Elucidation of intermediate (mobile) and slow (solidlike) protein motions in bovine lens homogenates by carbon-13 NMR spectroscopy

The motional dynamics of lens cytoplasmic proteins present in calf lens homogenates were investigated by two 13C nuclear magnetic resonance (NMR) techniques sensitive to molecular motion to further define the organizational differences between the cortex and nucleus. For the study of intermediate (mobile) protein rotational reorientation motion time scales [rotational correlation time (tau 0) range of 1-500 ns], we employed 13C off-resonance rotating frame spin-lattice relaxation, whereas for the study of slow (solidlike) motions (tau 0 greater than or equal to 10 microseconds) we used the solid-state NMR techniques of dipolar decoupling and cross-polarization. The frequency dependence of the peptide bond carbonyl off-resonance rotating frame spectral intensity ratio of the lens proteins present in native calf nuclear homogenate (42% protein) at 35 degrees C indicates the presence of a polydisperse mobile protein fraction with a tau 0,eff (mean) value of 57 ns. This mean value is consistent with the average value calculated from the known water-soluble nuclear lens protein polydispersity assuming a cytoplasmic viscosity 3 times that of pure water. Lowering the temperature to 1 degree C, a temperature which produces the cold cataract, results in an overall decrease in tau 0,eff to 43 ns, suggesting a selective removal of beta H-, LM-, and possibly gamma s-crystallins from the mobile lens protein population. The presence of solidlike or motionally restricted protein species was established by dipolar decoupling and cross-polarization. The fraction of motionally restricted protein in the nuclear region varied from 0.35 to 0.45 in the temperature range of 35-1 degree C. For native cortical homogenate (25% protein), the off-resonances rotating frame spectral intensity ratio frequency-dependent curves for the protein carbonyl resonance yielded tau 0,eff values of 34 and 80 ns at 35 and 1 degree C, respectively. Both values were reconciled with the known lens cortex soluble protein polydispersity using an assumed cytoplasmic viscosity 1.5 times that of pure water at the same temperature. Comparison of proton dipolar-decoupled and nondecoupled 13C NMR spectra of native cortical homogenate at 20 degrees C indicates the absence of significant contributions from slowly tumbling, motionally restricted species. This interpretation was confirmed by the failure to detect significant lens protein 13C-1H cross-polarization at this temperature. However, at 1 degree C, the fraction of solidlike protein was 0.15. Concentrated cortical homogenates at 20 degrees C (42% protein), by contrast, gave cross-polarization spectra with maximum absolute signal intensities 50-70% of native nuclear homogenates, but with similar magnetization parameters...     

High-resolution field-cycling NMR studies of a DNA octamer as a probe of phosphodiester dynamics and comparison with computer simulation

Phosphorus-spin longitudinal relaxation rates of the DNA duplex octamer [d(GGAATTCC)](2) have been measured from 0.1 to 17.6 T by means of conventional and new field-cycling NMR methods. The high-resolution field-cycling method is identical to a conventional relaxation experiment, except that after preparation the sample is moved pneumatically from its usual position at the center of the high-resolution magnet upward to a lower field above its normal position and then returned to the center for readout after it has relaxed for the programmed relaxation delay at the low field. This is the first measurement of all longitudinal relaxation rates R(1) of a nuclear species in a macromolecule over virtually the entire accessible magnetic field range. For detailed analysis, three magnetic field regions can be delineated: (i) dipolar relaxation dominates at fields below 2 T, (ii) chemical shift anisotropy (CSA) relaxation is roughly constant from 2 to 6 T, and (iii) a square-law increasing dependence is seen at fields higher than approximately 6 T due to internal motion CSA relaxation. The analysis provides a rotational correlation time (tau(r) = 4.1 +/- 0.3 ns) for the duplex at both 1.5 and 0.25 mM concentrations (of duplex) at 22 degrees C. For comparison, extraction of tau(r) in the conventional way from the ratio of T(1)/T(2) at 14 T yields 3.2 ns. The tau(r) discrepancy disappears when we exclude the contribution of internal motion from the R(1) in the ratio. The low-field dipolar relaxation provides a weighted inverse sixth power sum of the distances from the phosphorus to the protons responsible for relaxation. This average is similar for all phosphates in the octamer and similar to that in previous B-DNA structures (its inverse sixth root is about 2.40 A for two different concentrations of octamer). The CSA relaxation at intermediate field provides an estimate of the order parameter squared, S(c)(2), for each phosphorus. S(c)(2) is about 0.7-1, clearly different for different phosphate linkages in the octamer duplex. The increasing R(1) at high fields reflects CSA relaxation due to internal motions, for which a correlation time, tau(hf), can be approximately extracted with the aid of additional measurements at 14.0 and 17.6 T. We conclude that tau(hf) values are relatively large, in the range of about 150 ps. Insight into the motions leading to this correlation time was gained by a 28 ns molecular dynamics simulation of the molecule. S(2) and tau(s) (corresponding to tau(hf)) predicted by this simulation were in good agreement with the experimental values from the field-cycling data. Both the effect of Mg(2+) on the dynamic parameters extracted from (31)P relaxation rates and the field dependence of relaxation rates for several protons of the octamer were measured. High-resolution field cycling opens up the possibility of monitoring residue-specific dipolar interactions and dynamics for the phosphorus nuclei of diverse oligonucleotides.     

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 2. Evidence from phosphorus-31 NMR measurements.

31P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. 31P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T1) relaxation times of hydrated cardiolipin as a function of temperature describes a T1 minimum at around 25 degrees C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behavior of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T1 up to 75 degrees C. The enhancement in lipid phosphorus T1 relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T1 effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the "magic angle" was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.     

Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model

The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4-6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans-gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS)     

13C NMR and fluorescence analysis of tryptophan dynamics in wild-type and two single-Trp variants of Escherichia coli thioredoxin.

The rotational motion of tryptophan side chains in oxidized and reduced wild-type (WT) Escherichia coli thioredoxin and in two single-tryptophan variants of E. coli thioredoxin was studied in solution in the temperature range 20-50 degrees C from 13C-NMR relaxation rate measurements at 75.4 and 125.7 MHz and at 20 degrees C from steady-state and time-resolved trp fluorescence anisotropy measurements. Tryptophan enriched with 13C at the delta 1 and epsilon 3 sites of the indole ring was incorporated into WT thioredoxin and into two single-trp mutants, W31F and W28F, in which trp-28 or trp-31 of WT thioredoxin was replaced, respectively, with phenylalanine. The NMR relaxation data were interpreted using the Lipari and Szabo "model-free" approach (G. Lipari and A. Szabo. 1982. J. Amer. Chem. Soc. 104:4546-4559) with trp steady-state anisotropy data included for the variants at 20 degrees C. Values for the correlation time for the overall rotational motion (tau m) from NMR of oxidized and reduced WT thioredoxin at 35 degrees C agree well with those given by Stone et al. (Stone, M. J., K. Chandrasekhar, A. Holmgren, P. E. Wright, and H. J. Dyson. 1993. Biochemistry. 32:426-435) from 15N NMR relaxation rates, and the dependence of tau m on viscosity and temperature was in accord with the Stokes-Einstein relationship. Order parameters (S2) near 1 were obtained for the trp side chains in the WT proteins even at 50 degrees C. A slight increase in the amplitude of motion (decrease in S2) of trp-31, which is near the protein surface, but not of trp-28, which is partially buried in the protein matrix, was observed in reduced relative to oxidized WT thioredoxin. For trp-28 in W31F, order parameters near 1 (S2 > or = 0.8) at 20 degrees C were found, whereas trp-31 in W28F yielded the smallest order parameters (S2 approximately 0.6) of any of the cases. Analysis of time-resolved anisotropy decays in W28F and W31F yielded S2 values in good agreement with NMR, but gave tau m values about 60% smaller. Generally, values of tau e, the effective correlation time for the internal motion, were < or = 60 ps from NMR, whereas somewhat longer times were obtained from fluorescence. The ability of NMR and fluorescence techniques to detect subnanosecond motions in proteins reliably is examined.     

In vitro formation of organ specific proximate carcinogen of benzo(a)pyrene by rat homogenates

In order to determine the organ specific carcinogenicity of benzo(a)pyrene (B(a)P), its metabolites, formed in vitro by incubation with the homogenates from liver, lungs, kidneys, intestine and brain of rats, were isolated by TLC and spectroscopy. B(a)P was found to be converted into a number of metabolites by different tissue homogenates. The results showed that the proximate carcinogenic metabolite, 7,8-dihydro-7,8-dihydroxy B(a)P was formed only when rat lung and kidney homogenates were incubated with B(a)P in vitro. The UV spectral analysis also confirmed the formation of this metabolite only on incubation of B(a)P with rat lung and kidney homogenates. As the proximate carcinogenic metabolite was only formed by incubating B(a)P with the homogenates from target organs, its organ specific carcinogenicity may be explained.     

Backbone dynamics of barstar: a (15)N NMR relaxation study

Backbone dynamics of uniformly (15)N-labeled barstar have been studied at 32 degrees C, pH 6.7, by using (15)N relaxation data obtained from proton-detected 2D (1)H-(15)N NMR spectroscopy. (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and steady-state heteronuclear (1)H-(15)N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide (15)N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (tau(m)), the generalized order parameter (S(2)), the effective correlation time for internal motions (tau(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D( parallel)/D( perpendicular)) is 0.82 +/- 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, omega(H), and omega(N), where omega(H),(N) are proton and nitrogen Larmor frequencies. The value of tau(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The tau(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 +/- 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2 to 9.1 s(-1), indicating the presence of conformational averaging motions only for a small subset of residues.     

Molecular mobility and structure of elastin deduced from the solvent and temperature dependence of 13C magnetic resonance relaxation data.

13C relaxation parameters, T1, line width, and NOE, have been determined for backbone carbons of ligamentum nuchae elastin swollen by 0.15 M NaCl, 0.15 M NaCl-formamide, 0.15 M NaCl-ethanol, dimethyl sulfoxide, and formamide. The data have been analyzed in terms of (a) a single correlation time model and (b) a model employing a log-chi2 distribution of correlation times used by Schaefer (1973) to analyze solid cis-polyisoprene 13C relaxation data. Employing the latter mode, one obtains an approximately self-consistent quantitative analysis of all the elastin data. An average backbone correlation time, tau, of ca. 2 nsec is calculated for elastin swollen in the presence of polar organic solvents at 37 degrees, in approximate agreement with tau of 0.4 nsec obtained for bulk cis-polyisoprene at 35 degrees. The influence of solvent and temperature on elastin spectra indicate that the larger tau value (approximately 80 nsec) obtained for elastin swollen by 0.15 M NaCl at 37 degrees is a consequence of weak interchain polar and hydrophobic interactions, a result which is in accord with the reported viscoelastic behavior exhibited by water-swollen elastin at 37 degrees. The results obtained further suggest that Gly, Pro, and Val residues are significantly more mobile than Ala residues, which are located in the cross-link regions. Hence, the NMR data support the view that water-swollen elastin is composed of a network of mobile chains, except possibly in the cross-link regions.     

In vitro metabolism of the pro-carcinogen aflatoxin B1 by liver preparations of the calf, nurse shark and clearnose skate

1. Liver postmitochondrial supernatant preparations of calf, clearnose skate, and nurse shark were able to metabolize the fungal toxin aflatoxin B1 to various metabolites. 2. Calf liver produced aflatoxin M1 and Q1 as the major chloroform soluble metabolites, with small amounts of aflatoxicol formed during incubation. 3. Liver preparations of the elasmobranchs, however, produced aflatoxicol as the major chloroform soluble metabolite with no other metabolite being detected. 4. The water soluble metabolite profiles for the three species were also quite different with the tris diol adduct being produced to a much greater extent in calf liver preparations. 5. Aflatoxicol production by the elasmobranch liver homogenates was reversible with the skate reconverting a large amount (30%) of aflatoxicol to AFB1. The nurse shark, however, appeared to convert a portion of aflatoxicol to an unknown metabolite more polar than AFB1. 6. Calf liver DNA bound approximately 3 x more 3H-AFB1 than shark liver DNA.     

Effect of temperature and binding with copper ions on the motility of spin-labeled oxy- and methemoglobin subunits

Using the method for separate determination of correlation times of spin-labeled proteins (tau M) and labels (tau R) it has been shown that at temperatures below and about 25 degrees the mobility of oxy Hb subunits is higher than that of met Hb. From 30 degrees on oxy and met Hb show identical flexibility (tau M = 40 ns) in 0.01 M phosphate buffer (pH 7.3) containing 0.15 M NaCl. With a decline in pH from 7.3 to 6.4 the intramolecular mobility of met Hb subunits decreases. In the absence of 0.15 M NaCl at pH 7.3 (5 degrees C) met Hb becomes more flexible (tau M drops from 25 to 16 ns). Complex formation of beta-chains of oxy Hb with one Cu+2 ion at 20 degrees has a negligible bearing on the flexibility of the protein, whereas addition of second ion considerably enhances interaction between the subunits of the tetramer and decreases its flexibility (tau M rises from 17 to 30 ns).     

Adenosine triphosphate compartmentation in the rat heart: a 31P spin-lattice relaxation study

Longitudinal relaxation times (T1) of phosphorus compounds in the perfused rat heart and erythrocytes were measured using the 31P Driven-Equilibrium Single-Pulse Observation of T1 relaxation (DESPOT) method at 33 degrees C. Both creatine phosphate in the heart and the three phosphate groups of adenosine triphosphate (ATP) in erythrocytes showed single-exponential relaxation. The three phosphate groups of ATP in the heart, however, had two T1 components. The T1 values of the short and the long T1 components of the beta-phosphate of ATP were ca. 0.4 and 14 s, respectively. The fraction with the long T1 represented ca. 30% of the total ATP content. These results suggested that there were two major pools of intracellular ATP in the rat heart which could be determined by 31P NMR spectroscopy.     

Demonstration of different complexation modes between cobalt and 5' AMP, by direct NMR observation of the low-temperature complex

We report the first direct NMR observation of transition metal-nucleotide complexes. The phosphorus and proton spectra of a cobalt-5' AMP complex were observed in water, pH 7, -- 10 degrees C. This complex is different from the high temperature species : for instance its 31P chemical shift is -- 50 ppm, whereas the published value obtained indirectly for the high temperature form corresponds to ca. -- 1200 ppm. The -- 50 ppm complex is present in significant proportion at 20 degrees C and possibly at higher temperatures. Multiple complexation modes are also observed for Co-ATP.     

Effects of hypothermia on rat brain pHi and phosphate metabolite regulation by 31P-NMR

The effects of arterial alphastat regulation on brain intracellular pH (pHi) and several phosphate metabolites were assessed in anesthetized rats during hypothermia (28.6 +/- 0.2 degrees C) and normothermia (36.2 +/- 0.2 degrees C) by using 31P high-field (8.5 T) nuclear magnetic resonance (NMR). There were significant differences in pHi and metabolite ratios at the two temperatures under conditions of equal minute ventilation. During hypothermia, the brain pHi was 0.09 U higher, the phosphocreatine-to-inorganic phosphate (PCR/Pi) ratio 49% larger, and Pi-to-ATP 20% lower than at normothermia. These changes were fully reversible on warming the animal. The change in brain pHi/temperature was -0.011U/degrees C (95% confidence interval -0.007 to -0.016). The brain's ability to regulate its pHi and phosphate metabolism during hypercapnic acid-base stress was studied by using 10% CO2 ventilation. Hypothermic rats showed a larger fall in brain pHi (0.145 +/- 0.01 U, 7.15-7.01) with 10% CO2 than normothermic rats (0.10 +/- 0.02 U, 7.06-6.96). Similarly ventilated rats had a larger fall in arterial pH with 10% CO2 at hypothermia (0.36 +/- 0.04 U) than normothermia (0.24 +/- 0.01 U), so the delta brain pH/delta arterial pH was the same at both temperatures. The brain PCr-to-Pi ratio decreased approximately 20% during 10% CO2 breathing in both hypothermic and normothermic animals. Brain pHi and metabolite ratios returned to base line 30-50 min after CO2 washout in both groups. In summary, lowering body temperature while maintaining constant ventilation leads to changes in brain pHi and metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.     

Temperature dependence of kinetic parameters of single potential dependent K+ channels in mollusc neurons. Similarity in the action of potential and temperature]

Using the patch-voltage-clamp method on excised membrane fragments from molluscan neurones temperature dependences of kinetic parameters of the fast and slow K(+)-channels were investigated in the temperature range 1 to 40 degrees C. Temperature dependences of probability of the channel open state (P0) for the slow and fast K(+)-channels are, generally, opposite, that is P0 increases for the slow channel and decreases for the fast channel with temperature. Similar dependences characterize durations of single channel open intervals (tau 0) and burst durations (t(p)). Durations of interburst and interpulse intervals (respectively, t(i) and tau) decrease for the slow channel and increase, in contrast, for the fast channel with temperature. For the channels of both types temperature dependences of P0 (as for other parameters) are essentially nonmonotonous. There are two local extrema, at least: for the slow K(+)-channel-maximum at 15 degrees C (minimum for the fast channel) and minimum at 20-25 degrees C (maximum for the fast channel). In some cases the number of local extrema may be greater than two. Some similarity in the action of temperature and membrane potential on the kinetic parameters was observed. For the slow K(+)-channel P0, tau 0 and t p increase with temperature and membrane potential. For the fast channel these parameters decrease at the same conditions. Moreover, for the channels of both types temperature dependences of the kinetic parameters are slightly pronounced at the potentials where potential dependences of the parameters are least. As a whole, temperature measurements showed that there are, possibly, several points of structural transitions (similar to phase transitions) in the temperature range 0 to 40 degrees C. Primarily, the kinetic parameters are determined by these transitions.     

Multiple transients in the pre-steady-state of nucleoside hydrolase reveal complex substrate binding, product base release, and two apparent rates of chemistry

We have investigated the transient kinetics of the nucleoside hydrolase from Trypanosoma vivax (TvNH) at low temperatures (5 degrees C). Three novel absorbance transients (termed tau1, tau3, and tau4) were detected during multiple-guanosine turnover stopped-flow absorbance spectroscopy, in addition to a transient (tau2) that had been observed previously at 35 degrees C. At 5 degrees C, TvNH displays full-sites activity and not half-of-the-sites activity as is apparent at 35 degrees C. Both tau1 and tau2 are assigned to chemistry based on rapid-quench results. For tau1, the rate of chemistry is ca. 3000-fold faster than kcat (1-2 orders of magnitude greater than previous estimates). The pH dependencies of the burst amplitudes for tau1 and tau2 indicate that these transients arise from the formation of two different dimeric TvNH.substrate complexes and not from TvNH that contains kinetically asymmetric monomers. The saturating burst rates for tau1 and tau2 are surprisingly pH-independent, given the prominent role of acid-base chemistry in the proposed mechanism for TvNH. tau3 and tau4 are assigned to the substrate binding and base release processes, respectively, and shown to be equivalent to two fluorescence transients (tau3 and tau4, respectively) observed previously by stopped-flow methods at 35 degrees C. The rate of base release is shown to be an apparent rate. Together with steady-state product inhibition results, the data indicate that TvNH follows an ordered uni-bi kinetic mechanism with a TvNH.base dead-end complex, and not the rapid equilibrium random uni-bi mechanism proposed for other NHs. Two applicable kinetic models are presented and their implications for future mechanistic studies discussed.     

The effects of an acute temperature change on the metabolic recovery from exhaustive exercise in luvenile Atlantic salmon (Salmo salar)

This research examined the influence of acute changes of water temperature on the recovery processes following exhaustive exercise in juvenile Atlantic salmon (Salmo salar). White muscle phosphocreatine (PCr), ATP, lactate, glycogen, glucose, pyruvate, plasma lactate, and plasma osmolality were measured during rest and at 0, 1, 2, and 4 h following exhaustive exercise in fish acclimated and exercised at 12 degrees C and acutely exposed to either 6 degrees C or 18 degrees C water during recovery. An acute exposure to 6 degrees C water during the recovery period resulted in a severe reduction of metabolic recovery in salmon. However, metabolites such as muscle PCr and ATP and plasma lactate recovered very quickly (2-4 h) in fish acutely exposed to 18 degrees C during recovery. Overall, differences exist when postexercise metabolite levels are compared between acclimated fish and those fish acutely exposed to different water temperatures (either higher or lower). Taken together, the findings of the acute experiments suggest that at some point following exercise fish may seek warmer environments to speed the recovery process. However, the relationship between behavioural thermoregulation and recovery following exhaustive exercise in fish is not well understood.     

Dynamics of supercoiled and linear pTZ18U plasmids observed with a long-lifetime metal-ligand complex

The metal-ligand complex, [Ru(bpy)2(dppz)]2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) (Ru-BD), was used as a spectroscopic probe for studying nucleic acid dynamics. The Ru-BD complex displays a long lifetime of over 100 ns and a molecular light switch property upon DNA binding due to shielding of its dppz ligand from water. To further show the usefulness of this luminophore (Ru-BD) for probing DNA dynamics, we examined its intensity and anisotropy decays when intercalated into supercoiled and linear pTZ18U plasmids using frequency-domain fluorometry with a light-emitting diode (LED) as the modulated light source. Compared to the supercoiled plasmids with an average intensity decay time of 120.8 ns at 25 degrees C, we obtained somewhat longer lifetimes for the linear plasmids ((tau) = 141.4 ns at 25 degrees C), suggesting a more efficient shielding from water by the linear plasmids. The anisotropy decay data also showed longer rotational correlation times for the linear plasmids (495 and 35 ns at 25 degrees C) as compared to the supercoiled plasmids (412 and 27 ns at 25 degrees C). The slow and fast rotational correlation times appear to be consistent with the bending and torsional motions of the plasmids, respectively. The anisotropy values were quite similar, although the values of the supercoiled plasmids were slightly higher in both the steady-state and anisotropy decay measurements. These results indicate that Ru-BD can be applied in the study of both bending and torsional dynamics of nucleic acids.     

Low ambient temperature increases food intake and dropping production, leading to incorrect estimates of hormone metabolite concentrations in European stonechats

Non-invasive methods to measure steroid hormone metabolites in bird droppings or mammalian feces have become very popular. However, the accuracy of these measurements may be affected by many factors. Here, we use the stonechat (Saxicola torquata) as a passerine bird model to test whether differences in ambient temperature affect food intake and dropping production and whether these changes lead to measurement artefacts in hormone metabolite concentrations. In addition, we tested for diurnal patterns in hormone metabolites. We held European stonechats in climate chambers and subjected them to two different long-term ambient temperature regimes, +5 degrees C and +22 degrees C. As expected, food intake and dropping production was higher at +5 degrees C than at +22 degrees C. Plasma concentrations of corticosterone and testosterone did not differ between different ambient temperature regimes. However, corticosterone and testosterone metabolite concentrations (in ng/g) were significantly lower at +5 degrees C than at +22 degrees C. When we measured the rate of hormone metabolite excretion (in picogram per hour) instead of the concentration, there was no difference between treatment groups. Thus, the measurement of hormone metabolite concentrations can be flawed because, depending on the treatment, similar amounts of hormone metabolites can be excreted into very different amounts of droppings. In conclusion, hormone metabolite concentration measurements are sensitive to changes in ambient temperature and probably any other factor that alters metabolic rates. Any study involving systematic changes in metabolism--i.e., during molt, migration, hibernation, egg production, or seasonal comparisons--needs to take these caveats into account.     

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of ligand-dependent changes in cellular location

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.