with a relatively broad and concentration dependent endothermic transition. The delipidated enzyme shows an endothermic denaturation temperature at 331.3 K. When the delipidated cytochrome c oxidase was treated with chymotrypsin, a lowered thermodenaturation temperature was observed. When the delipidated cytochrome c oxidase was reconstituted with asolectin to form a functionally active enzyme complex, the thermodenaturation shifted to a higher temperature, with a sharper transition thermogram. The increase in thermotransition temperature and enthalpy change of thermodenaturation of the asolectin-reconstituted enzyme is directly proportionate to the amount of asolectin used, up to 0.5 mg asolectin per mg protein. The thermotransition temperature and enthalpy changes of thermodenaturation for the phospholipid-reconstituted cytochrome c oxidase are affected by the phospholipid headgroup and the fatty acyl groups. Among phospholipids with the same acyl moiety but different head groups, phosphatidylethanolamine was found to be more effective than phosphatidylcholine in protecting cytochrome c oxidase from thermodenaturation. An exothermic transition thermogram was observed for delipidated cytochrome c oxidase embedded in phospholipid vesicles formed with phospholipids containing unsaturated fatty acyl groups. The increase in exothermic transition temperature and exothermic enthalpy change of thermodenaturation of the oxidase-cytochrome c-cytochrome c oxidase complex destabilized cytochrome c but not cytochrome c oxidase toward thermodenaturation.     

Protein elasticity based on conformations of sequential polypeptides: The biological elastic fiber

Fibrous elastin of the biological elastic fiber is a cross-linked condensed state in which there is roughly one-half polypeptide and one-half water. The precursor protein tropoelastin, a chemical fragmentation product -elastin, and a sequential polypeptide (l·Val¹-l·Pro²-Gly³-l·Val⁴-Gly⁵) _n , which is a prominent primary structural feature of tropoelastin, are each soluble in all proportions in water at 20°C. On heating to physiological temperatures, each undergoes aggregation and forms a dense viscoelastic phase, which as the fiber itself, is about 60% water. This reversible heat-elicited condensed phase is called the coacervate. Circular dichroism studies show coacervation to be a process of increasing intramolecular order. Electron microscopy (light, scanning, and transmission) shows coacervation to be a process of increasing order intermolecularly. Thus a rise in temperature between 20 and 40°C results in an increase in order of the polypeptide. Coacervation is an inverse temperature transition, and the condensed state is anisotropic at the molecular level. Thermoelasticity studies in water on bovine ligamentum nuchae fibrous elastin and on -irradiation cross-linked polypentapeptide coacervates show increases in elastomeric force,f, over the same 20–40°C temperature range in which the inverse temperature transition gives rise to the coacervate, and the constancy off/T with temperature, once the transition is effectively completed, suggests a high-entropy component to the elastomeric force. Thus the data argue for an anisotropic-entropic elastomer.Detailed conformational studies on the polypentapeptide result in the development of a -spiral conformation in which there are regularly recurring -turns in loose helical array (a structure that forms on raising the temperature) and in which there are recurring dynamic suspended segments that are the focal point of large, low-energy oscillatory motions called librations. The structure gives rise to a librational entropy mechanism of elasticity wherein the amplitudes of the rocking motions become damped on stretching. This perspective is substantiated by dielectric relaxation studies on the coacervate state and by characterization of synthetic analogs of the polypentapeptide. Dielectric relaxation studies on a concentrated state of about 60% water show the development of a regular structure over the same temperature range as for the development of the coacervate state, and the development of the regular structure with increasing temperature is seen to parallel the development of elastomeric force with increasing temperature. Increasing elastomeric force coincides with increasing regularity of structure! Synthetic analogs of the polypentapeptide, designed to interfere with the librational processes of the suspended segment, impair elastic function, and an analog that makes the -turn more rigid results in increased elastic modulus. This development of a librational entropy mechanism for protein elasticity is a departure from the kinetic theory of rubber elasticity, the random network perspective that has dominated the traditional view of biological elasticity for the past several decades. The new perspective opens the way to insightful consideration of new elastomeric biomaterials with numerous biomedical applications.