Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study

The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, v_j, within the lipid matrix. At T = 35°C a value of v_j = 1.6 · 10³ s^?1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and charge of the lipids we obtain jump frequencies between 0.8 · 10⁸ s^?1 and 1.5 · 10⁸ s^?1 at T = 35°C. The results are compared with jump frequencies yielded in model membranes.The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{min at}\text{T = 37°}\text{C}$) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 100}\text{min at}\text{T = 37°}\text{C}$.The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 23°C and T = 33°C we observed an additional fluidity change at T = 38°C in erythrocyte ghosts. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.     

Raman spectroscopy of the milk globule membrane and triglycerides

The acyl chain mobilities of the lipids of bovine milk fat globules and the component triglycerides have been determined by Raman spectroscopy as a function of temperature from -100°C to 80°C. A broad transition is observed centered about 2–6°C. The C-H and C-C stretching bands in the spectra of liquid and crystalline triglycerides are used comparatively to show that the lipids of the milk globule membrane are 30–40% more ordered than the lipids of the intact milk fat globules at 20°C. Synthetic triglyceride melts, quenched rapidly, are used to illustrate the effect of the thermal history of a sample on lipid order as determined spectroscopically.Strong infrared amide I and amide II bands at 1646 and 1543 cm^?1, respectively, indicate that the protein conformation of the globule membrane is not characterized by extensive regions of beta-sheet structure. Raman spectra of the globule triglycerides indicate cis unsaturation of $\text{39 ± 5\%}$ by comparison to triolein and trielaidin.     

Thermal phase transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relation to chilling injury

The phase behaviour of leaf polar lipids from three plants, varying in their sensitivity to chilling, was investigated by differential scanning calorimetry. For the lipids from mung bean (Vigna radiata L. var. Berken), a chilling-sensitive plant, a transition exotherm was detected beginning at $\text{10 ± 2°}\text{C}$. No exotherm was evident above 0°C with polar lipids from wheat (Triticum aestivum cv. Falcon) or pea (Pisum sativum cv. Massey Gem), plants which are insensitive to chilling. The enthalpy for the transition in the mung bean polar lipids indicated that only about 7% w/w of the lipid was in the gel phase at ?8°C. The thermal transition of the mung bean lipids was mimicked by wheat and pea polar lipids after the addition of 1 to 2% w/w of a relatively high melting-point lipid such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol or dimyristoylphosphatidylcholine. Analysis of the polar lipids from the three plants showed that a dipalmitoylphosphatidylglycerol was present in mung bean (1.7% w/w) and pea (0.3% w/w) but undetected in wheat, indicating that the transition exotherm temperature of 10°C in mung bean, 0°C in pea and about ?3°C in wheat correlates with the proportion of the high melting-point disaturated component in the polar lipids. The results indicate that the transition exotherm, observed at temperatures above 0°C in the membranes of chilling-sensitive plants, could be induced by small amounts of high melting-point lipids and involves only a small proportion of the membrane polar lipids.     

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373–377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues ($\text{n = 3}$ through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by ²²Na permeability measurements, differential scanning calorimetry, fluorescence polarization, ³¹P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with $\text{n = 2}$ or 3 exhibit a sharp transition at 46°C or 49°C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with $\text{n > 3}$ exhibit a main transition between 38 and 43°C with enthalpies $\text{< 10}\text{kcal/mol}$ and after prolonged cooling (more than 3 days at 4°C) a broad endotherm at about $\text{20 ± 3°}\text{C}$ with enthalpies $\text{> 4}\text{kcal/mol}$. These same dispersions display a permeability peak at 20–25°C and a second increase in ²²Na efflux in the temperature range 30–40°C. The results of ³¹P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentanol-phosphocholine ($\text{n = 2}$) bilayers have restricted rotation below the main phase transition temperature.     

The effect of alterations in the physical state of the membrane lipids on the ability of Acholeplasma laidlawii B to grow at various temperatures

The physical state of the membrane lipids, as determined by fatty acid composition and environmental temperature, has a marked effect on both the temperature range within which Acholeplasma laidlawii B cells can grow and on growth rates within the permissible temperature ranges. The minimum growth temperature of 8 °C is not defined by the fatty acid composition of the membrane lipids when cells are enriched in fatty acids giving rise to gel to liquid-crystalline membrane lipid phase transitions occurring below this temperature. The elevated minimum growth temperatures of cells enriched in fatty acids giving rise to lipid phase transitions occurring at higher temperatures, however, are clearly defined by the fatty acid composition of the membrane lipids. The optimum and maximum growth temperatures are also influenced indirectly by the physical state of the membrane lipids, being significantly reduced for cells supplemented with lower melting, unsaturated fatty acids. The temperature coefficient of growth at temperatures near or above the midpoint of the lipid phase transition is 16 to 18 $\text{kcal}\text{mol}$, but this value increases abruptly to 40 to 45 $\text{kcal}\text{mol}$ at temperatures below the phase transition midpoint. Both the absolute rates and temperature coefficients of cell growth are similar for cells whose membrane lipids exist entirely or predominantly in the liquid-crystalline state, but absolute growth rates decline rapidly and temperature coefficients increase at temperatures where more than half of the membrane lipids become solidified. Cell growth ceases when the conversion of the membrane lipid to the gel state approaches completion, but growth and replication can continue at temperatures where less than one tenth of the total lipid remains in the fluid state. An appreciable heterogeneity in the physical state of the membrane lipids can apparently be tolerated by this organism without a detectable loss of membrane function.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

Seasonal and temperature-related changes in mitochondrial membranes associated with torpor in the mammalian hibernator Spermophilus richardsonii

Seasonal variations in the thermal response of liver mitochondrial membranes from Richardson's ground squirrels (Spermophilus richardsonii) were determined by measuring succinate-cytochrome $\text{c}$ reductase activity and spin label motion over a temperature range of 2 °C to 35 °C. For seven summer animals from the field the Arrhenius-type plots for enzyme activity and spin label motion were biphasic indicating a transition in structure and function at $\text{22 + 2.3°C}\text{and}\text{23 ± 1.9°C}$, respectively; typical of homeothermic mammals. For 12 winter animals maintained at 19°C, the transition in structure and function was lowered to $\text{12 ± 1.1°C}$ and $\text{13 ± 1.4°C}$, respectively. The transition for 5 of 11 winter animals which were kept at 4°C and maintained normal activity and body temperature was similar to animals maintained at 19°C, while for the other six the transition was further lowered to less than 4°C. The transition for seven winter animals which were in deep hibernation was less than 4°C. The results for liver mitochondria show that lowering of the transition in membrane structure and function occurs as a two-stage process of about 10 deg. C for each stage and that the lowering is a requisite for hibernation rather than a response to the low-body temperatures experienced during hibernation.     

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.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe³⁺, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 °C, while that for phosphatidylserine spin label had only one transition at 30 °C.When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogeneous fluidity. Mg²⁺ or $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ prevented the hemolysis-induced spectral changes. Ca²⁺ did not prevent the homogenization and acted antagonistically to Mg²⁺. The heterogeneity preservation by Mg²⁺ was nullified by trypsin, pronase or $\text{N-}\text{ethylmaleimide}$ added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg²⁺ was dependent on hemolysis temperature, starting to decrease at 18 °C and vanishing at 40 °C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus

The hydrolysis of d-erythro beef brain sphingomyelin and $\text{d,l}\text{-erythro}\text{-N-}\text{palmitoylsphingomyelin}$ dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30°C for beef brain sphingomyelin and 41°C for N-palmitoylsphingosinephosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.     

The effect of A13+ on the physical properties of membrane lipids in Thermoplasma acidophilum.

Using Electron Paramagnetic Resonance Spectroscopy, Al³⁺ was shown to produce a dramatic decrease of membrane lipid fluidity on the microorganism $\text{Thermoplasma}\text{acidophilum}$ at a pH > 2. The ability of Al³⁺ to alter lipid fluidity was enhanced with increasing pH (from 3 to 5). At pH 4, 10^?2 M Al³⁺ increased the lower lipid phase transition by 39°C, and a detectable change was observed with AlCl₃ concentrations as low as 10^?5 M. The ability of Al³⁺ to increase the lower lipid phase transition temperature of $\text{T.}\text{acidophilum}$ is the largest of any cation/lipid interaction yet reported.     

Effects of temperature and molecular interactions on the vibrational infrared spectra of phospholipid vesicles

Infrared spectra were obtained as a function of temperature for a variety of phospholipid/water bilayer assemblies (80% water by weight) in the 3000-950 cm^?1 region. Spectral band-maximum frequency parameters were defined for the 2900 cm^?1 hydrocarbon chain methylene symmetric and asymmetric stretching vibrations. Temperature shifts for these band-maximum frequencies provided convenient probes for monitoring the phase transition behavior of both multilamellar liposomes and small diameter single-shell vesiclesof dipalmitoyl phosphatidylcholine/water dispersions. As examples of the effects of bilayer lipid/cholesterol/water (3 : 1 mol ratio) and lipid/cholesterol/amphotericin B/water (3 : 1 : 0.1 mol ratios) vesicles were examined using the methylene stretching frequency indices. In comparison to the pure vesicle form, the transition width of the lipid/cholesterol system increased by nearly a factor of two (to 8°C) while the phase transition temperature remained approximately the same (41° C). For the lipid/cholesterol/amphotericin B system, the phase transition temperature increased by about 4.5° C (to 45.5°C) with the transition width increasing by nearly a factor of four ($\text{to}\text{≈ 15°}\text{C}$) above that of the pure vesicles. The lipid/cholesterol/amphotericin B data were interpreted as reflecting the formation below 38°C of a cholesterol/amphotericin B complex whose dissociation at higher temperature (38–60°C range) significantly broades the gel-liquid crystalline phase transition.     

Chloroplast membrane damage during freezing: the lipid phase

In order to study the effect of freeze damage to chloroplast membranes microviscosity of spinach thylakoids was probed by stearic acid spin labels. Changes in ESR parameters have been determined either as a function of temperature or during freezing at ?15 °C as a function of time. An empirical parameter $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ (ratio of height of a low field line component h₊ over height of the central line h₀) proved to be very sensitive to minute changes in membrane structure.In cryoprotected chloroplast membranes Arrhenius plot breaks indicative of phase changes are observed at +15 and ?10 °C. Breaks in the Arrhenius plots were not observed in vesicles prepared from chloroplast lipids by sonication. Instead, a melting zone was indicated below ?30 °C.Freeze damage of thylakoids during storage at ?15 °C is reflected in an increase of $\text{h}{}_{\mathrm{+}}\text{h}{}_0$ and a decrease in central line width W₀. At +20 °C, differences between the ESR parameters of active as compared to freeze-damaged membranes could be detected, if the osmolarity of the suspending medium exceeded 200 mosm. The observed changes in line shapes are interpreted as an increase in mobility and/or orientation of the lipids following the swelling of thylakoids. They do not indicate a disorganization of the lipid phase. Sedimentation experiments indicated that the freeze-damaged swollen membranes still exhibited osmotic responses. It is suggested that freezing which is known to dissociate proteins from the membranes altered the charge distribution of the membranes leading first to membrane swelling and finally, by the opening of hydrophilic channels, to membrane collapse.     

Effects of gaseous anaesthetics and inert gases on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine was studied under high pressures of helium (340 atm), nitrogen (340 atm), nitrous oxide (43 atm), cyclopropane (4.4 atm) and $\text{n-}\text{propane}$ (8.2 atm), using a turbidimetric technique. Helium and nitrogen increased the transition temperature by 0.021 and 0.006°C/atm, respectively, compared with 0.024°C/atm for hydrostatic pressure. Nitrous oxide reduced the transition by 0.58°C/atm. The hydrocarbon gases spread the transition width and lowered the transition temperature with increasing effect at higher doses. Comparisons with other membrane probes are made and the concentration of gases in the bilayer which lower the transition temperature by 1°C are estimated, in mol%: He, 10.2; N₂, 13.2; N₂O, 9.04; $\text{n-}\text{C}{}_3\text{H}{}_8$, 6.3 and cyclopropane, 12.8.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.     

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramylpentapeptide translocase

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by $\text{phospho}\text{-N-}\text{acetylmuramyl}$ (MurNAc)-pentapeptide translocase (UDP-MurNAc-Ala-γ dGlu-Lys-dAla-dAla undecaprenyl phosphate phospho-MurN Acpentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30°C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}$ derivatives of 12-and 16-ketostearic acid intercalated into membranes from this organism define the lower ($\text{T}{}_1\text{= 16–22°}\text{C}$) and upper ($\text{T}{}_{\mathrm{<mtext>h</mtext>}}\text{= 30°}\text{C}$) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurN Ac-pentapeptide translocase has a significant effect on the catalytic activity of this enzyme.     

The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.