Production and energy flow in the benthic community of a Texas pond

Energy flow (A = R + P; assimilation = respiration + production) and life-history/density-biomass cycles were followed for 1 year in a mayfly (Brachycercus sp.) population and two chironomid (Procladius sp. and Tendipes decorus) populations in a 0.94 ha pond located in north-central Texas. Temperature (5, 10, 15, 20, 25 and 30°C) effects on R and patterns of metabolic compensation/acclimatization were determined for the mayfly and chironomids. Chironomid and mayfly populations dominated the nine benthic macroinvertebrates identified in the pond, with greatest abundances in winter and winter-spring respectively. Annual energy flow was 51 kcal m^–2 (R = 40 kcal, P = 11 kcal or 1.9 g) in Brachycercus sp., 23 kcal m^–2 (R = 5 kcal, P = 18 kcal or 2.4 g) in Procladius sp. and 74 kcal m^–2 (R = 17 kcal, P = 57 kcal or 6.0 g) in T. decorus. Each species metabolically compensated partially to temperatures en countered during their life histories.     

H nuclear magnetic resonance study of restricted internal rotation of N-6,N-6-dimethyladenine in aqueous solution.

Kinetics of internal rotation about the C(6)-N(6) bond of N-6,N-6-dimethyladenine (M2-6A) was investigated by -1H nuclear magnetic resonance line-shape analysis of the methyl resonances (220 MHz). Rates of rotation were determined for M2-6A deuterated at N(1) and for neutral M2-6A. Activation parameters for monodeuterated M2-6A at 22 degrees are Ea = 13.8kcal/mol, log A = 12.6, incrementG++=14.9 kcal/mol, incrementH++ = 13.1 kcal/mol, incrementS++ = minus 5.8 eu; for neutral M2-6A: Ea = 15.5 kcal/mol, log A = 14.9, incrementG++ = 12.6 kcal/mol, incrementH++ = 14.9 kcal/mol, incrementS++ =7.8 eu. Vertical stacking of bases interferes with internal rotation of the dimethylamino group.     

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

The thermodynamics of bovine and porcine insulin and proinsulin association determined by concentration difference spectroscopy

Difference spectroscopy was used to determine the equilibrium constants and thermodynamic parameters for the monomer-dimer association of bovine and porcine insulin and bovine proinsulin at pH 2.0 and 7.0. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -6.6 kcal/mol, -18 cal/mol-deg, and -12 kcal/mol, respectively. Porcine insulin behaved similarly to bovine insulin in its dimerization properties in that delta G degree 25, delta S degree, and delta H degree were found to be -6.8 kcal/mol, -14 cal/mol-deg, and -11 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -7.2 kcal/mol, -16 cal/mol/deg, and -12 kcal/mol, respectively. At pH 7.0 delta G degree 25, delta S degree, and delta H degree for dimerization of porcine insulin were -6.7 kcal/mol, -11.6 cal/mol-deg, and -10 kcal/mol, respectively. The similarity in the thermodynamic parameters of both insulin species at the different pH's suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range. The dimerization of both insulin species is under enthalpic control. This may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine proinsulin were found to be -5.3 kcal/mol, -26 cal/mol-deg, and -13 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of proinsulin were -5.9 kcal/mol, -4.2 cal/mol-deg, and -7.2 kcal/mol, respectively. Although the presence of the C-peptide on proinsulin does not drastically affect the overall free energy change of dimer formation (as compared to insulin), the other thermodynamic parameters are rather drastically altered. This may be because of electrostatic interactions of groups on the C-peptide with groups on the B-chain which are near the subunit contact site in the insulin dimer.     

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I,V, L,N, A,and K

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.     

Interactions of troponin subunits: free energy of binary and ternary complexes

We have determined the free energy of formation of the binary complexes formed between skeletal troponin C and troponin T (TnC.TnT) and between troponin T and troponin I (TnT.TnI). This was accomplished by using TnC fluorescently modified at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine for the first complex and TnI labeled at Cys-133 with the same probe for the other complex. The free energy of the ternary complex formed between troponin C and the binary complex TnT.TnI [TnC.(TnT.TnI)] was also measured by monitoring the emission of 5-(iodoacetamido)eosin attached to Cys-133 of the troponin I in TnT.TnI. The free energies were -9.0 kcal.mol-1 for TnC.TnT, -9.2 kcal.mol-1 for TnT.TnI, and -8.7 kcal.mol-1 for TnC.(TnT.TnI). In the presence of Mg2+ the free energies of TnC.TnT and TnC.(TnT.TnI) were -10.3 and -10.9 kcal.mol-1, respectively; in the presence of Ca2+ the corresponding free energies were -10.6 and -13.5 kcal.mol-1. Mg2+ and Ca2+ had negligible effect on the free energy of TnT.TnI. From these results the free energies of the formation of troponin from the three subunits were found to be -16.8 kcal.mol-1, -18.9 kcal.mol-1, and -21.6 kcal.mol-1 in the presence of EGTA, Mg2+, and Ca2+, respectively. Most of the free energy decrease caused by Ca2+ binding to the Ca2+-specific sites is derived from stabilization of the TnI-TnC linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Assessment of physical activity in older individuals: a doubly labeled water study.

We compared the accuracy of two physical activity recall questionnaires and a motion detector in 45- to 84-yr-old women (n = 35) and men (n = 32), using doubly labeled water (DLW) in conjunction with indirect calorimetry as the criterion measure. Subjects were administered the Yale Physical Activity Survey (YPAS) and Minnesota Leisure Time Physical Activity Questionnaire (LTA). Physical activity energy expenditure was determined over a 10-day period by using a Caltrac uniaxial accelerometer and DLW in conjunction with indirect calorimetry. In older women, Minnesota LTA (386 +/- 228 kcal/day) and Caltrac (379 +/- 162 kcal/day) underestimated physical activity by approximately 55% compared with DLW (873 +/- 244 kcal/day). No difference was observed between daily physical activity measured by the YPAS (863 +/- 447 kcal/day) and DLW in older women. In older men, Minnesota LTA (459 +/- 288 kcal/day) and Caltrac (554 +/- 242 kcal/day) underestimated daily physical activity by approximately 50-60% compared with DLW (1,211 +/- 429 kcal/day). No difference was found between physical activity measured by the YPAS (1,107 +/- 612 kcal/day) and DLW in older men. Despite no difference in mean physical activity levels between YPAS and DLW in women and men, Bland and Altman (Lancet 1: 307-310, 1986) analyses demonstrated poor concordance between DLW and YPAS (i.e., limits of agreement = -1,310-1,518 kcal/day). Our data suggest that the Minnesota LTA recall and Caltrac uniaxial accelerometer may significantly underestimate free-living daily physical activity energy expenditure in older women and men. Although the YPAS compares favorably with DLW on a group basis, its use as a proxy measure of individual daily physical activity energy expenditure may be limited in older women and men.     

Weekly changes in basal metabolic rate with eight weeks of overfeeding

Objective : The contribution of basal metabolic rate (BMR) to weight gain susceptibility has long been debated. We wanted to examine whether BMR changes in a linear fashion with overfeeding. Our hypothesis was that BMR does not increase linearly with 1000‐kcal/d overfeeding in lean healthy subjects over 8 weeks. The null hypothesis states that BMR increases linearly with 1000‐kcal/d overfeeding in lean healthy subjects. Research Methods and Procedures : Initially, 16 lean healthy sedentary subjects completed 2 weeks of weight maintenance feeding at the General Clinical Research Center. The subjects were then overfed by 1000 kcal/d over 8 weeks. BMR was measured under standard conditions each week using indirect calorimetry. Results : Baseline BMR was 1693 ± 154.5 kcal/d. BMR increased from 1711 ± 201.3 kcal/d at week 1 of overfeeding to 1781 ± 171.65 kcal/d at the second week of overfeeding (p = 0.05). BMR fell during the third week of overfeeding to 1729 ± 179.5 kcal/d (p = 0.05). After 5 weeks of overfeeding, BMR reached a plateau. Thereafter, there was no further change. Comparison of BMR with weeks of overfeeding was significantly different compared with the linear model (p < 0.05). Discussion : Increases in BMR in lean sedentary healthy subjects with 1000‐kcal/d overfeeding are not linear over 8 weeks. There seems to be a short‐term increase in BMR in the first 2 weeks of overfeeding that is not representative of longer‐term changes.     

Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae

The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of ?12.8, ?11.9 and ?11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (?9.2 kcal/mol), Indinavir (?8.2 kcal/mol) and Travoprost (?8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from ?63.85 kcal/mol to ?34.57 kcal/mol for MTB LipU and ?71.33 kcal/mol to ?23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.     

An AB initio investigation of molecules with a disulfide bond: (HS)2, (CH3S)2 and cystine

ab initio Calculations at the Gaussian-70 STO-3G and 4-31G basis levels have been carried out for (HS)2 and (CH3S)2. Cystine was investigated at the STO-3G level. The STO-3G energy minimized geometry agrees well with experiments for (HS)2 and (CH3S)2. The barriers to internal rotation are predicted to be (at the 4.31G level): (HS)2, cis 8.5 kcal, trans 3.03 kcal; (CH3S)2, cis 18.47 kcal, trans 6.04 kcal.     

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.     

Role of the minor energetic determinants of chicken egg white lysozyme (HEWL) to the stability of the HEWL.antibody scFv-10 complex

Seven of the 13 non-glycine contact amino acids in the hen (chicken) egg white lysozyme (HEWL) epitope for antibody Fab-10 each contribute < or =0.3 kcal/mol to the change in free energy (DeltaDeltaG(D)) from wild type (WT) when replaced by alanine (nullspots), and three others each give (0.7 < DeltaDeltaG(D) < or = 1. 0) kcal/mol (warm spots) (Rajpal et al. Protein Sci 1998;7:1868-1874). The low DeltaDeltaG(D) values introduced by alanine mutations present an opportunity to explore accurately their cumulative effects, as the sum of the combined DeltaDeltaG(D) values is not so large as to destabilize the complex beyond the range of accurate measurement. Substitution of six of the seven null spot residues by alanine leads to a cumulative DeltaDeltaG(D) = 2.25 +/- 0.04 kcal/mol, whereas the sum of the six individual changes is only -0.36 +/- 0.32 kcal/mol. The triple warm spot mutation generates a DeltaDeltaG(D) = 5.11 +/- 0.06 kcal/mol versus DeltaDeltaG(D) = 2.52 +/- 0.22 kcal/mol for the sum of the three individuals. The non-additivity in the individual DeltaDeltaG(D) values for the alanine mutations may indicate that these residues provide a conformationally stabilizing effect on the hot spot residues, each of which exhibits DeltaDeltaG(D) > 4.0 kcal/mol on alanine substitution.     

A calorimetric study of the thermotropic behavior of pure sphingomyelin diastereomers

The phase-transition properties of sphingomyelins were investigated in detail with totally synthetic, chemically and stereochemically pure (2S,3R)-(N-stearoylsphingosyl)-1-phosphocholine (D-erythro-C18-SPM) (1) and the corresponding 2S,3S isomer (L-threo-C18-SPM) (2). Heating scans of an unsonicated dispersion of 1 right after hydration showed a main transition (I) at 44.7 degrees C (delta H = 6.8 kcal/mol). Upon incubation at 20-25 degrees C a second transition (II) appeared at 36.0 degrees C (delta H = 5.7 kcal/mol). The two gel phases were designated as G alpha and G beta phases, respectively. The G beta phase was also metastable and relaxed to a third gel phase (G gamma) upon incubation below 10 degrees C. Conversion of the G gamma phase to the liquid-crystalline phase occurred via two new endotherms at 33.4 degrees C (2.6 kcal/mol) (III) and 43.6 degrees C (8.0 kcal/mol) (IV) as well as a main transition at 44.7 degrees C (9.5 kcal/mol). Possible interpretations have been proposed to account for the observed phase transitions. The L-threo isomer 2 showed similar thermotropic behavior to dipalmitoylphosphatidylcholine (DPPC): a "main transition" at 44.2 degrees C (6.0 kcal/mol), a "pretransition" at 43.1 degrees C (1.8 kcal/mol), and upon incubation at 7 degrees C for 2 weeks, a very broad "subtransition" at ca. 35 degrees C. The results are substantially different from previous studies of sphingomyelins using mixtures of stereoisomers. Mixing of 1 with 2, 1 with DPPC, and 2 with DPPC removed the metastability of the gel phase and resulted in a single transition.     

Low molecular mass pectate lyase from Fusarium moniliforme: similar modes of chemical and thermal denaturation

A low molecular mass pectate lyase from Fusarium moniliforme was unfolded reversibly by urea and Gdn-HCl at its optimum pH of 8.5, as monitored by intrinsic fluorescence, circular dichroism, and enzymatic activity measurements. Equilibrium unfolding studies yielded a deltaG(H(2)O) of 1.741 kcal/mol, D1/2 of 2.3M, and m value of 0.755kcal/molM with urea and a deltaG(H(2)O) of 1.927kcal/mol, D1/2 of 1.52M, and m value of 1.27 kcal/molM with Gdn-HCl as the denaturant. Thermal denaturation of the pectate lyase at, pH 8.5, was also reversible even after exposure to 75 degrees C for 10 min. Thermodynamic parameters calculated from thermal denaturation curves at pH values from 5.0 to 8.5 yielded a deltaCp of 0.864kcal/(molK). The deltaG(25 degrees C) at, pH 8.5, was 2.06kcal/mol and was in good agreement with the deltaG(H(2)O) values obtained from chemical denaturation curves. There was no exposure of hydrophobic pockets during chemical or thermal denaturation as indicated by the inability of ANS to bind the pectate lyase.     

Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase.

The subunit composition, metal content, substrate-analogue binding and thermal stability of Aspergillus flavus uricase were determined. A. flavus uricase is a tetramer and contains no copper, iron or any other common prosthetic group. Analytical-gel-filtration and equilibrium-dialysis experiments showed one binding site per subunit for urate analogues. The free energy of xanthine binding was -30.5 kJ (-7.3 kcal)/mol of subunit by equilibrium dialysis and -30.1 kJ (-7.2 kcal)/mol of subunit by microcalorimetry. The enthalpy change for xanthine binding was -15.9 kJ (-3.8 kcal)/mol of subunit when determined from the temperature-dependence of the equilibrium constant and -18.0 kJ (-4.3 kcal)/mol of subunit when measured microcalorimetrically. The thermal inactivation rate of A. flavus uricase increases as protein concentration is decreased. This concentration-dependent instability is not due to subunit dissociation.     

Doubly labeled water measurement of human energy expenditure during strenuous exercise

The energy expenditures (EE) of 23 adult male Marines were measured during a strenuous 11-day cold-weather field exercise at 2,200- to 2,550-m elevation by both doubly labeled water (2H2 18O, DLW) and intake balance methods. The DLW EE calculations were corrected for changes in baseline isotopic abundances in a control group that did not receive 2H2 18O. Intake balance EE was estimated from the change in body energy stores and food intake. Body energy-store changes were calculated from anthropometric [-1,574 +/- 144 (SE) kcal/day] and isotope dilution (-1,872 +/- 293 kcal/day) measurements made before and after the field exercise. The subjects kept daily logbook records of ration consumption (3,132 +/- 165 kcal/day). Mean DLW EE (4,919 +/- 190 kcal/day) did not differ significantly from intake balance EE estimated from food intake and either anthropometric (4,705 +/- 181 kcal/day) or isotope dilution (5,004 +/- 240 kcal/day) estimates of the change in body energy stores. The DLW method can be used with at least the same degree of confidence as the intake balance method to measure the EE of active free-living humans.     

The Effect of Protonation and Electrical Interactions on the Stereochemistry of Retinal Schiff Bases

Based on quantumchemical MNDOC calculations it is shown that the ground-state properties of a retinal Schiff base depend sensitively on its protonation state and charge environment. This is exemplified for the equilibrium geometry, for the distribution of partial charges and, in particular, for the thermal isomerization barriers around the π-bonds. It is demonstrated that a protein, by protonating the retinal Schiff base and by providing one or two negative ions in its environment, can reduce double-bond isomerization barriers from 50 kcal/mol for the unprotonated compound to ~ 5 kcal/mol and can increase single bond barriers from 5 kcal/mol to ~ 20 kcal/mol. Thereby, the specific location of the ions relative to the polyene chain of the protonated retinal Schiff base determines the barrier heights. The results explain the ground-state isomerization reactions of retinal observed in bacteriorhodopsin and in squid retinochrome.