Temperature sensitivity of cyclic-adenosine-3':5'-monophosphate-binding proteins, activity of protein kinases and the regulation of cell growth.

A clone of neuroblastoma cells has been selected for its ability to survive and multiply at 40 degrees C. This temperature-resistant clone, like clones of neuroblastoma cells selected for resistance to dibutyryladenosine 3':5'-monophosphate (Bt2-Ado-3':5'-P) showed an increased tumorogenicity in animals and an increased saturation density at 37 degrees C. The Ado-3':5'-P-binding proteins and Ado-3':5'-P-dependent protein kinases from the temperature-resistant and non-resistant cells have been partially purified by chromatography on a DEAE-cellulose column. The Ado-3':5'-P-binding proteins from temperature-resistant cells were more sensitive to temperature than the binding proteins from non-resistant cells. After incubation of binding proteins from resistant cells at 37 degrees C, the specific activity of Ado-3':5'-P-binding to proteins was decreased about 50% and the apparent association constant (Ka) for Ado-3':5-p-binding was decreased from 7.4 X 10(7)M-1 to 4.4 x 10(7)M-1. There was no such decrease with binding proteins from non-resistant cells. A decrease in the activity of binding proteins from the temperature-resistant cells, but not of those from non-resistant cells, was also found when the proteins were stored at 2 degrees C. Treatment with 2-mercaptoethanol made binding proteins from the resistant cells less temperature-sensitive. In the absence of added Ado-3:5-P the protein kinase activity from the temperature-resistant cells was about 50% of the activity from non-resistant cells. Kinase activity was increased by addition of Ado-3:5-P and there was a greater increase with kinases from resistant cells. The maximum protein kinase activity was found in the presence of 10muM Ado-3':5'-P for the temperature-resistant cells and 0.1 muM Ado-3':5'-P for the non-resistant cells. The results indicate that the temperature sensitivity of Ado-3':5'-P-binding proteins, and the activity of protein kinase from cells selected for resistance to high temperature, are similar to those of cells selected for resistance to Bt2-Ado-3':5'-P. It is suggested that the temperature sensitivity of Ado-3':5'-P-binding proteins and the activity of Ado-3':5'-P-dependent protein kinases are involved in the regulation of malignancy and of cell growth at different temperatures.     

Entropic elastic processes in protein mechanisms. I. Elastic structure due to an inverse temperature transition and elasticity due to internal chain dynamics

Numerous physical characterizations clearly demonstrate that the polypentapeptide of elastin (Val1-Pro2-Gly3-Val4-Gly5)n in water undergoes an inverse temperature transition. Increase in order occurs both intermolecularly and intramolecularly on raising the temperature from 20 to 40 degrees C. The physical characterizations used to demonstrate the inverse temperature transition include microscopy, light scattering, circular dichroism, the nuclear Overhauser effect, temperature dependence of composition, nuclear magnetic resonance (NMR) relaxation, dielectric relaxation, and temperature dependence of elastomer length. At fixed extension of the cross-linked polypentapeptide elastomer, the development of elastomeric force is seen to correlate with increase in intramolecular order, that is, with the inverse temperature transition. Reversible thermal denaturation of the ordered polypentapeptide is observed with composition and circular dichroism studies, and thermal denaturation of the crosslinked elastomer is also observed with loss of elastomeric force and elastic modulus. Thus, elastomeric force is lost when the polypeptide chains are randomized due to heating at high temperature. Clearly, elastomeric force is due to nonrandom polypeptide structure. In spite of this, elastomeric force is demonstrated to be dominantly entropic in origin. The source of the entropic elastomeric force is demonstrated to be the result of internal chain dynamics, and the mechanism is called the librational entropy mechanism of elasticity. There is significant application to the finding that elastomeric force develops due to an inverse temperature transition. By changing the hydrophobicity of the polypeptide, the temperature range for the inverse temperature transition can be changed in a predictable way, and the temperature range for the development of elastomeric force follows. Thus, elastomers have been prepared where the development of elastomeric force is shifted over a 40 degrees C temperature range from a midpoint temperature of 30 degrees C for the polypentapeptide to 10 degrees C by increasing hydrophobicity with addition of a single CH2 moiety per pentamer and to 50 degrees C by decreasing hydrophobicity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carbon-13 NMR relaxation studies demonstrate an inverse temperature transition in the elastin polypentapeptide

Carbon-13 NMR longitudinal relaxation time and line-width studies are reported on the coacervate concentration (about 60% water by weight) of singly carbonyl carbon enriched polypentapeptides of elastin: specifically, (L-Val1-L-[1-13C]Pro2-Gly3-L-Val4-Gly5)n and (L-Val1-L-Pro2-Gly3-L-Val4-[1-13C]Gly5)n. On raising the temperature from 10 to 25 degrees C and from 40 to 70 degrees C, carbonyl mobility increases, but over the temperature interval from 25 to 40 degrees C, the mobility decreases. The results characterize an inverse temperature transition in the most fundamental sense of temperature being a measure of molecular motion. This transition in the state of the polypentapeptide indicates an increase in order of polypeptide on raising the temperature from 25 degrees C to physiological temperature. This fundamental NMR characterization corresponds with the results of numerous other physical methods, e.g., circular dichroism, dielectric relaxation, and electron microscopy, that correspondingly indicate an increase in order of the polypentapeptide both intramolecularly and intermolecularly for the same temperature increase from 25 to 40 degrees C. Significantly with respect to elastomeric function, thermoelasticity studies on gamma-irradiation cross-linked polypentapeptide coacervate show a dramatic increase in elastomeric force over the same interval that is here characterized by NMR as an inverse temperature transition. The temperature dependence of mobility above 40 degrees C indicates an activation energy of the order of 1.2 kcal/mol, which is the magnitude of barrier expected for elasticity.     

Adenosine cyclic 3',5'-monophosphate uptake and regulation of membrane protein kinase in intact human erythrocytes

The uptake of adenosine cyclic 3',5'-monophosphate (cAMP) and stimulation of membrane-associated protein kinase in mature human erythrocytes were investigated. cAMP transport across the membrane was temperature dependent, and cAMP binding to the isolated membrane had less temperature dependence. More than 99% of the [3H]-cAMP taken up by erythrocytes was nonmembrane bound. Maximal stimulation of membrane protein kinase and maximal occupancy of membrane cAMP binding sites by extracellular cAMP cccurred at 30 degrees C within 30 min after initiation of the incubation of erythrocytes with cAMP. The concentration of extracellular cAMP that gave half-maximal stimulation of membrane protein kinase was 5.4 X 10-4 M, a value consistent with the concentrations of cAMP (5.2 X 10-4 M) found to occupy half-maximally the membrane cAMP binding sites in erythrocytes. Extracellular cAMP and to a lesser extent guanosine cyclic 3',5'-monophosphate and inosine cyclic 3',5'-monophosphate stimulated membrane protein kinase in erythrocytes. The cAMP uptake by human erythrocytes as well as cAMP binding to membranes in the erythrocyte was blocked by an inhibitor [4,4'-bis(isothiocyano)stilbene-2,2-disulfonate] of the anion channel. These studies indicate that cAMP can be transported across membranes into human erythrocytes and can bind to membranes to activate membrane protein kinase. It appears that there is a shared transport channel for cAMP and anion transport.     

13C NMR studies of the thermal properties of a model high density lipoprotein. Apolipoprotein A-I-dimyristoylphosphatidylcholine complex

The most abundant lipid and protein components of human plasma high density lipoproteins are phosphatidylcholine and apolipoprotein A-I (A-I). Under appropriate conditions, A-I spontaneously associates with dimyristoylphosphatidylcholine (DMPC) to quantitatively form a lipid-protein complex with a DMPC/A-I molar ratio of 100:1. Differential scanning calorimetry of this complex reveals two broad thermal transitions centered at approximately 27 and 72 degrees C. 13C NMR spectra of the complex have been obtained above, at, and below the lower transition temperature. The 13C resonance arising from the 3' carbon of the fatty acyl chains is a doublet, split by approximately 0.2 ppm, suggesting that the 3' carbon nuclei occupy two magnetically inequivalent sites. By replacing the sn-2 fatty acyl chain with myristate selectively 13C-enriched at carbon 3', we have shown that the splitting is, in fact, a result of magnetic inequivalence of the two sites and have assigned the lower field resonance to the 3' carbon nucleus of the sn-2 chain. The temperature dependence of the NMR relaxation rates indicates that the endothermic transition at 27 degrees C is associated with increased motional freedom for the phospholipids within this complex. The temperature dependence of the fatty acyl chain methylene 13C chemical shifts suggests that the population of gauche conformers increases above the transition temperature. These dynamic and conformational changes are characteristic of gel----liquid crystalline phase transitions observed in pure phospholipid systems. For the DMPC-A-I complex at 37 degrees C, the chemical shifts of the fatty acyl C 4'- 11' methylene envelope and of the C 7' and C 13' resonances occur significantly downfield from the corresponding chemical shifts for the DMPC vesicle. These results suggest that the apoprotein rigidifies the acyl chains by increasing their number of trans conformers.     

Effects of light deprivation on RNA synthesis, accumulation of guanosine 3''(2'')-diphosphate 5''-diphosphate, and protein synthesis in heat-shocked Synechococcus sp. strain PCC 6301, a cyanobacterium.

The rate of total RNA synthesis, the extent of guanosine 3'(2')-diphosphate 5'-diphosphate (ppGpp) accumulation, and the pattern of protein synthesis were studied in light-deprived and heat-shocked Synechococcus sp. strain PCC 6301 cells. There was an inverse correlation between the rate of total RNA synthesis and the pool of ppGpp, except immediately after a temperature shift up, when a parallel increase in the rate of RNA synthesis and accumulation of ppGpp was observed. The inverse correlation between RNA synthesis and ppGpp accumulation was more pronounced when cells were grown in the dark. Heat shock treatment (47 degrees C) had an unexpected effect on ppGpp accumulation; there was a fairly stable level of ppGpp under heat shock conditions, which coincided with a stable steady-state rate of RNA synthesis even in the dark. We found that the pattern of dark-specific proteins was altered in response to heat shock. The transient synthesis of several dark-specific proteins was abolished by an elevated temperature (47 degrees C) in the dark; moreover, the main heat shock proteins were synthesized even in the dark. This phenomenon might be of aid in the study of cyanobacterial gene expression.     

Inactivation and proteolysis of heat-sensitive adenylate kinase of Escherichia coli CR341 T28

Adenylate kinase from Escherichia coli K12 (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. Denaturation by heat above 60 degrees C of pure or crude preparations of adenylate kinase from both strains of bacteria was shown to be "reversible" if the enzyme was converted to the random coiled state by guanidinium chloride after heat treatment. Like other small monomeric proteins, adenylate kinase refolded rapidly to the native active state by dilution of guanidinium chloride. Adenylate kinase from the mutant strain was irreversibly inactivated by exposure of crude extracts at 40 degrees C. This inactivation is due to proteolysis which follows thermal denaturation (or transconformation) of mutant adenylate kinase at 40 degrees C. ATP, P1, P5-di(adenosine 5')-pentaphosphate, and anti-adenylate kinase antibodies protected the thermosensitive adenylate kinase in crude extracts against denaturation and proteolysis at 40 degrees C.     

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.     

Cold denaturation of myoglobin

The stability of the structure of sperm whale metmyoglobin has been studied in various solutions, in the temperature range -8 degrees C to 100 degrees C, by scanning microcalorimetry, light absorption, circular dichroism, nuclear magnetic resonance spectroscopy and viscosimetry. It has been shown that in 10 mM-sodium acetate solutions (pH 3.5 to 3.9) the protein molecule undergoes a reversible conformational transition into a non-compact disordered state not only when the solution is heated above room temperature but also when it is cooled. In this state the protein does not have a tertiary structure, although it retains some residual ellipticity, which may be caused by the fluctuating alpha-helical conformation of the unfolded polypeptide chain. The disruption of the native protein structure both on cooling (cold-denaturation) and on heating (heat-denaturation) proceeds in an "all-or-none" manner, with a significant and similar increase of the protein heat capacity, but with inverse enthalpic and entropic effects: the enthalpy and entropy of the protein molecule decrease during cold-denaturation and increase during heat-denaturation.     

Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.     

Effects of acclimation temperature on enzymatic capacities and mitochondrial membranes from the body wall of the earthworm Lumbricus terrestris

Many ectotherms respond to low temperature by adjusting capacities of enzymes from energy metabolism, restructuring membrane phospholipids and modulating membrane fluidity. Although much is known about the temperature biology of earthworms, it is not known to what extent earthworms employ compensatory changes in enzymatic capacities and membrane physical properties after exposure to low temperature. We examined activities of enzymes from glycolysis and central oxidative pathways as well as fluidity and phospholipid fatty acid composition of mitochondrial membranes prepared from the body wall of the temperate oligochaete Lumbricus terrestris after a one month acclimation to 5 degrees and 15 degrees C. No compensation occurs in central pathways of oxidative metabolism since activities of cytochrome-c oxidase and citrate synthase, when measured at a common temperature, are similar for 5 degrees C and 15 degrees C-acclimated animals. In contrast, activity of pyruvate kinase is elevated 1.3-fold after acclimation to 5 degrees C. Mitochondrial membranes display inverse compensation with respect to temperature (membranes from 5 degrees C animals are more ordered than membranes from 15 degrees C animals). Our results, in combination with earlier reports, indicate that routine metabolism in L. terrestris may be maintained at reduced temperatures with little or no change in enzymatic capacities and inverse compensation of mitochondrial membranes.     

Sequence dependence of conformations of self-complementary duplex tetradeoxynucleotides containing cytosine and guanine

The four self-complementary tetradeoxynucleotides which contain only cytosine and guanine are 5'-d-(CpGpCpG)-3', 5'-d(CpCpGpG)-3', 5'-d(GpCpGpC)-3', and 5'-d(GpGpCpC)-3'. The Raman spectra of aqueous solutions (about 0.05 M in monomer) of these tetranucleotides at pH 7 and 2 degrees C show clearly that these self-complementary tetranucleotides form double-stranded duplex structures of the canonical B type when the NaCl concentration is 0.5 M NaCl. If the temperature is raised to 50 degrees C, the Raman spectra show that in each case the double-helical B form melts in a non-cooperative way to a disordered single-chain form. On the other hand, if the salt concentration is raised to saturation, the Raman spectrum of only one of these four tetranucleotide solutions at 2 degrees C is changed in any substantial way. The Raman spectrum of the tetranucleotide 5'-d(CpGpCpG)-3' at 2.2 degrees C and at 4 M or higher salt concentration strongly resembles that of double-helical Z-form poly(dC-dG) taken under similar conditions. We conclude that the tetramer 5'-d(CpGpCpG)-3' is the only self-complementary double-helical tetranucleotide containing only cytosine and guanine in which the B-Z transition can be induced by increasing the salt concentration. This tetramer has several types of stacking interactions which differ markedly from stacking interactions in the other tetramers and may account for the enhanced stabilization of its Z conformation.     

Motion and surface accessibility of spin-labeled lipids in a model lipoprotein containing cholesteryl oleate, dimyristoylphosphatidylcholine, and apolipoprotein E

A series of spin-labeled phosphatidylcholines (PCs) and cholesteryl esters (CEs) bearing the paramagnetic 2,2-dimethyloxazolidinyl-1-oxy (doxyl) group at fatty acyl carbon C5', C12', or C16' were used to study acyl chain motions in the polar surface shell and hydrophobic core domains of microemulsion (ME) particles containing cholesteryl oleate and dimyristoylphosphatidylcholine (DMPC), and of particles with apolipoprotein E (apoE) bound to their surfaces. Electron paramagnetic resonance data obtained with the doxyl-labeled PCs indicated a gradient of motion in the ME surface monolayer similar to that observed with the same probes in a bilayer. The 5- and 12-doxyl-CEs clearly demonstrated a higher degree of order for the cholesteryl ester rich core than the corresponding doxyl-PCs showed for the phospholipid-rich surface over the entire range 10-60 degrees C. The temperature dependencies of spectra of the 16-doxyl-CE in the core and PC in the surface of the ME were almost identical, suggesting that there was no sharp boundary between core and surface domains. None of the probes detected either the surface phospholipid transition (31 degrees C) or the cholesteryl ester core transition (46 degrees C) measured previously by differential scanning calorimetry and 13C nuclear magnetic resonance. Binding of apoE to spin-labeled DMPC vesicles increased the order of the 5'-position of the sn-2 acyl chain over the range 15-33 degrees C; the thermal transition was broadened and its midpoint elevated. The effect of protein binding was not as striking for the ME particles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature- and cyclic nucleotide-induced phase transitions of Histoplasma capsulatum.

The transition from yeast to mycelia of Histoplasma capsulatum could be accomplished by shifting the temperature of incubation from 37 to 25 degrees C. It was accompanied by many changes in cellular metabolism, including changes in respiration, intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels, and activities of two enzymes specific for the yeast phase, cystine reductase (EC 1.6.4.1) and cysteine oxidase (EC 1.13.11.20). Even at 37 degrees C, the yeast to mycelial transition could be induced by cAMP and agents which raise the intracellular levels of cAMP (theophylline, acetylsalicylic acid, prostaglandin E1, and nerve growth factor). During this morphogenesis the same pattern of changes occurred as in the temperature-induced transition. Therefore, these changes were not simply dependent on a shift in temperature, but rather were part of the process of the phase transition.     

Effects of temperature, aurintricarboxylic acid and cibacron blue on 2',5'-oligoadenylate binding protein (RNase L) activity in rabbit reticulocyte lysates

The binding of p3A4,3'-32P [pCp] to rabbit reticulocyte RNase L can be displaced by the trimer and tetramer triphosphates of 2',5'-oligoadenylates (2-5A). Using assay conditions of protein synthesis, 2-5A trimer or tetramer triphosphates are shown to be equally effective when the displacement is done at 4 degrees C (on ice). In contrast, at 30 degrees C, the tetramer triphosphates still displace whereas the trimer triphosphates become ineffective. When lysates are preincubated at temperature ranging from 4 degrees-37 degrees C, the same results are obtained even when the subsequent displacement is done on ice. Incubation temperature also significantly affects the ability of metabolically stable dyes cibacron blue and aurintricarboxylic acid to inhibit RNase L binding activity. Taken together, these results suggest that rabbit reticulocyte RNase L may assume multiple conformations which are differentially affected by various forms of 2-5A or other compounds.     

Physical properties of apoprotein B in mixed micelles with sodium deoxycholate and in a vesicle with dimyristoyl phosphatidylcholine

Apoprotein B, the major apoprotein of normal human low density lipoprotein (LDL) was solubilized with sodium deoxycholate (NaDC). The protein was recombined with the phospholipid dimyristoyl phosphatidylcholine (DMPC) to produce a complex of DMPC-apoB (4:1 w/w). (Biochemistry. 22: 3170-3178. 1983). Carboxyfluorescein and [3H]dextran entrapment studies show the DMPC-apoB 4:1 (w/w) complex to encapsulate an aqueous volume of 0.17 microliter/mumol of DMPC. From the chemistry and morphology of the complex and the evidence that the complex possesses an encapsulated volume, the most appropriate structural model for this assembly is that of a phospholipid single bilayer vesicle into which apoB is incorporated. Differential scanning calorimetry (DSC) and circular dichroic spectroscopy (CD) were used to investigate the physical properties of apoB in the mixed micellar complex with NaDC and in the vesicular DMPC-apoB complex. CD studies of apoB in NaDC mixed micelles show that apoB exhibits a similar secondary structure as apoB of native LDL over the temperature range 5-30 degrees C. Reversible structural changes occur between 30 and 50 degrees C. However, above 50 degrees C, disruption of the micellar particle and endothermic protein unfolding and denaturation occur with a Tmax of 52 degrees C and an enthalpy of 0.22 cal/g apoB, as shown by DSC. The DMPC-apoB complex exhibits a reversible thermal transition centered at 24 degrees C (delta H = 3.34 Kcal/mol DMPC) which is associated with the order-disorder transition of the hydrocarbon chains of DMPC. An endothermic transition occurs over the range 53-70 degrees C (delta H = 2.09 cal/g apoB) which, as shown by CD and turbidity study, corresponds to protein unfolding-denaturation and particle disruption. CD shows that apoB in the vesicular environment undergoes a series of conformational changes. The major alterations occur over the temperature range of the order-disorder transition of the phospholipid. Between 37-60 degrees C, the conformation is similar to that observed in native LDL.     

Microcalorimetric study of iodized and noniodized cells and C-phycocyanin of Spirulina platensis

It was shown that eight stages of transition are observed in the heating process of Spirulina platensis cells in temperature range 5-140 degrees C. The first stage covers the temperature range 5-53 degrees C with maximum approximately 45 degrees C. The heat evolved in this temperature range is equal to 380 +/- 20 J/g of dry biomass, it does not change at scanning rate lower than 0.083 degrees C/min and belongs, mainly, to cell respiration in a stationary regime, in the dark. It was shown that endotherm approximately 66 degrees C belongs to denaturation of C-phycocyanin which denaturates in solutions with Td = 64.2 degrees C, deltaHd = 34.7 +/- 2.1 J/g and for it deltaHd(cal)/deltaH(V.H) is equal to 10.8 +/- 1.2. The endotherms with Td equal to 58 and 88 degrees C are connected with denaturation of phycobilisome proteins and endotherm with Td = 48 degrees C and deltaHd = 4.2J/g of dry biomass-with denaturation of protein which, apparently, is connected with cell respiration.     

Conformational changes in single-strand DNA as a function of temperature by SANS

Small-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5'-ATGCTGATGC-3', in sodium phosphate buffer solution at 10 degrees C temperature increments from 25 degrees C to 80 degrees C. Cylindrical, helical, and random coil shape models were fitted to the SANS measurements at each temperature. All the shapes exhibited an expansion in the diameter direction causing a slightly shortened pitch from 25 degrees C to 43 degrees C, an expansion in the pitch direction with a slight decrease in the diameter from 43 degrees C to 53 degrees C, and finally a dramatic increase in the pitch and diameter from 53 degrees C to 80 degrees C. Differential scanning calorimeter scans of the sequence in solution exhibited a reversible two-state transition profile with a transition temperature of 47.5 +/- 0.5 degrees C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60 +/- 3 kJ mol(-1) that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47 +/- 1 degrees C was also obtained from ultraviolet optical density measurements of strand melting scans of the single-strand DNA. This transition corresponds to unstacking of the bases of the sequence and is responsible for the thermodynamic discrepancy between its binding stability to its complementary sequence determined directly at ambient temperatures and determined from extrapolated values of the melting of the duplex at high temperature.     

Catalytic and thermodynamic properties of the urocanate hydratase reaction.

Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.