Purification and characterization of caprine kidney uricase, possessing novel kinetic and thermodynamic properties

Purified uricase from a caprine kidney, possessed K _m and V _max values of 1.1 mg ml⁻¹ and 3512 IU (mg protein)⁻¹ for uric acid hydrolysis, respectively. The optimum temperature and pH for catalytic activity were 40 °C and 8.5, respectively. The activation energy for formation of ES complex was 13.6 kJ mol⁻¹. Enthalpy (ΔH*), entropy of activation (ΔS*) and Gibbs free energy demand of uricase inactivation were 62.8 kJ mol⁻¹, −102 J mol⁻¹ K⁻¹ and 104.3 kJ mol⁻¹, respectively. Gibbs free enrgy demand for substrate binding and transition state stabilization were also determined which were comparable with those for themostable enzymes.     

Kinetics of lipase catalyzed isoamyl acetate synthesis at high hydrostatic pressure in hexane

Lipase-catalyzed synthesis of isoamyl acetate in hexane at 10–250 MPa at 80°C and 1–100 MPa at 40°C resulted in activation volumes of −12.9 ± 1.7 and −21.6 ± 2.9 cm³ mol⁻¹, respectively. Increasing pressure from 10 to 200 MPa resulted in approximately 10-fold increase in V _max at both 40 and 80°C. Pressure increased the K _m from 2.4 ± 0.004 to 38 ± 0.78 mM at 40°C. In contrast, at 80°C the pressure did not affect the K _m.     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH₄)₂SO₄ precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca²⁺ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol⁻¹ (44.30 kJ mol⁻¹). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k _cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K _m and k _cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×10³ min⁻¹, respectively.     

Production,extraction, and thermodynamics protease partitioning from Aspergillus tamarii Kita UCP1279 using PEG/sodium citrate aqueous two-phase systems

Abstract

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 2⁴, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (M_PEG), concentrations of PEG (C_PEG) and citrate (C_CIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°_m (?10.89?kJ mol^?1), ΔH_m (?5.0?kJ?mol^?1) and partition ΔS_m (19.74?J mol^?1 K^?1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40–50?°C and 9.0–11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Interaction of 3’-O-caffeoyl D-quinic acid with multisubunit protein helianthinin

Chlorogenic acid, 3’-O-caffeoyl D-quinic acid, is an inherent ligand present inHelianthus annuus L. The effect of pH on chlorogenic acid binding to helianthinin suggests that maximum binding occurs at pH 6.0. The protein-polyphenol complex precipitates as a function of time. The association constant of the binding of chlorogenic acid to helianthinin, determined by equilibrium dialysis, at 31°C has a value of 3.5 ± 0.1 × 10⁴M^−-1 resulting in a ΔG value of − 6.32 ± 0.12 kcal /mol. The association constantK _ais 1.0 ± 0.1 × 10⁴M⁻¹ as determined by ultraviolet difference spectral titration at 25°C with ΔG° of -5.46 ± 0.06 kcal/mol. From fluorescence spectral titration at 28°C, theK _avalue is 1.38 ± 0.1 × 1 0 4M⁻¹ resulting in a ΔG of − 5.70 ± 0.05 kcal/mol. The total number of binding sites on the protein are 420 ± 50 as calculated from equilibrium dialysis. Microcalorimetric data of the ligand-protein interaction at 23°C suggests mainly two classes of binding. The thermal denaturation temperature,T _mof the protein decreases from 76°C to 72°C at 1 × 10⁻³M chlorogenic acid concentration upon complexation. This suggests that the complexation destabilizes the protein. The effect of temperature onK _aof chlorogenic acid shows a nonlinear increase from 10.2°C to 45°C. Chemical modification of both lysyl and tryptophanyl residues of the protein decreases the strength of binding of chlorogenic acid. Lysine, tryptophan and tyrosine of protein are shown to be present at the binding site. Based on the above data, it is suggested that charge-transfer complexation and entropically driven hydrophobic interaction are the predominant forces that are responsible for binding of chlorogenic acid to the multisubunit protein, helianthinin. Publication No. 324.     

The influence of irradiance,photoperiod and temperature on the growth kinetics of three planktonic diatoms

The influence of irradiance, photoperiod and temperature was determined for the growth kinetics of the diatoms Aulacoseira subarctica, Stephanodiscus astraea and Stephanodiscus hantzschii and the results compared with those of cyanobacteria. Irradiance and photoperiod relationships were qualitatively similar to those for cyanobacteria in that: (1) growth rate (K) was proportionally greater under short photoperiods, with ratios of K under continuous light to K under 3:21 light:dark (LD) cycles of 1·50, 1·80 and 2·96 for A. subarctica, S. astraea and S. hantzschii respectively; (2) at subsaturating irradiances, K was proportional to irradiance and independent of temperature with a negligible predicted maintenance growth rate requirement. Apparent growth efficiencies (GE) at subsaturating irradiances were 0·26±0·03, 0·42±0·03 and 0·50±0·03 divisions mol^-1m² for A. subarctica, S. astraea and S. hantzschii with the values for Stephanodiscus species comparable to values for Oscillatoria species. Under a 3:21 LD cycle at 4 °C, light-saturated growth rates were 0·066±0·004, 0·197±0·033 and 0·285±0·018 divisions day^-1 for A. subarctica, S. astraea and S. hantzschii. S. hantzschii growth rate at 4 °C exceeded maximum Oscillatoria growth rates at 23 °C and the S. astraea growth rate at 4 °C was equivalent to O. agardhii growth rate at 20 °C. Temperature increases above 4 °C gave Q₁₀ values between 4 °C and 12 °C of 3·68, 2·39 and 1·92 for A. subarctica, S. astraea and S. hantzschii, but higher temperatures resulted in minor increases in K. S. astraea growth rate peaked at 16 °C, declining sharply at higher temperatures. February to March in situ growth rates in Lough Neagh, mean temperature 4·3 °C, showed that the A. subarctica in situ K of 0·058 divisions day^-1 was close to the laboratory K at 4 °C, but that S. astraea in situ K of 0·101 divisions day^-1 was lower than the laboratory K at 4 °C.     

Effect of NaCl on kinetics ofd-glucosamine uptake in yeasts differing in halotolerance

The initial rate ofd-glucosamine uptake by the non-halotolerant yeastSaccharomyces cerevisiae was approximately halved as the apparent half saturation constant (K_m) and the apparent maximum velocity (V_max) changed from 6.6mm to 16.4mm and from 22 μmol · g⁻¹ · min⁻¹ to 16 μmol · g⁻¹ · min⁻¹, respectively, when the salinity in the medium was increased from zerom to 0.68m NaCl. Corresponding changes in a high affinity transport system in the halotolerant yeastDebaryomyces hansenii were from 1.1mm to 4.6mm and from 3.1 μmol · g⁻¹ · min⁻¹ to 4.5 μmol · g⁻¹ · min⁻¹, implying a practically unchanged transport capacity. In 2.7m NaCl, K_m and V_max in this system were 24.5mm and 1.1 μmol · g⁻¹ · min⁻¹, respectively, representing a marked decrease in transport capability. Nevertheless, the degree of affinity in this extreme salinity must still be regarded as noteworthy. In addition to the high affinity transport system inD. hansenii, a low affinity system, presumably without relevance ind-glucosamine transport, was observed.     

The influence of ultraviolet-B radiation on the growth, pigment production and chlorophyll fluorescence of Norway spruce seedlings

Norway spruce (Picea abies (L.)Karst.) from seven seed sources was grown in a greenhouse with 8.3 and 14.7 kJ·m⁻²·d⁻¹ m UV-B_BE (biologically effective UV-B: 280–320 nm) irradiation, and with no supplemental irradiation as control. The seedlings total biomass (dry weight) and shoot growth decreased with high UV-B treatment but spruce from low elevation seed sources were more affected. The seedlings grown at the highest UV-B irradiance (14.7 kJ·m⁻²·d⁻¹) showed from 5 to 38% inhibition of total biomass and 15 to 70 % shoot growth inhibition. Norway spruce populations from higher altitude seed sources manifested greater tolerance to UV-B radiation compared to plants from low altitudes. Changes in phospholipids and protective pigments were also determined. The plants grown at the lower UV-B irradiance (8.3 kJ·m⁻²·d⁻¹) showed greater ability to concentrations UV-B-absorbing pigments then control plants. Chlorophyll a fluorescence parameter R_fd, (R_fd=(F_m-F_s)/F_s) showed a significant decrease in needles of UV-B treated plants and this correlated with the altitude of seed source. Exposure to UV-B affect levels of the ratio of variable to maximum fluorescence (F_v/F_m). Results from this study suggest that the response to increased levels of UV-B radiation is depended upon the ecotypic differentiation of Norway spruce and involved changes in metabolites in plant tissues.     

Temperature dependence of sediment-water exchange in Lake Grevelingen,SW Netherlands

Extended abstract Lake Grevelingen is a brackish water lake in the SW Netherlands. The lake has an area of 108 km², a mean depth of 5.3 m (maximum 48 m), a mean chlorinity of 13 to 16%0 Cl⁻, and a hydraulic residence time of about 8 years. Mass budget studies have shown a consistent seasonal pattern in the phosphorus sediment-water exchange in Lake Grevelingen (Kelderman 1980). From May to August a P mobilization from the sediment takes place, estimated atca. 12.5 mg P · m⁻² · day⁻¹. The sediment accumulatesca. 5.5 mg P · m⁻² · day⁻¹ during the rest of the year. Temperature may be an important factor in establishing this pattern. Sediment-water exchange was studied by means of laboratory experiments under specified conditions. Sediment cores (30 cm depth, 11 cm diameter) were taken at four stations in the lake, with sediment types varying from medium- to muddy sand (Fig. 1). The cores with overlying water (ca. 21) were placed in the dark at 5 °C in thermostatically controlled water baths. After a week's incubation time the temperature was slowly raised, such that after three weeks eight cores (four sediment types, duplicates) were at 5 °C, eight were at 10 °C, eight at 15 °C and eight at 20 °C. The same procedure was applied to the four control cores, containing lake water.     

Photosynthetic characteristics and physiological plasticity of an Aphanizomenon flos-aquae (Cyanobacteria, Nostocaceae) winter bloom in a deep oligo-mesotrophic lake (Lake Stechlin, Germany)

In winter of 2009/2010, Aphanizomenon flos-aquae bloomed in the ice and snow covered oligo-mesotrophic Lake Stechlin, Germany. The photosynthesis of the natural population was measured at eight temperatures in the range of 2–35°C, at nine different irradiance levels in the range of 0–1,320 μmol m⁻² s⁻¹ PAR at each applied temperature. The photoadaptation parameter (I _k) and the maximum photosynthetic rate (P _max) correlated positively with the temperature between 2 and 30°C, and there was a remarkable drop in both parameters at 35°C. The low I _k at low temperatures enabled the active photosynthesis of overwintering populations at low irradiance levels under ice and snow cover. The optimum of the photosynthesis was above 20°C at irradiances above 150 μmol m⁻² s⁻¹. At lower irradiance levels (7.5–30 μmol m⁻² s⁻¹), the photosynthesis was the most intensive in the temperature range of 2–5°C. The interaction between light and temperature allowed the proliferation of A. flos-aquae in Lake Stechlin resulting in winter water bloom in this oligo-mesotrophic lake. The applied 2°C is the lowest experimental temperature ever in the photosynthesis/growth studies of A. flos-aquae, and the results of the P–I and P–T measurements provide novel information about the tolerance and physiological plasticity of this species.     

An alkaline protease from fresh fruiting bodies of the edible mushroom <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis>

A protease was purified from fresh fruiting bodies of the edible mushroom Pleurotus citrinopileatus. The isolation procedure included ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on CM-cellulose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease demonstrated a single band with a molecular mass of 28 kDa. The protease showed an optimal pH at 10 and an optimal temperature at 50°C. The activity of the protease was not affected by EDTA, indicating that it is not a metalloprotease. The protease exhibited a higher activity in the presence of K⁺ and Li⁺, but its activity was potently inhibited by Al³⁺, Cu²⁺, and Hg²⁺ ions. It manifested a K _m of 3.44 mg/ml and a V _max of 0.139 mg ml⁻¹ min⁻¹. It was devoid of ribonuclease and antifungal activities.     

Production of β-galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg⁻¹) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg⁻¹). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co²⁺, Mn²⁺, Fe²⁺ and Zn²⁺ had strong inhibitory effects on the enzyme activity. The K _m and V _max for p-nitrophenyl-β- _D-galactopyranoside and o-nitrophenyl-β - _D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min⁻¹ mg⁻¹ respectively. The K _m and V _max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min⁻¹ mg⁻¹. Received 10 March 1997/ Accepted in revised form 17 July 1997     

Behavioral responses of Colorado potato beetle larvae to combinations of temperature and insolation, under field conditions

In short-term field trials at combinations of ambient temperature (°C) and insolation (W·m⁻²), larval Colorado potato beetles (Leptinotarsa decemlineata [Say] [Coleoptera: Chrysomelidae]) were observed after their release on the adaxial surface of leaflets on potato plants (Solanum tuberosum L. Solanaceae). The larvae either began feeding or moved under the leaflet; mean interval from release to expression of these behaviors (2.9±0.05 min [n =358]) was independent of air temperature and insolation. Proportion of larvae moving under the leaflet increased logistically with both air temperature and insolation. A 1 W·m⁻² change in insolation (P) evoked the same effect on this proportion as a 0.0838 °C change in air temperature (T _a ), so the two quantities were combined as T^*=T _a +P·0.0838 °C/(W·m⁻²), which has units of °C. The proportion of larvae moving under the leaflet increased logistically with T^*. In 1-day field trials we monitored air temperature, insolation and proportion of larvae under the leaflet, and compared the latter to predictions from the logistic regression derived from the short-term trials. Consistently more larvae occurred under leaflets than predicted from the logistic regression; this bias diminished as T^* increased until at T^*≥40 °C, observed and predicted proportions were equal. This pattern of deviation from the predictions of the logistic regression is consistent with a thermoregulatory strategy in which larvae move away from hostile conditions, rather than seek optimal conditions.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Quercetin and daidzein β-apo-14’-carotenoic acid esters as membrane antioxidants

Abstract

Esterification by β-apo-14’-carotenoic acid was found to have opposite effects on antioxidant activity of quercetin (at B4’, B3’ hydroxyl) as of daidzein (at A7 hydroxyl) in phosphatidylcholine liposomes. The daidzein ester had increased activity, while quercetin had a significant decreased activity. Quantum mechanical calculations using density function theory (DFT) indicate a modest decrease in bond dissociation enthalpy, BDE, for (weakest) hydrogen–oxygen phenolic bond in daidzein from 368.4 kJ·mol^{? 1} to 367.7 kJ·mol^{? 1} compared to a significant increase in quercetin from 329.5 kJ·mol^{? 1} to 356.6 kJ·mol^{? 1} upon derivatization. These opposite changes in tendency for hydrogen atom transfer from phenolic groups to lipid radicals combined with an increase in A-to-B dihedral angle from 0.0° to 36.4° and in dipole moment from 0.40 D to 6.01 D for quercetin upon derivatization, while less significant for daidzein (36.4°–36.7° and 3.26 D–7.87 D, respectively), together provide a rationale for the opposite effect of esterification on antioxidation.     

An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

PHYSIOLOGICAL RESPONSES OF ANABAENA VARIABILIS (CYANOPHYCEAE) TO INSTANTANEOUS EXPOSURE TO VARIOUS COMBINATIONS OF LIGHT INTENSITY AND TEMPERATURE1

Light intensity and temperature interactions have a complex effect on the physiological process rates of the filamentous bluegreen alga Anabaena variabilis Kütz. The optimum temperature for photosynthesis increased with increasing light intensity from 10°C at 42 μE·m^?2·s^?1 to 35°C at 562 μE·m^?2·s^?1. The light saturation parameter, I_K, increased with increasing temperatures. The maximum photosynthetic rate (2.0 g C·g dry wt.^?1·d^?1) occurred at 35°C and 564 μE·m^?2·s^?1. At 15°C, the maximum rate was 1.25 g C·g dry wt.^?1·d^?1 at 332 μE·m^?2·s^?1. The dark respiration rate increased exponentially with temperature. Under favorable conditions of light intensity and temperature the percent of extracellular release of dissolved organic carbon was less than 5% of the total C fixed. This release increased to nearly 40% under combinations of low light intensity and high temperature. A mathematical model was developed to simulate the interaction of light intensity and temperature on photosynthetic rate. The interactive effects were represented by making the light-saturation parameters a function of temperature.     

Effects of Hyperthermia on Intracellular Chloride

Hyperthermia induces transient changes in [Na⁺] _i and [K⁺] _i in mammalian cells. Since Cl⁻ flux is coupled with Na⁺ and K⁺ in several processes, including cell volume control, we have measured the effects of heat on [Cl⁻] _i using the chloride indicator, MQAE, with flow cytometry. The mean basal level of [Cl⁻] _i in Chinese hamster ovary cells was 12 mm. Cells heated at 42.0° or 45.0°C for 30 min had about a 2.5-fold increase in [Cl⁻] _i above unheated control values when measured immediately after heating. There was about a 3-fold decrease in [Na⁺] _i under the same conditions, as measured by Sodium Green. The magnitude of the increase in [Cl⁻] _i depended upon time and temperature. The [Cl⁻] _i recovered in a time-dependent fashion to control values by 30 min after heating. When cells were heated at 45.0°C for 30 min in the presence of 1.5 mm furosemide, the heat-induced [Cl⁻] _i increase was completely blocked. Since furosemide inhibits the Na⁺/K⁺/2Cl⁻ cotransporter, Cl⁻ channels, and even Cl⁻HCO₃ exchange, these ion transporters may be involved in the heat-induced increase in [Cl⁻] _i . Received: 15 June 1995/Revised: 9 April 1996