Thermal stability and thermodynamic analysis of native and methoxypolyethylene glycol modified trypsin

Four methoxypolyethylene glycols (MPEG, molecular masses 350, 750, 2000 and 5000 Da), each activated by nitrophenyl chloroformate, were used to modify trypsin. Compared with the native trypsin, the MPEG-modified trypsin was more stable against temperature between 30°C and 70°C, longer chain of MPEG moiety corresponding to higher thermal stability. The T for the native and the modified trypsin (0.4 mg ml^–1) was increased from 47°C to 66°C. The stabilization effect caused by MPEG modification was the result of decreasing in both the autolysis rate and the thermal denaturation rate. The thermodynamic analysis of the thermal denaturation process showed that the activation free energy (G^*) of the native and the modified trypsin at 60°C was increased from 102.9 to 109.3 kJ mol^–1; the activation enthalpy (H^*) was increased from 57.4 to 86.9 kJ mol^–1; the activation entropy (S^*) was increased from –136 to –67 J molK^–1. A possible explanation for the decreased thermal denaturation rate caused by MPEG modification was also discussed.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Oxygen isotope fractionation during respiration for different temperatures ofT. utilis andE. coli K12

Summary The temperature dependence of the oxygen isotope fractionation factor during respiration has been examined for two different microorganisms, namelyTorulopsis utilis andEscherichia coli K12 representing a yeast and a bacterium, respectively. The investigation covered a temperature range of 18° C, that is from 16° C to 34° C forT. utilis and from 19° C to 37° C forE. coli K12. Within this temperature range the fractionation factor ofT. utilis increases by 0.18; an insignificant change ( _{10° C} = 0.063;r = 0.067), whereas withE. coli K 12 an increase of 1.12; has been observed ( _{10° C} = 0.6;r = 0.55).     

Deuterium isotope effects and fractionation factors of hydrogen-bonded A:T base pairs of DNA

Deuterium isotope effects and fractionation factors of N1...H3–N3 hydrogen bonded Watson–Crick A:T base pairs of two DNA dodecamers are presented here. Specifically, two-bond deuterium isotope effects on the chemical shifts of ¹³C2 and ¹³C4, ²¹³C2 and ²¹³C4, and equilibrium deuterium/protium fractionation factors of H3, , were measured and seen to correlate with the chemical shift of the corresponding imino proton, _H3. Downfield-shifted imino protons associated with larger values of ²¹³C2 and ²¹³C4 and smaller values, which together suggested that the effective H3–N3 vibrational potentials were more anharmonic in the stronger hydrogen bonds of these DNA molecules. We anticipate that ²¹³C2, ²¹³C4 and values can be useful gauges of hydrogen bond strength of A:T base pairs.     

Tetramethyl Rhodamine Methyl Ester (TMRM) is Suitable for Cytofluorometric Measurements of Mitochondrial Membrane Potential in Cells Treated with Digitonin

A new method for cytofluorometric analysis of mitochondrial membrane potential has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells thus represents only the probe accumulated in mitochondria. Fibroblasts and cybrids were used as a model cell systems and optimal conditions for digitonin treatment and staining by TMRM were described. The TMRM signal collapsed by valinomycin, KCN and antimycin A and FCCP titration was used to gradually lower and characterise the stability of . The method is suitable for sensitive measurement of in different types of cultured cells.     

Characterization of the light induced reversible changes in the chiral macroorganization of the chromophores in chloroplast thylakoid membranes. Temperature dependence and effect of inhibitors

We investigated the temperature dependence and inhibitor sensitivity of the light-induced reversible changes in the circular dichroism (CD) of chloroplast thylakoid membranes. Earlier, these changes, which originate from structural changes affecting the chiral macroorganization of the chromophores, were thought to be driven by photochemically generated proton and/or ion gradients in the thylakoids [Garab et al. (1988) Biochemistry 27: 2430]. However, more recently, these changes have been shown to be largely independent of the photochemical activity of thylakoids, and CD has been observed in lamellar aggregates of the light harvesting chlorophyll a/b complex (LHC II) of Photosystem II [Barzda et al. (1996) Biochemistry 35: 8981]. Here, we show that in thylakoids (i) CD is gradually and substantially decelerated upon gradually decreasing the temperature from 33 °C to 2 °C, and abruptly disappears above 35–37 °C; (ii) CD is inhibited with nigericin with I₅₀ 1 M, which is about 10 times higher than the I₅₀for the transmembrane pH; (iii) CD can be inhibited with dicyclohexylcarbodiimide that blocks proton binding at the lumenal side of LHC II; (iv) quinone antagonists, such as antimycin-A and myxothiazol, inhibit CD without noticeably affecting the electron and proton transport, and the chiral macroorganization of the chromophores in the dark. We conclude that CD is conditioned but not driven by the photochemical activity of the membranes, and the structural changes are given rise by a physical mechanism previously unrecognized in thylakoids, thermooptic effect described for liquid crystals. We discuss the possible link between the deactivation(s) of the excess excitation energy and CD, the light-induced changes in the chiral macroorganization of the chromophores of the photophysical apparatus in thylakoids.     

Optomotorische Untersuchung des visuellen systems einiger Augenmutanten der Fruchtfliege Drosophila

Summary The optical properties of the compound eye of Drosophila have been analysed using the optomotor reactions of flies with normal and mutant eye pigmentation. The stimulus was provided by cylindrical patterns with different periodic intensity distributions rotating at different speeds. The response consists of a torque about the vertical axis and was recorded under conditions of fixed flight. (Maximum reaction is about 0.04 dyn · cm). The transfer characteristics of the optical system are determined by the inter-ommatidial angle , influencing the resolving power and by the width of the visual field of single ommatidia , influencing the response at high spatial frequencies. The values = 4.6° and = 3.5° are obtained from stimulus-response experiments with Drosophila. They are independent of the presence of screening pigments. Differences in the response of flies with strong (+, se), weak (w ^a), and missing (w) pigmentation can be explained by the increased amount of scattered light in the pigment-deficient eyes. The overall intensities in the equally illuminated receptors are expected to be in the ratio 11825, respectively. The perception of motion depends only on the temporal, not on the spatial phase relations between periodic intensity variations in neighbouring ommatidia. Therefore the inhomogeneous distribution of the inter-ommatidial angle changes the resolving power in different parts of the eye without changing the response to motion. Different simultaneous stimuli of equal strength in different parts of the eye are averaged in the perceptive system of Drosophila according to the number of ommatidia in these parts.     

Cyclosporin A-sensitive decrease in the transmembrane potential across the inner membrane of liver mitochondria induced by low concentrations of fatty acids and Ca2+

At low Ca²⁺ concentrations the pore of the inner mitochondrial membrane can open in substates with lower permeability (Hunter, D. R., and Haworth, R. A. (1979) Arch. Biochem. Biophys., 195, 468-477). Recently, we showed that Ca²⁺ loading of mitochondria augments the cyclosporin A-dependent decrease in transmembrane potential () across the inner mitochondrial membrane caused by 10 M myristic acid but does not affect the stimulation of respiration by this fatty acid. We have proposed that in our experiments the pore opened in a substate with lower permeability rather than in the classic state (Bodrova, M. E., et al. (2000) IUBMB Life, 50, 189-194). Here we show that under conditions lowering the probability of classic pore opening in Ca²⁺-loaded mitochondria myristic acid induces the cyclosporin A-sensitive decrease and mitochondrial swelling more effectively than uncoupler SF6847 does, though their protonophoric activities are equal. In the absence of P_i and presence of succinate and rotenone (with or without glutamate) cyclosporin A either reversed or only stopped decrease induced by 5 M myristic acid and 5 M Ca²⁺. In the last case nigericin, when added after cyclosporin A, reversed the decrease, and the following addition of EGTA produced only a weak (if any) increase. In P_i-containing medium (in the presence of glutamate and malate) cyclosporin A reversed the decrease. These data show that the cyclosporin A-sensitive decrease in by low concentrations of fatty acids and Ca²⁺ cannot be explained by specific uncoupling effect of fatty acid. We propose that: 1) low concentrations of Ca²⁺ and fatty acid induce the pore opening in a substate with a selective cation permeability, and the cyclosporin A-sensitive decrease results from a conversion of to pH gradient due to the electrogenic cation transport in mitochondria; 2) the ADP/ATP-antiporter is involved in this process; 3) higher efficiency of fatty acid compared to SF6847 in the Ca²⁺-dependent pore opening seems to be due to its interaction with the nucleotide-binding site of the ADP/ATP-antiporter and higher affinity of fatty acids to cations.     

Protonmotive force in Brevibacterium flavum: the lack of intracellular pH homeostasis

Brevibacterium flavum 22LD-P cells were shown to maintain a transmembrane pH gradient (pH) from 0.6 to 1.8–2 units and a transmembrane electric potential difference () from 0 to 200 mV depending on the pH and ionic composition of the incubation medium, grwoth substrate and concentration of cells. decreased from 120–140 mV to 0 when medium pH was lowered from neutral to 5.0–5.5 and increased to 180–200 mV when medium pH was raised to 8–9 in cells utilizing acetate or endogenous substrate. Cells growing on sucrose, kept around 100–120 mV at neutral as well as acidic medium pH. Intracellular pH in the acetate utilizing or endogenously respiring cells was maintained with the range of 8.9 to 5.5 at medium pH ranging from 9.1 to 4.0, respectively. Sucrose grown cells were able to maintain a more stable intracellular pH. Endogenously respiring cells in potassium phosphate buffer at high biomass concentrations maintained larger pH and relatively smaller , than the same cells in diluted suspensions. Cells in sodium phosphate buffer possessed larger and almost no pH, but was still dependent on biomass concentration.The lack of intracellular pH homeostasis and the collapse of at acid medium pH are discussed in the context of cell membrane proton permeability.     

Gas exchange and carbon isotope discrimination in lichens: Evidence for interactions between CO2-concentrating mechanisms and diffusion limitation

The characteristics of gas exchange and carbon isotope discrimination were determined for a number of lichen species, representing contrasting associations between fungal (mycobiont) and photosynthetic (photobiont) organism. These parameters were evaluated with regard to the occurrence of any CO₂-concentrating mechanism (CCM) expressed specifically by the green algal (phycobiont) or cyanobacterial (cyanobiont) partner. Carbon isotope discrimination () fell into three categories. The highest , found in lichens comprising a phycobiont plus cyanobacteria limited to pockets in the thallus (known as cephalodia), ranged from 24 to 28, equivalent to a carbon isotope ratio (¹³C) of around -32 to-36 vs. Pee Dee Belemnite (PDB) standard. Further evidence was consistent with CO₂ supply to the carboxylating system entirely mediated by diffusion rather than a CCM, in that thallus CO₂ compensation point and online instantaneous were also high, in the range normally associated with C₃ higher plants. For lichens consisting of phycobiont or cyanobiont alone, organic material formed two distinct ranges around 15 (equivalent to a ¹³C of -23%.). Thallus compensation point and instantaneous were lower in the cyanobiont group, which also showed higher maximum rates of net photosynthesis, whether expressed on the basis of thallus dry weight, chlorophyll content or area. These data provide additional evidence for the activity of a CCM in cyanobiont lichens, which only show photosynthetic activity when reactivated with liquid water. Rates of net CO₂ uptake were lower in both phycobiont associations, but were relatively constant across a wide working range of thallus water contents, usually in parallel with on-line . The phycobiont response was consistent whether photosynthesis had been reactivated with liquid water or water vapour. The effect of diffusion limitation could generally be seen with a 3–4 decrease in instantaneous at the highest water contents. The expression of a CCM in phycobiont algae, although reduced compared with that in cyanobacteria, has already been related to the occurrence of pyrenoids in chloroplasts. In view of the inherent requirement of cyanobacteria for some form of CCM, and the smaller pools of dissolved inorganic carbon (DIC = CO₂ + HCO _inf3 ^su– + CO _inf3 ^su2– ) associated with phycobiont lichens, it appears that characteristics provide a good measure of the magnitude of any CCM, albeit tempered by diffusion limitation at the highest thallus water contents.Abbreviations ANOVA analysis of variance - CCM CO₂-concentrating mechanism - cyanobiont cyanobacterium - DIC CO₂ + HCO _inf3 ^su– + CO _inf3 ^su2– (dissolved inorganic carbon) - photobiont photosynthetic organism present in the association - phycobiont green alga - phycobiont + cephalodia green algae + cyanobacteria in cephalodia - Pmax maximum photosynthetic rate - PPFD photosynthetic photon flux density, 400–700 nm - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - carbon isotope discrimination () - ¹³C carbon isotope ratio () We would like to thank Dr. Enrico Brugnoli (CNR, Porano, Italy) and E.C. Smith (University of Newcastle) for many helpful discussions. Dr. Kristin Palmqvist (Department of Plant Physiology, University of Umeå, Sweden) kindly provided the samples of Peltigera apthosa. In particularly, Cristina Máguas would like to thank to Prof. Fernando Catarino (University of Lisbon) for his support throughout this study. Cristina Máguas has been supported by JNICT-Science Programme studentship (BD/153/90-RN).     

Effect of chemical modifiers of amino acid residues on proton conduction by the H+-ATPase of mitochondria

The effect of chemical modifiers of amino acid residues on the proton conductivity of H⁺-ATPase in inside out submitochondrial particles has been studied. Treatment of submitochondrial particles prepared in the presence of EDTA (ESMP) with the arginine modifiers, phenylglyoxal or butanedione, or the tyrosine modifier, tetranitromethane, caused inhibition of the ATPase activity. Phenylglyoxal and tetranitromethane also caused inhibition of the anaerobic release of respiratory H⁺ in ESMP as well as in particles deprived of F₁ (USMP). Butanedione treatment caused, on the contrary, acceleration of anaerobic proton release in both particles. The inhibition of proton release caused by phenylglyoxal and tetranitromethane exhibited in USMP a sigmoidal titration curve. The same inhibitory pattern was observed with oligomycin and withN,N-dicyclohexylcarbodiimide. In ESMP, relaxation of H⁺ exhibited two first-order phases, both an expression of the H⁺ conductivity of the ATPase complex. The rapid phase results from transient enhancement of H⁺ conduction caused by respiratory H⁺ itself. Oligomycin,N,N-dicyclohexylcarbodiimide, and tetranitromethane inhibited both phases of H⁺ release, and butanedione accelerated both. Phenylglyoxal inhibited principally the slow phase of H⁺ conduction. In USMP, H⁺ release followed simple first-order kinetics. Oligomycin depressed H⁺ release, enhanced respiratory H⁺, and restored the biphasicity of H⁺ release. Phenylglyoxal and tetranitromethane inhibited H⁺ release in USMP without modifying its first-order kinetics. Butanedione treatment caused biphasicity of H⁺ release from USMP, introducing a very rapid phase of H⁺ release. Addition of soluble F₁ to USMP also restored biphasicity of H⁺ release. A mechanism of proton conduction by F _o is discussed based on involvement of tyrosine or other hydroxyl residues, in series with the DCCD-reactive acid residue. There are apparently two functionally different species of arginine or other basic residues: those modified by phenylglyoxal, which facilitate H⁺ conduction, and those modified by butanedione, which retard H⁺ diffusion.     

Response of effective quantum yield of photosystem 2 to in situ temperature in three alpine plants

The response of effective quantum yield of photosystem 2 (F/F_m) to temperature was investigated under field conditions (1 950 m a.s.l.) in three alpine plant species with contrasting leaf temperature climates. The in situ temperature response did not follow an optimum curve but under saturating irradiances [PPFD >800 µìmol(photon) m^–2s^–1] highest F/F_m occurred at leaf temperatures below 10°C. This was comparable to the temperature response of antarctic vascular plants. Leaf temperatures between 0 and 15°C were the most frequently (41 to 56%) experienced by the investigated species. At these temperatures, F/F_m was highest in all species (data from all irradiation classes included) but the species differed in the temperature at which F/F_m dropped below 50% (Soldanella pusilla >20°C, Loiseleuria procumbens >25°C, and Saxifraga paniculata >40°C). The in situ response of F/F_m showed significantly higher F/F_m values at saturating PPFD for the species growing in full sunlight (S. paniculata and L. procumbens) than for S. pusilla growing under more moderate PPFD. The effect of increasing PPFD on F/F_m, for a given leaf temperature, was most pronounced in S. pusilla. Despite the broad diurnal leaf temperature amplitude of alpine environments, only in S. paniculata did saturating PPFD occur over a broad range of leaf temperatures (43 K). In the other two species it was half of that (around 20 K). This indicates that the setting of environmental scenarios (leaf temperature×PPFD) in laboratory experiments often likely exceeds the actual environmental demand in the field.This revised version was published online in March 2005 with corrections to the page numbers.     

Temperature induced variation in the distribution of different types of muscle fibre in the goldfish (Carassius auratus)

Summary Goldfish (Carassius auratus L.) were acclimated to environmental temperatures of 3 °C, 18 °C and 31 °C for a period of three months. Cytochemical techniques were used to study the metabolism and myofibrillar ATPase activities of individual muscle fibres. Fish muscle is composed of three basic fibre types each with distinct contractile and metabolic characteristics. Cold acclimation resulted in a shift to a more aerobic type of metabolism, particularly in the red and pink fibres. In addition, environmental temperature was found to affect the size and relative distribution of the different fibre types in the myotome. The total number of pink and red fibres increased significantly with cold acclimation. Mechanisms of environmentally-induced adaptation of muscle fibre phenotype are discussed.In addition to changes in the metabolism and distribution of muscle-fibre types, biochemical studies have provided evidence for different kinetic forms of Mg²⁺Ca²⁺ myofibrillar ATPase at different environmental temperatures. Activities of myofibrillar ATPase assayed at 31 °C were 2–3 times higher in fish acclimated to the higher environmental temperature. Activation enthalpy (H ) of the ATPase was also signficantly reduced in the cold adapted enzyme. Reduction of H in the cold acclimated ATPase is thought to reduce the temperature sensitivity of the activation process thus partly compensating for the reduced cell temperature.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Basis of the Relationship between Ash Content in the Flag Leaf and Carbon Isotope Discrimination in Kernels of Durum Wheat

The relationship between ash content and carbon isotope discrimination () was studied in durum wheat (Triticum durum Desf.) grown in a Mediterranean region (Northwest Syria) under three different water regimes (hereafter referred to as environments). In two of these environments, 144 genotypes were cultivated under rain-fed conditions. In the third environment, 125 genotypes were cultivated under irrigation. Ash content was measured in the flag leaf about 3 weeks after anthesis, whereas was analysed in mature kernels. Total transpiration of the photosynthetic tissues of the culm contributing, from heading to maturity, to the filling of kernels was also estimated. Leaf ash content, expressed either on dry matter or leaf area basis or as total ash per blade, correlated positively (p< 0.001) with in the three environments. However, this relationship was not the result of a positive correlation across genotypes between and tissue water content. Moreover, only a small part of the variation in across genotypes was explained by concomitant changes in ash content. When all genotypes across the three environments were plotted, and ash content followed a non-linear relationship (r ² = 74), with tending to a plateau as the ash content increased. However, for the set of genotypes and environments combined, total ash content per leaf blade was positively and linearly related (r ² = 0.76) with the accumulated culm transpiration. The non-linear nature of the relationship between ash content and is sustained by the fact that culm transpiration also showed a non-linear relationship with kernel . Therefore, differences in leaf ash content between environments, and to a lesser extent between genotypes, seem to be brought about by variations in accumulated transpiration during grain formation.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

Theoretical stability diagram of solvent-containing black lipid films

Recently, we have developed an analytical, semi-microscopic theory for the macroscopic behavior of a solvent-containing black lipid film subjected to an electric cross film voltage, . Here we employ the theoretical expressions derived for the disjoining pressure, _D, the film elasticity, _F, and the film tension, _F, to construct the stability diagram of the film, in the _D-. Depending on its state (_D, ), the film is stable or is prone to squeezing or bending deformations. For a monooleate film we show how the destruction of the plane film due to a periodic thickness fluctuation (squeezing) is facilitated by two mechanisms: i) lowering of _D at fixed ; ii) lowering of at fixed _D, provided that the film is in a stable state characterized by _D<–7.03×10³ dyne/cm² and >0 mV. Bending of a low tension film (single interface tension _s 0.025 dyne/cm¹) can be achieved only for >170 mV and _D > –8.7 × 10⁴ dyne/cm². Finally, we demonstrate the existence of a marginal state ( _D ⁰ , ⁰) where the film is predicted to exhibit strong fluctuations both in the squeezing and in the bending mode.     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .     

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, H^app, of the binding of Mn²⁺-ions to goat -lactalbumin as a function of temperature. The observed H^app can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat -lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (H, G, S, C_p) into the binding and conformational contributions. By circular dichroism we showed that NH ₄ ⁺ -ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na⁺ and K⁺: the binding constant equals 98 ± 9 M^–1.Abbreviations BLA bovine -lactalbumin - GLA goat -lactalbumin - HLA human -lactalbumin - CD circular dichroism Offprint requests to: H. Van DaelDeceased     

Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cerevisiae

Summary Excision of pyrimidine dimers and interstrand DNA crosslinks was examined in the deletion mutants rad7-1, rad23-1, and rad7-1 rad23-1. These mutants remove pyrimidine dimers and crosslinks much less efficiently than the RAD ⁺ strains; only 30–60% of pyrimidine dimers and 25–40% of crosslinks are removed even after prolonged incubation. The rad7 and rad23 mutations may represent defects in protein factors which increase the efficiency of the nicking enzyme complex or make chromatin more accessible to the nicking activity.