Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability

Interactions of the solutes glycine betaine (GB) and urea with mononucleosomal calf thymus DNA in aqueous salt solutions are characterized by vapor pressure osmometry (VPO). Analysis of osmolality as a function of solute and DNA concentration yields the effect of the solute on the chemical potential, mu(2), of the DNA. Although both GB and urea generally are nucleic acid denaturants and therefore must interact favorably with the nucleic acid surface exposed upon melting, VPO demonstrates that neither interacts favorably with duplex DNA. Addition of GB greatly increases mu(2) of DNA, indicating that the average local concentration of GB in the vicinity of the double helix is much less than its bulk concentration. By contrast, addition of urea has almost no effect on mu(2) of duplex DNA, indicating that the average local concentration of urea in the vicinity of duplex DNA is almost the same as in bulk solution. Qualitatively, we conclude that the nonuniform distribution of GB occurs primarily because duplex DNA and GB prefer to interact with water rather than with each other. Comparison with thermodynamic data for the interaction of GB with various protein surfaces (Felitsky et al., Biochemistry, 43, 14732-14743) shows that GB is excluded primarily from anionic DNA surface and that the hydration of anionic DNA phosphate oxygen surface (>or approximately 17 H(2)O per nucleotide or >or approximately 0.22 H(2)O A(-)(2)) involves at least two layers of water. From analysis of literature data for effects of urea and of GB on DNA melting, we propose that urea is an effective nonspecific nucleic acid denaturant because of its favorable interactions with the polar amide-like surface of G, C, and especially T or U bases exposed in denaturation, whereas GB is a specific GC denaturant because of its favorable interaction with G and/or C surface in the single-stranded state.     

Ontogeny of rabbit proximal tubule urea permeability

Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (P(urea)) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25 degrees C. The PCT P(urea) was also lower in the neonates than the adults (37 degrees C: 45.4 +/- 10.8 vs. 88.5 +/- 15.2 x 10(-6) cm/s, P < 0.05; 25 degrees C: 28.5 +/- 6.9 vs. 55.3 +/- 10.4 x 10(-6) cm/s; P < 0.05). The activation energy for PCT P(urea) was not different between the neonatal and adult groups. BLMV P(urea) was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV P(urea) was not different from adult BLMV P(urea) at 37 degrees C [1.14 +/- 0.05 x 10(-6) vs. 1.25 +/- 0.05 x 10(-6) cm/s; P = not significant (NS)] or 25 degrees C (0.94 +/- 0.06 vs. 1.05 +/- 0.10 x 10(-6) cm/s; P = NS). There was no effect of 250 microM phloretin, an inhibitor of the urea transporter, on P(urea) in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower P(urea) in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.     

Thermodynamics of interactions of urea and guanidinium salts with protein surface: relationship between solute effects on protein processes and changes in water-accessible surface area.

To interpret effects of urea and guanidinium (GuH(+)) salts on processes that involve large changes in protein water-accessible surface area (ASA), and to predict these effects from structural information, a thermodynamic characterization of the interactions of these solutes with different types of protein surface is required. In the present work we quantify the interactions of urea, GuHCl, GuHSCN, and, for comparison, KCl with native bovine serum albumin (BSA) surface, using vapor pressure osmometry (VPO) to obtain preferential interaction coefficients (Gamma(mu3)) as functions of nondenaturing concentrations of these solutes (0-1 molal). From analysis of Gamma(mu3) using the local-bulk domain model, we obtain concentration-independent partition coefficients K(nat)(P) that characterize the accumulation of these solutes near native protein (BSA) surface: K(nat)(P,urea)= 1.10 +/- 0.04, K(nat)(P,SCN(-)) = 2.4 +/- 0.2, K(nat)(P,GuH(+)) = 1.60 +/- 0.08, relative to K(nat)(P,K(+)) identical with 1 and K(nat)(P,Cl(-)) = 1.0 +/- 0.08. The relative magnitudes of K(nat)(P) are consistent with the relative effectiveness of these solutes as perturbants of protein processes. From a comparison of partition coefficients for these solutes and native surface (K(nat)(P)) with those determined by us previously for unfolded protein and alanine-based peptide surface K(unf)(P), we dissect K(P) into contributions from polar peptide backbone and other types of protein surface. For globular protein-urea interactions, we find K(nat)(P,urea) = K(unf)(P,urea). We propose that this equality arises because polar peptide backbone is the same fraction (0.13) of total ASA for both classes of surface. The analysis presented here quantifies and provides a physical basis for understanding Hofmeister effects of salt ions and the effects of uncharged solutes on protein processes in terms of K(P) and the change in protein ASA.     

The exclusion of glycine betaine from anionic biopolymer surface: why glycine betaine is an effective osmoprotectant but also a compatible solute

Paradoxically, glycine betaine (N,N,N-trimethyl glycine; GB) in vivo is both an effective osmoprotectant (efficient at increasing cytoplasmic osmolality and growth rate) and a compatible solute (without deleterious effects on biopolymer function, including stability and activity). For GB to be an effective osmoprotectant but not greatly affect biopolymer stability, we predict that it must interact very differently with folded protein surface than with that exposed in unfolding. To test this hypothesis, we quantify the preferential interaction of GB with the relatively uncharged surface exposed in unfolding the marginally stable lacI helix-turn-helix (HTH) DNA binding domain using circular dichroism and with the more highly charged surfaces of folded hen egg white lysozyme (HEWL) and bovine serum albumin (BSA) using all-gravimetric vapor pressure osmometry (VPO) and compare these results with results of VPO studies (Hong et al. (2004), Biochemistry, 43, 14744-14758) of the interaction of GB with polyanionic duplex DNA. For these four biopolymer surfaces, we observe that the extent of exclusion of GB per unit of biopolymer surface area increases strongly with increasing fraction of anionic oxygen (protein carboxylate or DNA phosphate) surface. In addition, GB is somewhat more excluded from the surface exposed in unfolding the lacI HTH and from the folded surface of HEWL than expected from their small fraction of anionic surface, consistent with moderate exclusion of GB from polar amide surface, as predicted by the osmophobic model of protein stability (Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963). Strong exclusion of GB from anionic surface explains how it can be both an effective osmoprotectant and a compatible solute; analysis of this exclusion yields a lower bound on the hydration of anionic protein carboxylate surface of two layers of water (>or=0.22 H(2)O A(-)(2)).     

Comparative transport efficiencies of urea analogues through urea transporter UT-B

Expression of urea transporter UT-B confers high urea permeability to mammalian erythrocytes. Erythrocyte membranes also permeate various urea analogues, suggesting common transport pathways for urea and structurally similar solutes. In this study, we examined UT-B-facilitated passage of urea analogues and other neutral small solutes by comparing transport properties of wildtype to UT-B-deficient mouse erythrocytes. Stopped-flow light-scattering measurements indicated high UT-B permeability to urea and chemical analogues formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with P(s)>5.0 x 10(-6) cm/s at 10 degrees C. UT-B genetic knockout and phloretin treatment of wildtype erythrocytes similarly reduced urea analogue permeabilities. Strong temperature dependencies of formamide, acetamide, acrylamide and butyramide transport across UT-B-null membranes (E(a)>10 kcal/mol) suggested efficient diffusion of these amides across lipid bilayers. Urea analogues dimethylurea, acryalmide, methylurea, thiourea and methylformamide inhibited UT-B-mediated urea transport by >60% in the absence of transmembrane analogue gradients, supporting a pore-blocking mechanism of UT-B inhibition. Differential transport efficiencies of urea and its analogues through UT-B provide insight into chemical interactions between neutral solutes and the UT-B pore.     

Graft copolymerization of ethyl acrylate onto cellulose using ceric ammonium nitrate as initiator in aqueous medium

Ceric ammonium nitrate (CAN) in the presence of nitric acid has been used as efficient initiator for graft copolymerization of the ethyl acrylate onto cellulose at 35.0 +/- 0.1 degrees C. Graft copolymerization of ethyl acrylate onto cellulose has taken place through the radical initiation process. The graft yield and other grafting parameters have been evaluated by varying concentration of ethyl acrylate from 2.5 x 10(-1) to 15.0 x 10(-1) mol dm(-3) and ceric ammonium nitrate from 5.0 x 10(-3) to 25.0 x 10(-3) mol dm(-3) at constant concentration of the nitric acid (8.0 x 10(-2) mol dm(-3)). The rate of graft copolymerization has shown 1.5 order with respect to the concentration of the ceric ammonium nitrate. The graft copolymerization data obtained at different temperatures were used to calculate the energy of activation, which has been found to be 28.9 kJ mol(-1) within the temperature range from 20 to 50 degrees C. The effect of addition of cationic and anionic surfactants on graft copolymerization has also been studied. On the basis of the experimental observations, reaction steps have been proposed and a suitable rate expression for graft copolymerization has been derived.     

Urea and water permeability in dogfish (Squalus acanthias) gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.     

Application of the local-bulk partitioning and competitive binding models to interpret preferential interactions of glycine betaine and urea with protein surface

Two thermodynamic models have been developed to interpret the preferential accumulation or exclusion of solutes in the vicinity of biopolymer surface and the effects of these solutes on protein processes. The local-bulk partitioning model treats solute (and water) as partitioning between the region at/or near the protein surface (the local domain) and the bulk solution. The solvent exchange model analyzes a 1:1 competition between water and solute molecules for independent surface sites. Here we apply each of these models to interpret thermodynamic data for the interactions of urea and the osmoprotectant glycine betaine (N,N,N-trimethylglycine; GB) with the surface exposed in unfolding the marginally stable lacI HTH DNA binding domain. The partition coefficient K(P) quantifying accumulation of urea at this protein surface (K(P) approximately equal 1.1) is only weakly dependent on urea concentration up to 6 M urea. However, K(P) quantifying exclusion of GB from the vicinity of this protein surface increases from 0.83 (extrapolated to 0 M GB) to 1.0 (indicating that local and bulk GB concentrations are equal) at 4 M GB (activity > 40 M). We interpret the significant concentration dependence of K(P) for GB, predicted to be general for excluded, nonideal solutes such as GB, as a modest (8%) attenuation of the GB concentration dependence of solute nonideality in the local domain relative to that in the bulk solution. Above 4 M, K(P) for the interaction of GB with the surface exposed in protein unfolding is predicted to exceed unity, which explains the maximum in thermal stability observed for RNase and lysozyme at 4 M GB (Santoro, M. M., Liu, Y. F., Khan, S. M. A., Hou, L. X., and Bolen, D. W. (1992) Biochemistry 31, 5278-5283). Both thermodynamic models provide good two-parameter fits to GB and urea data for lacI HTH unfolding over a wide concentration range. The solute partitioning model allows for a full spectrum of attenuation effects in the local domain, encompasses the cases treated by the competitive binding model, and provides a somewhat better two-parameter fit of effects of high GB concentration on lacI HTH stability. Parameters of this fit should be applicable to isothermal and thermal unfolding data for all proteins with similar compositions of surface exposed in unfolding.     

Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B.

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U(osm)) was lower (1532 +/- 71 versus 2056 +/- 83 mosM/kg H(2)O, mean +/- S.E., p < 0.001). After 24 h of water deprivation, U(osm) (in mosM/kg H(2)O) was 2403 +/- 38 in UT-B null mice and 3438 +/- 98 in wild-type mice (p < 0.001). Plasma urea concentration (P(urea)) was 30% higher, and urine urea concentration (U(urea)) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U(urea)/P(urea) ratio (61 +/- 5 versus 124 +/- 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.     

Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor.

Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid-induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence. All four probes revealed an acid-denatured state below pH 3 which resembled a typical molten globule. The pH transition is not two-state as the CD data at 222 nm deviated from all other probes. Urea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an apparent DeltaGU app H2O of 31 +/- 3 kJ.mol-1 when extrapolated to zero denaturant concentration. However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentration (in 10-fold range) even though size-exclusion chromatogryphy (SEC) revealed a dimer-monomer dissociation prior to MIF unfolding. Refolding experiments were performed starting from acid, guanidinium-Cl and urea-denatured states. The kinetics were multiphasic with at least two folding intermediates. The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s-1 (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol-1.     

Rate and equilibrium constants for protein unfolding and refolding determined by hydrogen exchange-mass spectrometry

Studies of protein unfolding and refolding may help us understand the more general problem of protein folding. Recent studies from this laboratory demonstrated that the unfolding and refolding of a large protein, rabbit muscle aldolase (M(r) 157 kDa), can be studied by combining amide hydrogen exchange and mass spectrometry. Results of these studies indicated that aldolase has three unfolding domains which likely unfold sequentially. Urea was used to increase the populations of partially unfolded states which were labeled with deuterium following a brief exposure to D(2)O. Electrospray ionization mass spectra of both the intact protein and its peptic fragments had multiple envelopes of isotope peaks from which the populations of unfolded forms were determined. The present study extends the previous investigations to include different urea concentrations and kinetic modeling of data taken as the system approaches equilibrium. Analysis of these results gives rate and equilibrium constants describing the unfolding and refolding processes characteristic of aldolase destabilized in urea. The change in solvent-accessible surface, which has been used as a reaction coordinate for protein folding, is estimated from the dependence of the equilibrium and rate constants on the concentration of urea.     

Reactivity of halides with triplets of 6-chloro and 6-bromopicolinic acids: contrasting effects of bromide and chloride.

The reactivity of Br(-) and Cl(-) with triplet of anionic 6-chloropicolinic acid (pH = 5.4) and with triplets of 6-chloro and 6-bromopicolinic acids in zwitterionic forms (pH = 0.9) was studied by laser flash photolysis and steady-state irradiation. Br(-) was found to trap the three triplets. Triplet lifetime measurements gave quenching rate constants of 8 x 10(8) mol(-1) dm(3) s(-1) for the zwitterion of 6-chloropicolinic acid and of 3.4 x 10(5) mol(-1) dm(3) s(-1) for the anionic counterpart. No secondary transient species were observed indicating that the charge transfer intermediates are subject to dissipative processes. Cl(-) trapped triplet of zwitterions only, and reactions were found to be associated with a high quantum yield of radicals. The photolysis of 6-bromopicolinic acid photolysis was drastically enhanced by Cl(-), 6-chloropicolinic acid being produced with a chemical yield of about 90%. The 6-bromo-2-carboxypyridinyl radical could be characterized (lambda(max)/nm = 318 with shoulder at 370 nm and epsilon/mol(-1) dm(3) cm(-1) = 8100).     

Ion concentration and temperature dependence of DNA binding: comparison of PurR and LacI repressor proteins.

Purine repressor (PurR) binding to specific DNA is enhanced by complexing with purines, whereas lactose repressor (LacI) binding is diminished by interaction with inducer sugars despite 30% identity in their protein sequences and highly homologous tertiary structures. Nonetheless, in switching from low- to high-affinity DNA binding, these proteins undergo a similar structural change in which the hinge region connecting the DNA and effector binding domains folds into an alpha-helix and contacts the DNA minor groove. The differences in response to effector for these proteins should be manifest in the polyelectrolyte effect which arises from cations displaced from DNA by interaction with positively charged side chains on a protein and is quantitated by measurement of DNA binding affinity as a function of ion concentration. Consistent with structural data for these proteins, high-affinity operator DNA binding by the PurR-purine complex involved approximately 15 ion pairs, a value significantly greater than that for the corresponding state of LacI (approximately 6 ion pairs). For both proteins, however, conversion to the low-affinity state results in a decrease of approximately 2-fold in the number of cations released per dimeric DNA binding site. Heat capacity changes (DeltaC(p)) that accompany DNA binding, derived from buried apolar surface area, coupled folding, and restriction of motional freedom of polar groups in the interface, also reflect the differences between these homologous repressor proteins. DNA binding of the PurR-guanine complex is accompanied by a DeltaC(p) (-2.8 kcal mol(-1) K(-1)) more negative than that observed previously for LacI (-0.9 to -1.5 kcal mol(-1) K(-1)), suggesting that more extensive protein folding and/or enhanced structural rigidity may occur upon DNA binding for PurR compared to DNA binding for LacI. The differences between these proteins illustrate plasticity of function despite high-level sequence and structural homology and undermine efforts to predict protein behavior on the basis of such similarities.     

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19(lacO3/lacOs)), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOS and lacO3 were 5.7 +/- 0.3 x 10(-11) M and 4.1 +/- 0.2 x 10(-11) M respectively, which compare favorably with literature reports of 5 x 10(-10)-1 x 10(-9) M for native lacO1 and 1-1.2 x 10(-10) M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.     

Solution studies on heme proteins: subunit structure, dissociation, and unfolding of Lumbricus terrestris hemoglobin by the ureas.

The subunit structure, dissociation, and unfolding of the hemoglobin of the earthworm, Lumbricus terrestris, were investigated by light scattering molecular weight methods and changes in optical rotatory dispersion (at 233 nm) and absorption in the Soret region. Urea and the alkylureas, methyl-, ethyl-, propyl-, and butylurea, were employed as the reagents to cause both dissociation and unfolding of the protein. Analysis of the light scattering data suggests that the dissociation patterns as a function of hemoglobin concentration in the various dissociating solvents can be described in quantitative terms, either as an equilibrium mixture consisting of parent duodecamers and hexamers of 3 x 10(6) and 1.5 x 10(6) molecular weight (in 1-3 M urea, 1-2 M methyl- and ethylurea, and 1 M propylurea), as a mixture of hexamers and monomers, the latter with a molecular weight of 250000 (i.e., in 4 M urea), or as a mixture of all three species of duodecamers, hexamers, and monomers, seen in 2 M propylurea. Parallel studies by optical rotation and absorption measurements indicate that there is little or no unfolding of the subunits at urea and alkylurea concentrations where complete dissociation to hexamers and extensive dissociation to monomers can be achieved. Further splitting of the monomers (A subunits) to smaller fragments of one-third to one-quarter of the molecular weight of the monomers (B subunits) is seen in the presence of 7 and 8 M urea (pH 7) and in alkaline urea to propylurea solutions. Analysis of the dissociation data of duodecamers to monomers, based on equations used in studies of the urea and amide dissociation of human hemoglobin A from our laboratory, suggests few urea and alkylurea binding sites at the areas of hexamer contacts in the associated duodecameric form of L. terrestris hemoglobin. This suggests that hydrophobic interactions are not the dominant forces that govern the state of association of L. terrestris hemoglobin relative to polar and ionic interactions. The unfolding effects of the ureas, at concentrations above the dissociation transitions, are closely similar to their effects on other globular proteins, suggesting that hydrophobic interactions play an important role in the maintenance of the folded conformation of the subunits. Use of the Peller-Flory equation, with binding constants based on free energy transfer data of hydrophobic amino acid side chains and denaturation data used in previous denaturation studies, gave a relatively good acount of the observed denaturation midpoints obtained with the various ureas supporting these conclusions.     

Pulse radiolysis studies of beta-carotene in oxygenated DMSO solution. Formation of beta-carotene radical cation

The spectroscopic and kinetic characteristics of beta-carotene radical cation (beta-carotene(.+)) were studied by pulse radiolysis in aerated DMSO solution. The buildup of beta-carotene(.+) with k(1) = (4.8 +/- 0.2) x 10(8) dm(3) mol(-1) s(-1) [lambda(max) = 942 nm, epsilon = (1.6 +/- 0.1) x 10(4) dm(3) mol(-1) cm(-1)] results from an electron transfer from beta-carotene to DMSO(.+). The beta-carotene(.+) species decays exclusively by first-order reaction, k = (2.1 +/- 0.1) x 10(3) s(-1), probably by two processes: (1) at low substrate concentration by hydrolysis and (2) at high concentrations also by formation of dimer radical cation (beta-carotene)(2)(.+). Under the experimental conditions, a small additional beta-carotene triplet-state absorption ((3)beta-carotene) in the range of 525 to 660 nm was observed. This triplet absorption is quenched by oxygen (k = 7 x 10(4) s(-1)), resulting in singlet oxygen ((1)O(2)), whose reactions can also lead to additional formation of beta-carotene(.+).     

Solute effects on mitochondria from an elasmobranch (Raja erinacea) and a teleost (Pseudopleuronectes americanus)

Varying osmolarity with sucrose/KCl media resulted in similar effects on the oxidation of glutamate by mitochondria isolated from the livers of an elasmobranch, Raja erinacea, and a teleost, Pseudopleuronectes americanus. In both species trimethylamine oxide (TMAO) inhibited mitochondrial oxidation of glutamate. Urea penetrated the inner mitochondrial membrane of both species and equilibrated with a ratio ureai/ureao of unity. Urea had little effect on the oxidation of glutamate in both species at concentrations as high as 760 mM. Addition of urea (urea/TMAO, 2:1) did not overcome the detrimental effects of TMAO in the mitochondria of either species. In the case of the elasmobranch, the osmolarity of the urea/TMAO media giving the optimal rate of respiration was hypoosmotic with respect to the intracellular osmolarity. The rate of glutamate oxidation steadily declined as osmolarity increased above this value. Assuming the osmotic profile obtained with the urea/TMAO (2:1) medium resembled most closely the in vivo situation, higher rates of oxidation or organic solutes at low osmolarity would help deplete the cell of these solutes and could contribute to cell volume regulation during hypoosmotic stress. It is suggested that two broad classes of intracellular solutes can be defined based on their effects on mitochondrial respiration. Solutes such as K+, C1-, and TMAO penetrate the inner mitochondrial membrane slowly or not at all. Increasing concentrations of these solutes result in lower rates of oxidation. This capacity may be important in regulating intracellular levels of organic solutes during osmotic stress. Solutes such as urea rapidly penetrate the cell and inner mitochondrial membrane reducing the mitochondrial volume changes associated with osmotic stress. The known detrimental effects of urea on protein structure may prevent its exclusive use as an intracellular osmotic effector.     

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.     

Adrenergic regulation of HSL serine phosphorylation and activity in human skeletal muscle during the onset of exercise

Skeletal muscle hormone-sensitive lipase (HSL) activity is increased by contractions and increases in blood epinephrine (EPI) concentrations and cyclic AMP activation of the adrenergic pathway during prolonged exercise. To determine the importance of hormonal stimulation of HSL activity during the onset of moderate- and high-intensity exercise, nine men [age 24.3 +/- 1.2 yr, 80.8 +/- 5.0 kg, peak oxygen consumption (VO2 peak) 43.9 +/- 3.6 ml x kg(-1) x min(-1)] cycled for 1 min at approximately 65% VO2 peak, rested for 60 min, and cycled at approximately 90% VO2 peak for 1 min. Skeletal muscle biopsies were taken pre- and postexercise, and arterial blood was sampled throughout exercise. Arterial EPI increased (P < 0.05) postexercise at 65% (0.45 +/- 0.10 to 0.78 +/- 0.27 nM) and 90% VO2 peak (0.57 +/- 0.34 to 1.09 +/- 0.50 nM). HSL activity increased (P < 0.05) following 1 min of exercise at 65% VO2 peak [1.05 +/- 0.39 to 1.78 +/- 0.54 mmol x min(-1) x kg dry muscle (dm)(-1)] and 90% VO2 peak (1.07 +/- 0.24 to 1.91 +/- 0.62 mmol x min(-1) x kg dm(-1)). Cyclic AMP content also increased (P < 0.05) at both exercise intensities (65%: 1.52 +/- 0.67 to 2.75 +/- 1.12, 90%: 1.85 +/- 0.65 to 2.64 +/- 0.93 micromol/kg dm). HSL Ser660 phosphorylation (approximately 55% increase) and ERK1/2 phosphorylation ( approximately 33% increase) were augmented following exercise at both intensities, whereas HSL Ser563 and Ser565 phosphorylation were not different from rest. The results indicate that increases in arterial EPI concentration during the onset of moderate- and high-intensity exercise increase cyclic AMP content, which results in the phosphorylation of HSL Ser660. This adrenergic stimulation contributes to the increase in HSL activity that occurs in human skeletal muscle in the first minute of exercise at 65% and 90% VO2 peak.