Glutathione increases the binding affinity of a bovine B12 trafficking chaperone bCblC for vitamin B12

Intracellular B₁₂ metabolism involves a B₁₂ trafficking chaperone CblC that is well conserved in mammals including human. The protein CblC is known to bind cyanocobalamin (CNCbl, vitamin B₁₂) inducing the base-off transition and convert it into an intermediate that can be used in enzyme cofactor synthesis. The binding affinity of human CblC for CNCbl was determined to be K_d = ≈6–16 μM, which is relatively low considering sub-micromolar B₁₂ concentrations (0.03–0.7 μM) in normal cells. In the current study, we discovered that the base-off transition of CNCbl upon binding to bCblC, a bovine homolog of human CblC, is facilitated in the presence of reduced form of glutathione (GSH). In addition, GSH dramatically increases the binding affinity for CNCbl lowering the K_d from 27.1 ± 0.2–0.24 ± 0.09 μM. The effect of GSH is due to conformational change of bCblC upon binding with GSH, which was indicated by limited proteolysis and urea-induced equilibrium denaturation of the protein. The results of this study suggest that GSH positively modulates bCblC by increasing the binding affinity for CNCbl, which would enhance functional efficiency of the protein.     

Hemoglobin senses body temperature

When aspirating human red blood cells (RBCs) into 1.3 μm pipettes (ΔP = −2.3 kPa), a transition from blocking the pipette below a critical temperature T _c = 36.3 ± 0.3°C to passing it above the T _c occurred (micropipette passage transition). With a 1.1 μm pipette no passage was seen which enabled RBC volume measurements also above T _c. With increasing temperature RBCs lost volume significantly faster below than above a T _c = 36.4 ± 0.7 (volume transition). Colloid osmotic pressure (COP) measurements of RBCs in autologous plasma (25°C ≤ T ≤ 39.5°C) showed a T _c at 37.1 ± 0.2°C above which the COP rapidly decreased (COP transition). In NMR T₁-relaxation time measurements, the T₁ of RBCs in autologous plasma changed from a linear (r = 0.99) increment below T _c = 37 ± 1°C at a rate of 0.023 s/K into zero slope above T _c (RBC T₁ transition). In conclusion: An amorphous hemoglobin–water gel formed in the spherical trail, the residual partial sphere of the aspirated RBC. At T _c, a sudden fluidization of the gel occurs. All changes mentioned above happen at a distinct T _c close to body temperature. The T _c is moved +0.8°C to higher temperatures when a D₂O buffer is used. We suggest a mechanism similar to a “glass transition” or a “colloidal phase transition”. At T _c, the stabilizing Hb bound water molecules reach a threshold number enabling a partial Hb unfolding. Thus, Hb senses body temperature which must be inscribed in the primary structure of hemoglobin and possibly other proteins. This article is dedicated to Ludwig Artmann who died on July 21, 2001 on a beautiful summer day during which we performed experiments far away. Ludwig Artmann was a man who encouraged us to be strong and to study hard no matter what were the costs.     

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.     

Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> Brot.)

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 μM 2,4-D + 0.03 mg l⁻¹ vitamin B₁₂ (MS1) or with 2.26 μM 2,4-D + 0.1 mg l⁻¹ vitamin B₁₂ (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar- and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 μM NAA, 8.88 μM BA and 50 mg l⁻¹ casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 μM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT _m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT _m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT _m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.     

Winter body temperature patterns in free-ranging Cape ground squirrel, Xerus inauris: no evidence for torpor

The body temperature (T _b) of Cape ground squirrels (Xerus inauris, Sciuridae) living in their natural environment during winter has not yet been investigated. In this study we measured abdominal T _b of eight free-ranging Cape ground squirrels over 27 consecutive days during the austral winter. Mean daily T _b was relatively stable at 37.0 ± 0.2°C (range 33.4 to 40.2°C) despite a marked variation in globe temperature (T _g) (range −7 to 37°C). Lactating females (n = 2) consistently had a significantly higher mean T _b (0.7°C) than non-lactating females (n = 3) and males. There was a pronounced nychthemeral rhythm with a mean active phase T _b of 38.1 ± 0.1°C and a mean inactive phase T _b of 36.3 ± 0.3°C for non-lactating individuals. Mean daily amplitude of T _b rhythm was 3.8 ± 0.2°C. T _b during the active phase closely followed T _g and mean active phase T _b was significantly correlated with mean active phase T _g (r ² = 0.3–0.9; P < 0.01). There was no evidence for daily torpor or pronounced hypothermia during the inactive phase, and mean minimum inactive phase T _b was 35.7 ± 0.3°C for non-lactating individuals. Several alternatives (including nocturnal huddling, an aseasonal breeding pattern and abundant winter food resources) as to why Cape ground squirrels do not employ nocturnal hypothermia are discussed.     

Modulation of Glutamate and Glycine Transporters by Niflumic,Flufenamic and Mefenamic Acids

Three fenamates—niflumic, flufenamic and mefenamic acids—were tested for effects on substrate-induced currents of glutamate and glycine transporters (EAAT1, EAAT2, GLYT1b and GLYT2a) expressed in Xenopus laevis oocytes. All fenamates inhibited EAAT1 currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 53 ± 4% (P < 0.001). EAAT2 currents were less sensitive, but 100 μM flufenamic acid inhibited the I _max by 34 ± 5% (P = 0.006). All fenamates inhibited GLYT1b currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 61 ± 1% (P < 0.001). At 100 μM, effects on the GLYT2a I _max were mixed: 13 ± 2% inhibition by flufenamic acid (P = 0.002), 30 ± 6% enhancement by niflumic acid (P = 0.002), and no effect by mefenamic acid. Minor effects on substrate affinity suggested non-competitive mechanisms. These data could contribute to the development of selective transport modulators.     

Decreased Brain Levels of Vitamin B12 in Aging,Autism and Schizophrenia

Many studies indicate a crucial role for the vitamin B₁₂ and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B₁₂ status in the brain across the lifespan has not been previously investigated. Vitamin B₁₂ (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B₁₂ status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.     

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe–S proteins contain a highly conserved all-β fold, which harbors a [2Fe–2S] Rieske center. We have identified a novel subtype of Rieske ferredoxins present in hyperthermophilic archaea, in which a two-cysteine conserved SKTPCX_(2–3)C motif is found at the C-terminus. We establish that in the Acidianus ambivalens representative, Rieske ferredoxin 2 (RFd2), these cysteines form a novel disulfide bond within the Rieske fold, which can be selectively broken under mild reducing conditions insufficient to reduce the [2Fe–2S] cluster or affect the secondary structure of the protein, as shown by visible circular dichroism, absorption, and attenuated total reflection Fourier transform IR spectroscopies. RFd2 presents all the EPR, visible absorption, and visible circular dichroism spectroscopic features of the [2Fe–2S] Rieske center. The cluster has a redox potential of +48 mV (25 °C and pH 7) and a pK _a of 10.1 ± 0.2. These shift to +77 mV and 8.9 ± 0.3, respectively, upon reduction of the disulfide. RFd2 has a melting temperature near the boiling point of water (T _m = 99 °C, pH 7.0), but it becomes destabilized upon disulfide reduction (ΔT _m = −9 °C, ΔC _m = −0.7 M guanidinium hydrochloride). This example illustrates how the incorporation of an additional structural element such as a disulfide bond in a highly conserved fold such as that of the Rieske domain may fine-tune the protein for a particular function or for increased stability.     

Thermal ecology of the Australian agamid Pogona barbata

This study compares the thermal ecology of male bearded dragon lizards (Pogona barbata) from south-east Queensland across two seasons: summer (1994–1995) and autumn (1995). Seasonal patterns of body temperature (T _b) were explored in terms of changes in the physical properties of the thermal environment and thermoregulatory effort. To quantify thermoregulatory effort, we compared behavioral and physiological variables recorded for observed lizards with those estimated for a thermoconforming lizard. The study lizards' field T _bs varied seasonally (summer: grand daily mean (GDM) 34.6 ± 0.6°C, autumn: GDM 27.5 ± 0.3°C) as did maximum and minimum available operative temperatures (summer: GDM T _max 42.1 ± 1.7°C, T _min 32.2 ± 1.0°C, autumn: GDM T _max 31.7 ± 1.2°C, T _min 26.4 ± 0.5°C). Interestingly, the range of temperatures that lizards selected in a gradient (selected range) did not change seasonally. However, P. barbata thermoregulated more extensively and more accurately in summer than in autumn; lizards generally displayed behaviors affecting heat load nonrandomly in summer and randomly in autumn, leading to the GDM of the mean deviations of lizards' field T _bs from their selected ranges being only 2.1 ± 0.5°C in summer, compared to 4.4 ± 0.5°C in autumn. This seasonal difference was not a consequence of different heat availability in the two seasons, because the seasonally available ranges of operative temperatures rarely precluded lizards from attaining field T _bs within their selected range, should that have been the goal. Rather, thermal microhabitat distribution and social behavior appear to have had an important influence on seasonal levels of thermoregulatory effort. Received: 28 April 1997 / Accepted: 29 December 1997     

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B₁ and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B₁. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B₁. Pyruvate was found to be the preferred co-substrate and amino group receptor (K _M = 490 μM at 10 μM hydrolyzed fumonisin B₁) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K _M = 1.1 μM and k _cat = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.     

Characterization of vitamin B12 compounds in the fruiting bodies of shiitake mushroom (Lentinula edodes) and bed logs after fruiting of the mushroom

This study determined the vitamin B₁₂ content in commercially available dried fruiting bodies of shiitake mushroom, Lentinula edodes. The vitamin B₁₂ contents in dried donko-type fruiting bodies with closed caps (5.61 ± 3.90 μg/100 g dry weight), did not significantly differ from those of dried koushin-type fruiting bodies with open caps (4.23 ± 2.42 μg/100 g dry weight). The bed logs after fruiting of the mushroom also contained the vitamin B₁₂ levels similar to that in the dried shiitake fruiting bodies. To determine whether the dried shiitake fruiting bodies and their bed logs contained vitamin B₁₂ or other corrinoid compounds that are inactive in humans, we purified corrinoid compounds using an immunoaffinity column and identified vitamin B₁₂ using vitamin B₁₂-dependent Escherichia coli 215 bioautograms and liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) chromatograms. Dried shiitake fruiting bodies rarely contained an unnatural corrinoid vitamin B₁₂[c-lactone] that is inactive in humans. Given that shiitake mushroom lacks the ability to synthesize vitamin B₁₂ de novo, the vitamin B₁₂ found in dried shiitake fruiting bodies must have been derived from the bed logs.     

The cranial arterio-venous temperature difference is related to respiratory evaporative heat loss in a panting species,the sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>)

Panting is a mechanism that increases respiratory evaporative heat loss (REHL) under heat load. Because REHL uses body water, it is physiologically and ecologically relevant to know under what conditions free-ranging animals use panting. We investigated whether the cranial arterio-venous temperature difference could provide information about REHL. We exposed sheep to environments varying in ambient dry bulb temperatures (Env 1: ~15°C, Env 2: ~25°C, Env 3: ~40°C, Env 4: ~40°C + infrared radiation) and measured REHL simultaneously with carotid arterial (T _car) and jugular venous (T _jug) blood temperatures, as well as brain (T _brain) and rectal (T _rec) temperatures. REHL increased significantly with ambient temperature, from 18.4 ± 4.5 W at Env 1 to 79.5 ± 12.6 W at Env 4 (P < 10⁻⁶). While there was no effect of environment on T _car (P = 0.7) or T _jug (P = 0.09), the difference between them (T _a-v = T _car − T _jug) increased from Env 1 to Env 2 (P = 0.04) and from Env 3 to Env 4 (P = 0.008). T _a-v reached a maximum of 0.7 ± 0.2°C at Env 4 and was positively correlated with REHL across environments (r ² = 0.78, F = 34.7, P < 10⁻³). Calculated cranial blood flow changed only from Env 2 to Env 3 (P = 0.002). The increase in REHL maintained homeothermy when dry heat loss decreased. While REHL could increase without generating an increase in T _a-v, any increase in T _a-v was always associated with an increase in REHL. We conclude that the cranial T _a-v provides useful information about REHL in panting animals.     

Influence of the Selected Antioxidants on the Stability of the Celsior Solution Used for Perfusion and Organ Preservation Purposes

The purpose of the following research was to improve the original Celsior solution in order to obtain a higher degree of stability and effectiveness. The solution was modified by the addition of selected antioxidants such as vitamin C, cysteine, and fumaric acid in the following concentrations: 0.1, 0.3, and 0.5 mmol/l. The solution’s stability was estimated using an accelerated stability test based on changes in histidine concentrations in the solution using Pauly’s method for determining concentrations. Elevated temperatures, the factor accelerating substances’ decomposition reaction rate, were used in the tests. The research was conducted at four temperatures at intervals of 10°C: 60 ± 0.2°C, 70 ± 0.2°C, 80 ± 0.2°C, and 90 ± 0.2°C. It was stated that the studied substances’ decomposition occurred in accordance with the equation for first-order reactions. The function of the logarithmic concentration (log%C) over time was revealed to be rectilinear. This dependence was used to determine the kinetics of decomposition reaction rate parameters (the rate constant of decomposition k, activation energy E _a, and frequency factor A). On the basis of these parameters, the stability of the modified solution was estimated at +5°C. The results obtained show that the proposed antioxidants have a significant effect on lengthening the Celsior solution’s stability. The best results were reached when combining two antioxidants: vitamin C and cysteine in 0.5 mmol/l concentrations. As a result, the Celsior solution’s stability was lengthened from 22 to 299 days, which is 13.5 times. Vitamin C at a concentration of 0.5 mmol/l increased the solution’s stability by 5.2 times (t ₉₀ = 115 days), cysteine at a concentration of 0.5 mmol/l caused a 4.4 times stability increase (t ₉₀ = 96 days), and fumaric acid at a concentration of 0.5 mmol/l extended the stability by 2.1 times (t ₉₀ = 48 days) in relation to the original solution.     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

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.     

Use of principal component analysis in interpretation of deoxyribonucleic acid relatedness

Principal component analysis (PCA) of published DNA-relatedness data showed the usefulness of this method in displaying relationships among closely related bacteria. Very similar ordinations were obtained when relative binding ratios (RBR) at 60°C or 75°C or ΔT _m values were used to form the data matrix. A curvilinear relationship and a (quasi) linear relationship were found, respectively, between 75°C and 60°C RBR and ΔT _m and 60°C RBR. These statistical relationships explain the similarity of PCA results using either measurement (60°C RBR, 75°C RBR, or ΔT _m). Use of PCA is suggested to delineate groups within a complex set of DNA-relatedness data. The level of ΔT _m within groups and between groups should help decide whether these groups are genospecies.