Prolindehydrogenase aus keimenden farnsporen

A soluble enzyme which converts proline to glutamic acid using NAD as coenzyme was isolated from young prothallia and spores of the fern Anemia phyllitidis. The purification was about 36-fold. The pH optimum is between 10·2 and 10·7; the K_m for proline is 4·6 × 10⁻⁴ M and for NAD 3·4 × 10⁻⁴ M. There are no multiple forms of this enzyme, as proved by gel electrophoresis.     

Isocitrate lyase in germinating spores of the fern Anemia phyllitidis

Isocitrate lyase was partially purified from germinating spores of the fern Anemia phyllitidis. The enzyme requires Mg²⁺ and thiol compounds for maximal activity and has a pH optimum between 6.5 and 7.5. The K_m of the enzyme for threo-Δ_s-isocitrate is 0.5 mM. Succinate inhibits the enzyme non-competitively (K_i. 1.8 mM). The increase of isocitrate lyase activity is closely correlated with the induction of the germination process. The fall of enzyme activity during germination is associated with the decline in triglyceride reserves.     

Characterization and constitutive expression of an acidic mesophilic endo-1,4-β-d-xylanohydrolase with high thermotolerance and catalytic efficiency in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl11 from Aspergillus niger, encoding a 188-residue xylanase of glycosyl hydrolase family 11, was constitutively expressed in Pichia pastoris. The recombinant Xyl11 exhibited optimal activity at pH 5.0 and 50 °C, and displayed more than 68 % of the maximum activity over the temperature range 35–65 °C and 33 % over the pH range 2.2–7.0. It maintained more than 40 % of the original activity after incubation at 90 °C (pH 5.0) for 10 min and more than 75 % of the original activity after incubation at pH 2.2–11.0 (room temperature) for 2 h. The specific activity, K _m and V _max of purified Xyl11 were 22,253 U mg^?1, 6.57 mg ml^?1 and 51,546.4 μmol min^?1 mg^?1. It could degrade xylan to a series of xylooligosaccharides and no xylose was detected. The recombinant enzyme with high stability and catalytic efficiency could work over wide ranges of pH and temperature and thus has the potential for various industrial applications.     

Kinetics of the reaction of pentacyanonitrosylferrate(II) with aliphatic amines

The reactions of pentacyanonitrosylferrate(II) with ethyl-, n-propyl-, n-butyl-, cyclohexyl- and benzylamines were studied in dilute aqueous solution at 8.6–9.6 pH and 15–35 °C. Nitrosation, diazotation and deamination processes take place in the reactions resulting in alcohols and N₂ gas as final products. On the basis of spectrophotometric and gasvolumetric experiments the rate law was determined as follows.v = k[RNH₂][Fe(CN)₅NO^2?] The dependence on pH was interpreted by the protonation equilibria of the amines. From the function of the logarithm of rate constants vs. reciprocal temperature, relatively small activation enthalpies (15–70 kJ mol^?1) and extremely high negative activation entropies [(?80) ? (?240) J K^?1 mol^?1] were found. The mechanism was interpreted by the analogy with nitrous acid diazotation.A parallel trend was observed between the rate constants at 25 °C and the basicity constants of the amines.     

Purification and characterization of two distinct acidic phytases with broad pH stability from Aspergillus niger NCIM 563

Aspergillus niger NCIM 563 produced two different extracellular phytases (Phy I and Phy II) under submerged fermentation conditions at 30°C in medium containing dextrin-glucose-sodium nitrate-salts. Both the enzymes were purified to homogeneity using Rotavapor concentration, Phenyl-Sepharose column chromatography and Sephacryl S-200 gel filtration. The molecular mass of Phy I and II as determined by SDS–PAGE and gel filtration were 66, 264, 150 and 148 kDa respectively, indicating that Phy I consists of four identical subunits and Phy II is a monomer. The pI values of Phy I and II were 3.55 and 3.91, respectively. Phy I was highly acidic with optimum pH of 2.5 and was stable over a broad pH range (1.5–9.0) while Phy II showed a pH optimum of 5.0 with stability in the range of pH 3.5–9.0. Phy I exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. Similarly Phy II was strongly inhibited by Ag⁺, Hg²⁺ (1 mM) metal ions and Phy I was partially inhibited. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF also indicated that both the proteins were totally different. The K _m for Phy I and II for sodium phytate was 2.01 and 0.145 mM while V _max was 5,018 and 1,671 μmol min^?1 mg^?1, respectively. The N-terminal amino acid sequences of Phy I and Phy II were FSYGAAIPQQ and GVDERFPYTG, respectively. Phy II showed no homology with Phy I and any other known phytases from the literature suggesting its unique nature. This, according to us, is the first report of two distinct novel phytases from Aspergillus niger.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Coordination abilities of tetrapeptides containing proline and tyrosine—A spectrophotometric and potentiometric study

The synthesis of three tetrapeptides, Gly-Pro-Gly-Tyr. Gly-Pro-Tyr-Gly. and Tyr-Pro-Gly-Gly, are described. All contain proline as the second amino acid subunit to act as a break point in metal complex formation.The proton and copper(II) complex formation constants have been measured at 22°C and l = 0.10 mol dm^?3 (KNO₃). The copper(II) complexes have also been studied spectrophotometrically over the pH range of 6–11 by absorption spectroscopy (800–200 nm), circular dichroism spectroscopy, and electron paramagnetic resonance spectroscopy. The experimental data have been combined to determine the complex species present as a function of pH and the coordination centers used.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Glutathione s-transferase from hevea brasiliensis

Glutathione (GSH) S-transferase can be detected in a variety of tissues of Hevea brasiliensis. Lyophilized powders prepared from 20 000 g supernatants of latex adjusted to pH 5.0 contain substantial amounts of GSH S-transferase activity which is stable at ? 20° for up to 6 months. The enzyme has a broad pH optimum between 8.5 and 9.5. The K_m values for GSH and 1-chloro-2,4-dinitrobenzene are in the range of 33–45 and 150–200,μM, respectively. The enzyme has a MW in the range of 47 000–50 000 and an isoelectic point of 4.3. Although it appears homogeneous on analytical polyacylamide disc gel electrophoresis (PAGE) and isoelectric focusing, it resolves into five forms on DEAE-Sephadex chromatography.     

l-Proline transport in Saccharomyces cerevisiae

Transport of l-proline into Saccharomyces cerevisiae K is mediated by two systems, one with a K_T of 31 μM and J_max of 40 nmol · s^?1 · (g dry wt.)^?1, the other with K_T > 2.5 mM and J_max of 150–165 nmol · s^?1 · (g dry wt.)^?1, The kinetic properties of the high-affinity system were studied in detail. It proved to be highly specific, the only potent competitive inhibitors being (i) l-proline and its analogs l-azetidine-2-carboxylic acid, sarcosine, d-proline and 3,4-dehydro-dl-proline, and (ii) l-alanine. The other amino acids tested behaved as noncompetitive inhibitors. The high-affinity system is active, has a sharp pH optimum at 5.8–5.9 and, in an Arrhenius plot, exhibits two inflection points at 15°C and 20–21°C. It is trans-inhibited by most amino acids (but probably only the natural substrates act in a trans-noncompetitive manner) and its activity depends to a considerable extent on growth conditions. In cells grown in a rich medium with yeast extract maximum activity is attained during the stationary phase, on a poor medium it is maximal during the early exponential phase. Some 50–60% of accumulated l-proline can leave cells in 90 min (and more if washing is done repeatedly), the efflux being insensitive to 0.5 mM 2,4-dinitrophenol and uranyl ions, to pH between 3 and 7.3, as well as to the presence of 10–100 mM unlabeled l-proline in the outside medium. Its rate and extent are increased by 1% d-glucose and by 10 μg nystatin per ml.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

Properties of a repressible alkaline phosphatase secreted by the wild-type strain 74a of neurospora crassa

A repressible extracellular alkaline phosphatase (with activity increasing steadily even up to pH 10.5) was purified from cultures of the wild-type strain 74A of Neurospora crassa, after growth on acetate and under limiting amounts of inorganic phosphate for 72 hr at 30°. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE) with or without sodium dodecyl sulphate (SDS). The MW was ca 172 000 and 82 000 as determined by Sephadex G-200 gel filtration and SDS-PAGE, respectively. The enzyme contained 23.6% neutral sugars, cations were not required for activity, and it was not inactivated by 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) at pH 8. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of 4-nitrophenyl disodium orthophosphate (PNP-P) at pH 9 (the K_m value and Hill coefficient were 2.2 × 10^?4 M and 0.95, respectively). It was also shown that, at pH 9, the apparent number of Pi bound per dimer molecule equalled one, with a K_i value of 7.0 × 10^?4 M. The secreted enzyme showed half-lives of 23.5, 49.0 and 23.5 min at, pH 5.4, 7.4 and 9.0, respectively, after thermal inactivation at 60°. At pH 5.4, the half-life value was quite similar, while the others were respectively 2 and 4 times greater than those previously described for the repressible alkaline phosphatase retained by the mycelium at pH 5.6 or secreted by ‘slime’ cells.     

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni²⁺). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH?6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K _m and k _cat values of 0.33 mM and 4.07 s^?1, respectively. Amounts of 2 mM Ca²⁺ and Co²⁺ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.     

Characterization flavanone 3β-hydroxylase expressed from <Emphasis Type="Italic">Populus euphratica</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in dihydroflavonol production

Flavanone 3β-hydroxylase plays very important role in the biosynthesis of flavonoids. A putative flavanone 3β-hydroxylase gene (Pef3h) from Populus euphratica was cloned and over-expressed in Escherichia coli. Induction performed with 0.1 mM IPTG at 20°C led to localization of PeF3H in the soluble fraction. Recombinant enzyme was purified by Ni-NTA affinity. The optimal activity of PeF3H was revealed at pH 7.6 and 35°C. The purified enzyme was stable over pH range of 7.6–8.8 and had a half-life of 1 h at 50°C. The activity of PeF3H was significantly enhanced in the presence of Fe²⁺ and Fe³⁺. The K _M and V _max for the enzyme using naringenin as substrate were 0.23 mM and 0.069 μmoles mg–1min-1, respectively. The K _m and V _max for eriodictyol were 0.18 mM and 0.013 μmoles mg^–1min^–1, respectively. The optimal conditions for naringenin bioconversion in dihydrokaempferol were obtained: OD₆₀₀ of 3.5 for cell concentration, 0.1 mM IPTG, 5 mM α-ketoglutaric acid and 20°C. Under the optimal conditions, naringenin (0.2 g/L) was transformed into 0.18 g/L dihydrokaempferol within 24 h by the recombinant E. coli with a corresponding molar conversion of 88%. Thus, this study provides a promising flavanone 3β-hydroxylase that may be used in biosynthetic applications.     

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA–dT)]2, [poly(dG–dC)]2, and calf thymus DNA

The interaction of the Cu(II) drugs CuL(NO₃) and CuL′(NO₃) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL′ is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL]⁺ and [CuL′]⁺) with [poly(dA–dT)]₂, [poly(dG–dC)]₂, and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 °C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA–dT)]₂, with a site size n = 6–7, whereas they act as external binders with [poly(dG–dC)]₂ and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL′]⁺ were slightly larger than with [CuL]⁺. The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL]⁺ and [CuL′]⁺, with a significative dose–response effect for [CuL′]⁺, which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH₂ group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.     

Lunularic acid decarboxylase from the liverwort Conocephalum conicum

Some properties of a preparation of an enzyme, lunularic acid decarboxylase, from the liverwort Conocephalum conicum are described. The enzyme is normally bound and could be solubilized with Triton X-100; at least some of the bound decarboxylase activity appears to be associated with chloroplasts. For lunularic acid the enzyme has K_m 8.7 × 10^?5 M (pH 7.8 and 30°). Some substrate analogues have been tested but no other substrate was found. Pinosylvic acid is a competitive inhibitor for the enzyme, K_i 1.2 × 10^?4 M (pH 7.8 and 30°). No product inhibition was observed. Lunularic acid decarboxylase activity has also been observed with a cell-free system from Lunularia cruciata.     

Expression of a family 10 xylanase gene from Aspergillus usamii E001 in Pichia pastoris and characterization of the recombinant enzyme

A cDNA gene (Auxyn10A), which encodes a mesophilic family 10 xylanase from Aspergillus usamii E001 (abbreviated to AuXyn10A), was amplified and inserted into the XhoI and NotI sites of pPIC9K^M vector constructed from a parent pPIC9K. The recombinant expression vector, designated pPIC9K^M-Auxyn10A, was transformed into Pichia pastoris GS115. All P. pastoris transformants were spread on a MD plate, and then inoculated on geneticin G418-containing YPD plates for screening multiple copies of integration of the Auxyn10A. One transformant expressing the highest recombinant AuXyn10A (reAuXyn10A) activity of 368.6 U/ml, numbered as P. pastoris GSX10A4-14, was selected by flask expression test. SDS-PAGE assay demonstrated that the reAuXyn10A was extracellularly expressed with an apparent M.W. of 39.8 kDa. The purified reAuXyn10A displayed the maximum activity at pH 5.5 and 50 °C. It was highly stable at a broad pH range of 4.5–8.5, and at a temperature of 45 °C. Its activity was not significantly affected by EDTA and several metal ions except Mn²⁺, which caused a strong inhibition. The K _m and V _max, towards birchwood xylan at pH 5.5 and 50 °C, were 2.25 mg/ml and 6,267 U/mg, respectively. TLC analysis verified that the AuXyn10A is an endo-β-1,4-d-xylanase, which yielded a major product of xylotriose and a small amount of xylose, xylotetraose, and xylopentose from birchwood xylan, but no xylobiose.     

Purification and characterization of a thermostable aliphatic amidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1

Amidases catalyze the hydrolysis of amides to free carboxylic acids and ammonia. Hyperthermophilic archaea are a natural reservoir of various types of thermostable enzymes. Here, we report the purification and characterization of an amidase from Pyrococcus yayanosii CH1, the first representative of a strict-piezophilic hyperthermophilic archaeon that originated from a deep-sea hydrothermal vent. An open reading frame that encoded a putative member of the nitrilase protein superfamily was identified. We cloned and overexpressed amiE in Escherichia coli C41 (DE3). The purified AmiE enzyme displayed maximal activity at 85 °C and pH 6.0 (NaH₂PO₄–Na₂HPO₄) with acetamide as the substrate and showed activity over the pH range of 4–8 and the temperature range of 4–95 °C. AmiE is a dimer and active on many aliphatic amide substrates, such as formamide, acetamide, hexanamide, acrylamide, and l-glutamine. Enzyme activity was induced by 1 mM Ca²⁺, 1 mM Al³⁺, and 1–10 mM Mg²⁺, but strongly inhibited by Zn²⁺, Cu²⁺, Ni²⁺, and Fe³⁺. The presence of acetone and ethanol significantly decreased the enzymatic activity. Neither 5 % methanol nor 5 % isopropanol had any significant effect on AmiE activity (99 and 96 % retained, respectively). AmiE displayed amidase activity although it showed high sequence homology (78 % identity) with the known nitrilase from Pyrococcus abyssi. AmiE is the most characterized archaeal thermostable amidase in the nitrilase superfamily. The thermostability and pH-stability of AmiE will attract further studies on its potential industrial applications.     

Estimation of intracellular pH in muscle of fishes from different thermal environments

A technique based on homogenisation of rapidly frozen tissue was used to investigate the regulation of intracellular pH (pH_i) in freshwater and marine fish from diverse environmental temperatures. The following species were held at ambient temperatures of ca. 1°C (Notothenia coriiceps; Antarctica), 5°C (Pleuronectes platessa, Myoxocephalus scorpius; North Sea), and 26°C (Oreochromis niloticus; African lakes). The effects of seasonal acclimatisation to 4, 11 and 18°C were also examined in rainbow trout in the winter, autumn and summer, respectively. Extracellular (whole blood) pH (pH_e) did not follow the constant relative alkalinity relationship, where pH⁺=pOH⁻ for any particular temperature, over a range of 1–26°C (overall δpH_e/δT=0.009±0.002 U °C⁻¹; P<0.001), apparently being regulated by ionic fluxes and ventilation. Intracellular pH (pH_i) was also regulated independently of pN(=0.5 pK water) in all species of fish examined. The inverse relationship between pH_i and environmental temperature gave an overall δpH_i/δT of −0.010±0.001 U °C⁻¹ (for both white and red muscle) and −0.004±0.003 U °C⁻¹ (cardiac muscle). However, between 1 and 11°C δpH_i/δT was much higher (P<0.001), −0.022±0.003 U °C⁻¹ (white muscle) and −0.022±0.004 U °C⁻¹ (red muscle). The possible adaptive roles for these different acid–base responses to environmental temperature variation among tissues and species, and the potential difficulties of estimating pH_i, are discussed.