Heat of hydrolysis of some phosphate compounds

The enzymatic hydrolysis of various phosphate compounds was carried out in an isothermic calorimeter. The following heats of reaction were found (average values): p-nitrophenyl phosphate, 4500 cal./mole; sodium pyrophosphate, 9000 cal./mole; POP bond of diphosphopyridine nucleotide, 13,000 cal./mole.     

Thermodynamic parameters for the helix-coil thermal transition of ribonuclease-S-peptide and derivatives from 1H-NMR data

Values for the thermodynamic quantities, ΔH° = 11.8 ± 2.0 Kcal/mole and ΔS° = 43.6 ± 6.0 e.u., of the 3-13 helix–coil equilibrium of isolated S-peptide (19 residue N-terminal fragment of ribonuclease A) in aqueous solution (3 m M, 1M NaCl, pD 5.4) have been determined from a joint analysis of the Thr 3γ, Ala 6β, Phe 8meta, and Phe 8para ¹H chemical shift vs temperature curves (?7 to 80°C) in several aqueous–trifluorethanol mixtures. Chemical shifts in the coil and in the helix have been determined for up to 16 protons belonging to the 3-13 fragment. Thermodynamic parameters have also been determined for C-peptide (13 residue fragment) and a number of S-peptide derivatives. From the variation of the values of the thermodynamic parameters at pD 2.5, 5.4, and 8.0, a quantitation of the two helix-stabilizing side-chain interactions can be made: (1) Δ(ΔH°) ? 5 Kcal/mole and Δ(ΔS°) ? 18 e.u. for the salt bridge Glu 2^? … Arg 10⁺ and (2) Δ(ΔH°) ? 3 Kcal/mole and Δ(ΔS°) = 9 e.u. for the one in which the His 12⁺ imidazolium group is involved, presumably a partial stacking with the Phe 8 side chain.     

Kinetic and Equilibrium Studies on d-Mannose-d-Fructose Isomerization Catalyzed by Mannose Isomerase from Streptomyces aerocolorigenes

The equilibrium constant of the isomerization reaction between d-mannose and d-fructose which is catalyzed by a mannose isomerase from Streptomyces aerocolorigenes was obtained by using three methods over the temperature range from 1 to 40°C.

It was found that the equilibrium constant was scarcely dependent on temperature, ΔH, the heat of the formation of d-fructose from d-mannose, being approximately zero.

The standard free energy change, ΔG, and the standard entropy change, ΔS, of the reaction were calculated from the equilibrium constants at various temperatures and ΔH. The values of ΔG and ΔS at 25°C were ?650 cal/mole and + 2.2 cal/deg·mole, respectively.

By combining these thermodynamic data with those obtained for the isomerization reaction between d-glucose and d-fructose reported in the previous paper, ΔH, ΔG and ΔS for the isomerization between d-mannose and d-glucose were indirectly obtained to be +2220 cal/mole, +830 cal/mole and +4.6 cal/deg·mole at 25°C, respectively.     

Binding of diclofenac sodium with bovine serum albumin at different temperatures, pH and ionic strengths

The study was designed to examine the binding of diclofenac sodium with bovine serum albumin (BSA) at different temperatures (20 degrees, 30 degrees and 40 degrees C), pH (6.4, 7.4 and 8.4) and ionic strengths (micro = 0.1, 0.2 and 0.3) by means of equilibrium dialysis method. The concentration of diclofenac sodium was maintained at wider range from 15 to 900 micromole/l and BSA concentration was maintained at 61.5 micromole/l. The data obtained were interpreted by nonlinear regression method using Graphpad prism software. The analysis showed that the interaction between diclofenac sodium with BSA results in two-site saturable binding. A decrease in association constant was observed with increasing temperature. The average standard free energy change (deltaGdegrees) value was -7.07 (site I) and -4.2 (site II) Kcal/mol. The standard enthalpy change (deltaHdegrees) and the standard entropy change (deltaSdegrees) were -7.8 Kcal/mole, -2.35 cal/mole (site I) and -7.4 Kcal/mole, -10.5 cal/mole (site II), respectively. The negative enthalpy change suggested the binding between diclofenac sodium and the binding sites of BSA were spontaneous and exothermic. The negative value of deltaHdegrees and deltaSdegrees indicated hydrogen bonding and van der Waal's force was the major mechanism for diclofenac sodium and BSA interaction. Increase in pH and ionic strength also caused decrease in association constant of diclofenac sodium and BSA binding.     

Preliminary Investigation on Protein Bodies of Soybean Seeds

With the purpose of separation and isolation of protein bodies from soybean, soybean seeds were homogenized in oil and fractionated by successively adjusting densities of extracts with carbon tetrachloride. Isolated protein bodies consist of about 10% nitrogen, 0.8% phosphorus, 8.5% sugar, 7% ash and 0.5% RNA. Over 93% of protein in the bodies is found as particle-bound protein which is insoluble in 15% Carbowax 6000 solution but soluble in 10% sodium chloride solution. Protein bodies in intact cells, isolated bodies and those treated with Carbowax 6000 solution were respectively observed by electron-microscopy.     

Enantiomeric selectivity of a lipase from Penicillium simplicissimum in the esterification of menthol in microemulsions

Summary Lipase from Penicillium simplicissimum catalyzes the stereospecific esterification of menthol with fatty acids. The studies on the specificity of this new lipase were carried out using (+), (-) and racemic menthol with water soluble enzyme entrapped in microemulsion systems stabilized with sodium(bis-2-ethylhexyl)sulfosuccinate (AOT) as surfactant, in isooctane. Microemulsions appear to be an effective and fast system for racemic resolution of alcohols.     

Hairpin Formation In d-AAGCTTAAGCTT Under High Salt Conditions Shows Unusual Properties

Abstract

The hairpin-duplex equillibria of the dodecamer d-AAGCTTAAGCTT and interaction of the duplex form with a pentapeptide, KGWGK, has been studied. UV thermal transitions are monophasic at low salt but biphasic at higher salt concentrations. At 10^?5M or less oligomer concentration biphasic melting curves persist till 900 mM NaCl. The d(Tm)/d log(Na⁺) for the duplex form is 12 °C and for the hairpin is 18 °C. The ΔH and ΔS values for duplex formation are low(-25 Kcal/mole and—59 Cal/mole respectively). KGWGK binds to the duplex form with a binding constant K = 3.4×10⁵M^?1measured from fluorescence quenching of tryptophan. These unusual results are markedly different from that reported for d-AGATCT- AGATCT (Biochemistry 31, 6241–6245) and are discussed in ternis of sequence dependence of loop folding and cruciform extrusion pathway of hairpin formation.     

Cis-Trans equilibrium and kinetic studies of acetyl-L-proline and glycyl-L-proline

By means of carbon-13 nmr (at 25 MHz) the trans/cis conformer ratio in glycyl-L -proline has been measured in aqueous (D₂O) solution over the temperature range 33–96°C. It is found that ΔH⁰ = ?4.2 kJ/mole and ΔS⁰ = ?9.7 J/mole/K. Measurements of the T₁ values for the proline ring carbons yielded values consistent with a fast puckering process involving both the β- and γ-carbons. Measurements of the rate of cis-trans conformational interconversion in glycyl-L -proline, using complete line-shape analysis for the glycyl α-carbon resonance, gave values for the trans → cis isomerization as follows: ΔH^≠ = 83.5 ± 0.2 kJ/mole; ΔS^≠ = 0.0 ± 10 J/mole/K. A more approximate determination from coalescence temperature observations gave a value of ΔG^≠ of 82.0 ± 0.4 kJ/mole for this process in acetyl-L -proline in aqueous solution. The presence of 12M NaSCN lowered this barrier by ca. 2.6 kJ/mole. Such measurements are relevant to present theoretical models of the denaturation-renaturation processes in proteins, in which proline residues may play a key role.     

Polypeptides. LVI. Effect of lithium bromide and of temperature on the conformation of a copolymer of glutamic acid and lysine

By using optical rotatory dispersion measurements, the helix content of poly Glu⁵⁰Lys⁵⁰ has been investigated and compared with that of poly Glu²⁰Lys²⁰Ala⁶⁰ in aqueous solutions. Measurements were made at pH 3 and at pH 8 in various concentrations of lithium bromide. Various factors affecting helix stabilization are considered and their perturbation by lithium bromide is related to the shape of the observed transition curves. A residual helix content of 12% in 8M LiBr, based upon a b₀ of +100 for a fully random conformation, was observed for poly Glu⁵⁰Lys⁵⁰ at pH 3 and 8. The loss of helix content of poly Glu⁵⁰Lys⁵⁰ as a function of temperature is also reported. ΔH is approximately ?6.9 kcal./mole for the overall transition, compared to ?6.5 kcal./mole for poly Glu²⁰Lys²⁰Ala⁶⁰. The midpoint of the broad transition is near 40°C. at pH 3, but much lower, at ?10 to 0°C., at pH 8. These results are discussed in terms of the stabilizing factors for the partial helix content of the polypeptides.     

Phylogenetic Analysis of Selected Menthol-Producing Species Belonging to the Lamiaceae Family

Menthol is an organic compound with diverse medicinal and commercial applications, and is made either synthetically or through extraction from mint oils. The aim of the present study was to investigate menthol levels in selected menthol-producing species belonging to the Lamiaceae family, and to determine phylogenetic relationships of menthol dehydrogenase gene sequence among these species. Three genus of Lamiaceae, namely Mentha, Salvia, and Micromeria, were selected for phytochemical and phylogenetic analyses. After identification of each species based on menthol dehydrogenase gene in NCBI, BLAST software was used for the sequence alignment. MEGA4 software was used to draw phylogenetic tree for various species. Phytochemical analysis revealed that the highest and lowest amounts of both essential oil and menthol belonged to Mentha spicata and Micromeria hyssopifolia, respectively. The species Mentha spicata and Mentha piperita, which were assigned to one cluster in the dendrogram, contained the highest amounts of essential oil and menthol while Micromeria species, which was in the distinct cluster and placed in the farther evolutionary distance, contained the lowest amount of essential oil and menthol. Phylogenetic and phytochemistry analyses showed that essential oil and menthol contents of menthol-producing species are associated with menthol dehydrogenase gene sequence.     

Breakage by hydrodynamic shear of the bonds between cohered ends of lambda-DNA molecules

The rate of breakage by hydrodynamic shear of the cohered ends of λ-DNA molecules has been observed for the circular monomers, joined half molecules, and joined quarter molecules, in a capillary apparatus with known flow parameters. The rate constant for breakage has been measured as a function of shear stress, temperature, ionic strength, and molecular length. There is a large temperature coefficient, with an activation energy of 120 ± 20 kcal./mole. The values of d ln k/dG, where k is the rate constant for breaking and G is shear gradient, in aqueous solution at 25°C. are about 3.8 ± 0.3 × 10^?4 see. The shear stresses needed for breakage of joined quarter molecules and of circular monomers, respectively, are about equal, and about half that needed for breakage of joined half molecules. The rate of breakage at a given shear stress increases with decreasing ionic strength, approximately as [Na⁺]^?1.6. Self-protection effects are not observed for opening of circular monomers at a DNA concentration of 5 μg./ml. but are observed for breakage of joined half molecules at concentrations down to 0.5 μg./ml. The large temperature coefficient which is approximately equal to that of the thermal dissociation of the cohered ends is interpreted to mean that shear breakage is a mechanically assisted thermal reaction in which the thermal fluctuations provide most of the free energy of activation for breakage. A detailed model for this interpretation is presented. The self-protection effect implies that those molecules which break are not average molecules but exceptional ones which, due to some fluctuation, are more fully extended in the flow field.     

Thermodynamic characterization of ethidium bromide binding to a unique site on yeast tRNAphe.

A self-consistent thermodynamic characterization of the binding of ethidium to yeast phenylalanine-specific tRNA at 25°C, pH 7.0, in 11 nM MgCl₂, 375 nM NaCl, and 25 mM sodium phosphate has been obtained. Two ethidium molecules bind per tRNA under these conditions. The stronger site has a dissociation constant equal to 1.9 ± 0.5 μM and ΔH_dis°′ = 12 ± 1 Kcal/mol, and the weaker sites has a dissociation constant equal to 24 ± 9 μM and ΔH_dis°′ = 8.9 ± 1.5 Kcal/mol. The average calorimetric ΔH_dis°′ for the to sites 10.6 ± 0.4 kcal/mol. The thermodynamics of binding to the stranger sites are most probably the thermodynamics of interaction between A·U (6) and A·U (7), the unique site identified by Jones and Kearns. The binding is enthalpically driven and classical hydrophobic interactions do not appear to be important in the binding reaction.     

A Raman and calorimetric investigation of 3′, 5′ GpG

The Raman spectra of guanylyl (3′-5′) guanosine (GpG) in solution in H₂O and D₂O at pH 3–7 have been recorded at various temperatures between 0 and 80°C. The results are consistent with the existence in the lower temperature range of stable aggregates formed by the stacking of GpG tetramers. The aggregates melt cooperatively near 60°C, which results in important changes in the spectra. Among these, a large increase in intensity of some of the bands assigned to the guanine residues shows that unstacking of the bases occurs at the melting. Also apparent in the spectra are changes in the intensity and frequency of band attributable to molecular groups involved in intermolecular hydrogen bonding between adjacent molecules in the complex. The melting temperature of GpG decreases by approximately 15°C upon lowering the concentration from 5 × 10^?2 to 5 × 10^?4M, as shown by Raman, calorimetric, CD, and uv measurements. The experimentally determined ΔH and ΔS for the melting transition are 9 Kcal/mol and 28 e.u./mol, respectively. The aggregation of GpG in 1.5 × 10^?3M solutions was found to be very slow. The half-time of the process, which roughly follows first-order kinetics, is approximately 3 min at 10°C and 21 min at 35°C. The negative energy of activation associated with this reaction (?143 Kcal) indicated that the process involves intermediates whose concentrations decrease the temperatures raised, thus slowing down the overall process. The rate of disaggregation of GpG upon dilution to very low concentration is also extremely slow, indicating that the GpG aggregates, once formed, are very stable.     

The effect of temperature on the activity of the adenylate cyclase system of liver plasma membranes

The rat liver adenylate cyclase system shows a discontinuity in the Arrhenius plots at 20°C in the nonstimulated activity (basal) with activation energies of 16 and 28 Kcal/mole. The discontinuity disappears when the enzyme is stimulated either by glucagon, sodium fluoride, 5′ guanylyl-imidodiphosphate or glucagon plus 5′ guanylyl-imidodiphosphate and the energy of activation was the same with all the compounds tested. If the activator was initially in contact with the membranes at 0°C the energy of activation was similar to that observed below the break (26 Kcal/mole) but it changed to that above the break if the compound contacted the membranes at temperatures above the break (22–24°C). We discuss the possibility of two different conformations of the enzyme; both conformations can be “frozen” by any of the compounds tested, “isolating” the enzyme from any subsequent physical change of the membrane due to temperature.     

Prodigiosin-25 C

The equilibrium constant of the isomerization reaction between d-glucose and d-fructose which is catalyzed by a. glucose isomerase from Streptomyces sp. was obtained by both methods of chemical analysis and of kinetic study over the temperature range of 25° to 70°C.

It was found that the formation of d-fructose from d-glucose was an endothermic reaction with the heat of the reaction, ΔH, of +2220 cal/mole. The standard free energy change, ΔG, and the standard entropy change, ΔS, associated with the isomeric change were found to be +180 cal/mole and + 6.8 cal/deg. mole at 25°C, respectively. The values of these thermodynamic quantities at other temperature are also summarized.     

Isolation of Allo-isoleucic Acid (α-Hydroxy-β-methylvaleric Acid) and β-Hydroxy-β-methylvaleric Acid from Turkish Tobacco Leaves

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity.

The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added.

After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol.

The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation. $u=114\times \frac{E_{t1}?E_{t2}}{t_2?t_1}$

Where E_t1 and E_t2 mean the optical density of the ethanol solution at 310 mμ prepared after t₁ and t₂ minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute.

The substrate tannic acid used in this determining method was purified. It was composed of one mole of glucose and nine moles of gallic acid, and eight moles of which formed four moles of m-digallic acid.     

Synthesis of chitin by particulate preparations from Aspergillus flavus

Cell-free extracts from Aspergillus flavus catalyzed the synthesis of chitin from UDP-GlcNAc. Most of the activity was associated with membrane-rich fractions whereas no activity was detected in the cell walls. Chitin synthetase was activated by fungal acid proteases; animal and plant proteases destroyed it. Upon incubation at 0 C and 28 C chitin synthetase was inactivated, probably by the action of proteases present in the particulate preparations. Maximal activity was obtained at pH 6.6–7.1 and 15 C. Arrhenius plot showed a biphasic curve with the transition at 7 C. E values were 3300 Kcal/mole above this temperature and 15500 Kcal/mole below it. The enzyme was activated by GlcNAc and required a divalent metal, the most active being Mg⁺⁺. By plotting v vs UDP-GlcNAc concentration a sigmoidal curve was obtained. Km calculated at high substrate concentrations was 20mm. Chitin synthetase was competitively inhibited by polyoxin D (Ki 6.5 m) and UDP (Ki 1.35mm), the latter giving complex kinetics.This work was supported by grants No. 020 and 847 of the Consejo Nacional de Ciencia y Tecnología, México.Part of this work was carried out while the authors were research visitors at the Department of Plant Pathology, University of California, Riverside.     

Some effects of differently-acting nematicides on the cereal cyst-nematode (Heterodera avenae) and on the appearance of ''scorch'' in spring wheat on light loamy sand

In fungitoxicity tests against Phytophthora cinnamomi on Chamaecyparis lawsoniana cv. Ellwoodii, a drench of furalaxyl (1000 mg a.i./l) applied to the compost in which 1-yr-old plants were growing, 1 wk before they were inoculated with 650 000 zoospores, controlled disease for at least 12 months. With an inoculum dose of 650 zoospores/plant, furalaxyl at 500 mg a.i./l controlled disease even when inoculation was 12 wk after fungicide treatment. Aluminium tris (ethyl phosphonate) (2000 mg a.i./l) applied as a drench 1 wk before inoculation with 650 000 zoospores/plant did not prevent root infection but delayed foliar symptoms for 9 months: the same treatment, using etridiazole (500 mg a.i./l) only slightly reduced disease incidence. When applied as a single drench 2 days before inoculation, prothiocarb (2000 mg a.i./l) and cuprammonium compounds (200 mg a.i./l) were much less effective than furalaxyl (1200 mg a.i./l), sodium ethyl phosphonate (1500 mg a.i./l), aluminium tris (ethyl phosphonate) (1500 mg a.i./l) or etridiazole (500 mg a.i./l). However, a drench of furalaxyl at 1000 mg a.i./l, aluminium tris (ethyl phosphonate) at 2000 mg a.i./l or etridiazole at 500 mg'a.i./l did not eradicate P. cinnamomi from compost containing infected root debris. Pre-planting drenching of the compost was ineffective. All fungicide treatments were non-phototoxic to 1-yr-old C. lawsoniana cv. Ellwoodii. These results are of special relevance to the control of P. cinnamomi on container-grown woody ornamentals.     

Protein-Vitamin E Relation and the Hemolysis Test

The blood cells of vitamin E deficient rats are hemolyzed by dialuric acid. The percent of hemolysis in the dialuric acid test was higher in rats fed a 5% casein diet than in pair-fed animals receiving 10 or 20% casein diet when a suboptimal amount of α-tocopherol (7 ~ 32 µg per day) was administered. With 1 mg per day of α-tocopherol there was no hemolysis at any protein level. The significance of the results is discussed.     

Consequences of Sequential Ca2+ Occupancy for the Structure and Dynamics of CalbindinD9K: Computational Simulations and Comparison to Experimental Determinations in Solution (10, 309, (1993))

Abstract

Due to the slow convergence of the atomic rms fluctuations with the length of time for which they are calculated, the entropic contribution to the cooperativity of Ca²⁺ binding to calbindin_D9k, discussed on pp. 329–330, has to be evaluated for a longer interval than used for the rms values given in Table 5. The value of ΔΔG obtained from the rms fluctuations given in Table 5 is about 0.5 Kcal/mole. For the longer interval of 179 ps the average rms values calculated for CAB₀, CAB₁ and CAB₂ (see original article for definitions) are 4.5 Å, 0.9 Å and 0.8 Å, respectively. The value of ΔΔG calculated from these rms fluctuations according to the equations in the the original article is about 1.0 Kcal/mole. It is noted that these trends do not affect the interpretation or conclusions regarding the significance of the configurational entropy for the cooperativity of binding in calbindin_D9k.