Catalytic Properties of Two Pyruvate Kinase Isoforms in Nordic Krill,Meganyctiphanes norvegica,With Respect to Seasonal Temperature Adaptation

Two isoforms of pyruvate kinase (PK I and PK II) were partly purified and characterized from the Nordic krill Meganyctiphanes norvegica. Both PK variants were present in summer and winter specimens with a tissue specificity in abdominal muscle (PK I) and cephalothorax (PK II). Obvious differences were found in chromatographic and kinetic characteristics. Enzymatic adaptations to low temperatures were found in PK I only, whereas PK II did not contribute to seasonal temperature adaptation. In winter specimens, the activation energy of PK I decreased significantly from 53.2 ± 1.5 to 50.2 ± 1.2 kJ·mol⁻¹. The affinity of PK I to phosphoenol-pyruvate was higher in winter (K_M = 0.024 ± 0.002 mmol·l⁻¹) compared to summer (K_M = 0.033 ± 0.003 mmol·l⁻¹). Both effects lead to an increased efficiency of this enzyme isoform in the cold. In contrast, K_M values of PK II showed no significant differences between summer (K_M = 0.181 ± 0.014 mmol·l⁻¹) and winter specimens (K_M = 0.193 ± 0.015 mmol·l⁻¹). The effects of cooperativity remained unchanged during the seasons with approximate values of n_Hill = 1.0 (PK I) and 1.5 (PK II). Fructose-1,6-bis-phosphate affected only PK II by shifting sigmoidal kinetics to hyperbolic curves resulting in a decrease of K_M to 0.027 mmol·l⁻¹. Further effectors were tested showing an inhibiting effect of oxalate on both isoforms with a reduction to 20% and 50% in PK I and PK II, respectively. Presumably, the ecophysiological effect of the capacity to regulate muscle PK is related to the necessity to increase motility during vertical migration and phases of feeding activity.     

Probing of the porcine serum lipoprotein surfaces by Mn(II) binding: an e.s.r. study

Mn(II) ions were used for probing the surfaces of porcine LDL₁, LDL₂ and HDL. From the intensity of the e.p.r. lines corresponding to the unbound Mn(II) the percentage of the ions bound to the lipoprotein surface is determined. From the titration curves the binding parameters, dissociation constant. K_d, and the number of binding sites, n, in all the three lipoproteins studied have been derived. There are at least two types of binding sites in each lipoprotein class. The ”weak’ binding sites are charaterized by approximately the same value of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(≈ 6.2 × 10}{}^{\mathrm{?3}}\text{mol l}{}^{\mathrm{?1}}$ and different values for n (n = 114 for LDL₁, n = 135 for LDL₂ and n = 28 for HDL). Similarly, for the ”strong’ binding sites K_d ≈ 1.6 × 10^?4 mol l^?1 and the number of binding sites is 15, 20 and 5 for LDL₁, LDL₂ and HDL respectively. It is concluded that the binding sites are probably located in the protein part of the lipoproteins and that they are mainly associated with the negatively charged amino acids.     

Binding of meso-tetra(4-sulfonatophenyl)porphine to haemopexin and albumin studied by spectroscopy methods

• 1.1. The interaction of haemopexin and albumin with TPPS₄ was studied by measuring the absorption and fluorescence spectra. Haemopexin was found to have one strong TPPS₄ binding center (K_a = 3 × 10⁷M⁻¹).
• 2.2. Haem-haemopexin complex appears to have no specific binding site for TPPS₄. Occupation of the specific binding center of haemopexin molecule by a haem abolishes TPPS₄ binding.
• 3.3. Albumin was found to possess one strong TPPS₄ binding center (K_a = 3 × 10⁶M⁻¹) besides two or three weak binding sites (K_a = 2 × 10⁵M⁻¹).
• 4.4. Haern-albumin complex possesses only one weak TPPS₄ binding site (K_a = 7 × lO⁵M⁻¹). These observations suggest identity of primary binding sites of TPPS₄ and haem on albumin molecule.

     

Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K_D = 25 ± 4 μM and K′_D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ?_b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ?_b = 3.5 ± 0.4 for tight binding sites and an average ?_b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s^?1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.     

Effects of dehydration and low temperatures on the oxidation of high-potential cytochrome c by photosynthetic reaction centers in Ectothiorhodospira shaposhnikovii

Effects of temperature and dehydration on the efficiency of electron transfer from membrane-bound high-potential cytochromes c_h to the reaction-center bacteriochlorophyll (P-890) in Ectothiorhodospira shaposhnikovii have been studied. A kinetic analysis of the cytochrome oxidation suggests that there are at least two conformational states of the c_h-P-890 complex, of which only one allows photoinduced electron transfer from cytochrome to P-890⁺. Lowering the temperature of dehydration leads to a change in the proportion of the populations in the two conformations. The observed 2-fold deceleration of cytochrome oxidation can be related only to the diminution of the amount of photoactive cytochromes per reaction center. The rate constant for the transfer of an electron from cytochrome c_h to bacteriochlorophyll is 2.8 · 10⁵ s⁻¹ and is independent of temperature and dehydration (as estimated within the accuracy of the experiments). The effects produced by low temperature and dehydration are completely reversible. The thermodynamic parameters of the transition of the cytochrome from the nontransfer to electron-transfer conformation were estimated. For room temperature (+ 20°C) in chromatophore preparations, ΔG = −5.4 kJ · M⁻¹, ΔH = 60 kJ · M⁻¹, ΔS = 0.22 kJ · M⁻¹ · K⁻¹. For Triton X-100 subchromatophore preparations, the absolute values of the above parameters are significantly lower: ΔG = −2.8 kJ · M⁻¹, ΔH = 18 kJ · M⁻¹, and ΔS = 0.075 kJ · M⁻¹ · K⁻¹. To a larger extent, the above parameters are diminished for chromatophore preparations in an 80% glycerol solution: ΔG = −1.7 kJ · M⁻¹, ΔH = 6 kJ · M⁻¹, ΔS = 0.025 kJ · M⁻¹ · K⁻¹. The data suggest the hydrophobic character of the forces that maintain the P-890-c_h complex in the electron-transfer conformation. The results obtained suggest that electron tunneling within the complex cannot occur until a specific conformational configuration of the complex is formed. The efficiency of cytochrome c_h oxidation is determined by the temperature, the degree of dehydration and the environmental conditions, whereas the transfer of an electron itself in the electron-transfer configuration is essentially independent of temperature and hydration.     

Carbohydrate adducts with zinc-group-metal ions. Interactions of β-d-fructose with Zn(II), Cd(II), and Hg(II) cations,and the effects of metal-ion coordination on the sugar isomer binding

Interaction of β-d-fructose with hydrated salts of zinc-group-metal has been studied in aqueous solution and solid adducts of the type M(d-fructose)X₂·nH₂O, where M = Zn(II), Cd(II), and Hg(II) ions, X = Cl⁻ or Br⁻, and n = 0–2, have been isolated, and characterized by means of F.t.-i.r. spectroscopy, X-ray powder diffraction, and molar conductivity measurements. The marked spectral similarities observed with the Mg(d-fructose)X₂·4 H₂O (X = Cl⁻ or Br⁻) compounds indicated that the Zn(II) and Cd(II) ions are six-coordinated, binding to two d-fructose molecules through O-2, O-3 of the first d-fructose, and O-4, O-5 of the second, as well as to two H₂O. The Hg(II) ion binds to two sugar moieties in the same fashion as do the Zn(II) and Cd(II) ions, resulting in four-coordination geometry around the mercury ion. The crystalline sugar is in the β-d-fructopyranose form, and the coordination of the of the Ca(II) ion takes place through the β-d-fructopyranose isomer, whereas the binding of the Mg(II), Zn(II), Cd(II), Hg(II), and UO²⁺₂ cations could be via the β-d-fructopyranose and the β-d-fructofuranose structures.     

Analyzing the structural and functional roles of residues from the ‘black’ and ‘gray’ clusters of human S100P protein

Two highly conserved structural motifs observed in members of the EF-hand family of calcium binding proteins. The motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif represents a cluster of three amino acids called cluster I (‘black’ cluster) and cluster II (‘grey’ cluster). Cluster I is more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II is noticeably less conserved and includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. In the human calcium binding S100 P protein, these ‘black’ and ‘gray’ clusters include residues F15, F71, and F74 and L33, L58, and K30, respectively. To evaluate the effects of these clusters on structure and functionality of human S100 P, we have performed Ala scanning. The resulting mutants were studied by a multiparametric approach that included circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probes. Spectrofluorimetric Ca2+-titration of wild type S100 P showed that S100 P dimer has 1–2 strong calcium binding sites (K₁ = 4 × 106 M⁻¹) and two cooperative low affinity (K₂ = 4 × 104 M⁻¹) binding sites. Similarly, the S100 P mutants possess two types of calcium binding sites. This analysis revealed that the alanine substitutions in the clusters I and II caused comparable changes in the S100 P functional properties. However, analysis of heat- or GuHCl-induced unfolding of these proteins showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Opposite to literature data, the F15 A substitution did not cause the S100 P dimer dissociation, indicating that F15 is not crucial for dimer stability. Overall, similar to parvalbumins, the S100 P cluster I is more important for protein conformational stability than the cluster II.     

Specific binding sites for d-α-tocopherol on human erythrocytes

Since vitamin E deficiency is associated with increased susceptibility of erythrocytes to hemolysis, we investigated the presence of tocopherol binding sites in human red blood cells. Erythrocytes were found to have specific binding sites for d-α-[³H]tocopherol with properties of receptors. Kinetic studies of binding demonstrated two binding sites: one with high affinity (equilibrium association constant K_a = 2.6·10⁷ M^?1), low capacity (7600 sites/cell) and the second with low affinity (K_a = 1.24·10⁶ M^?1), high capacity (150000 sites/cell). These sites are at least partly protein in nature.     

β-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

β-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [³H]dihydro-l-alprenolol ([³H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [³H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, β-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of B_max = 12.01 fmol/10⁶ cells (7200 sites/cell), K_d = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with l-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k₊ = 8.31·10⁷ M^?1 · min^?1 and k_? = 0.28 min^?1 from the association results, and k_? = 0.24 min^?1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; K_d, as $\text{(k}{}_{\mathrm{?}}\text{/k}{}_{\mathrm{+}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>n</mtext>}}$ was found to be 101 pM. Analysis of the equilibrium binding of [³H]DHA to rat and mouse living brain cells resulted in values of B_max = 13.04 fmol/10⁶ cells (7800 sites/cell), K_d = 43.85 pM and n = 2.52, and B_max = 8.08 fmol/10⁶ cells (4800 sites/cell), K_d = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the β-receptor in mammalian objects, too. The [³H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, K_d in the range 1–2 nM, and a B_max of 10³–10⁴ sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the β-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

Localization and pharmacological characterization of nicotinic-cholinergic binding sites in cockroach brain using α- and neuronal bungarotoxin

[¹²⁵I]α-Bungarotoxinisusedasaprobetostudythenicotinic-cholinergicreceptorinmembrane preparations of the cockroach brain. Binding is restricted mainly to particulate fractions of brain homogenates, is time dependent and is saturable above 2 nM with very low non-specific binding. Scatchard analysis indicates that binding is associated with a single affinity site (K_d = 1.09 nM) having a B_max of 8926 fmol/mg protein which is the highest concentration of binding sites yet reported in insects. Association kinetics are best fit by a mono-exponential model with a k_obs = 4.37 × 10⁻³s⁻¹. Dissociation is best described by a bi-exponential model giving dissociation constants of 1.18 × 10⁻⁵ and 9.94 × 10⁻⁵s⁻¹. The K_ds calculated from kinetic data are 0.029 and 0.25 nM suggesting the possibility of heterogeneous binding sites not detected by saturation studies. Displacement studies indicate that binding follows a nicotinic pharmacology and demonstrate the high affinity of methyllycaconitine and the anthelmintics, morantel and pyrantel. Displacement by neuronal bungarotoxin shows the presence of two distinct binding sites not differentiated by α-bungarotoxin. Autoradiographic studies show α-bungarotoxin to be binding to neuropile regions of the brain, to be displaced from these regions by agents effective in binding studies and demonstrate that the neuronal bungarotoxin binding sites can be regionally localized.     

Exchange rates of pyridine on ferro- and ferriprotoporphyrin (IX) dimethylester in chloroform.

Nuclear magnetic resonance line-widths data have been used to determine the rate of solvent exchange from the first coordination sphere of ferro-and ferriprotoporphyrin(IX) dimethylester (Fe-PPD) in pyridine/chloroform. The average values of kinetic parameters for pyridine (PY) exchange indicate an SN2 mechanism tor Fe(III)-PPD(ΔH^&;#; = 36 kJ · mol⁻¹ ; ΔS^&;#; = −53 J·mol⁻¹K⁻¹; T_M(298 K) = 0.07 msec) and an SNI mechanism for Fe(II)-PPD (ΔH^&;#; = 67 kJ·mol⁻¹; ΔS^&;#; = 42 J · mol⁻¹K⁻¹; T_M(298 K) = 0.06 msec). Parallel to the accelerated ligand exchange rate at rising temperatures a redistribution of the electrons causing a transition of the metal porphyrin from the low-spin state to the high-spin state is observed. Enthalpy and entropy of the thermodynamic equilibrium between low- and high-spin Fe-PPD have been determined from experimental values of the average magnetic moment. A mean lifetime of low-spin Fe(III)-PPD was estimated from line. widths changes (T_L→H(298 K)≈ 20 msec) and the corresponding activation parameters have been obtained (ΔH^&;#;_L→H(298 K) = 26 kJ · mol⁻¹; ΔS^&;#;_L→H(298K) = −125 J · mol⁻¹K⁻¹).     

The binding of acetic anhydride- and citraconic anhydride-modified human low-density lipoprotein to mouse peritoneal macrophages The evidence for separate binding sites

Human plasma low-density lipoprotein (LDL) was modified chemically with either the monocarboxylic acid derivative, acetic anhydride, or the dicarboxylic acid derivative, citraconic anhydride, reagents which react principally with the lysine residues of protein. The modifications increased the net negative charge on the LDL particles, with citraconyl-LDL displaying a greater negative charge than acetylated LDL. Neither the antigenic reactivity nor the overall gross protein/lipid composition of the LDL were affected by the modification procedures, although a small reduction in the total cholesterol content was observed. The altered LDL species lost the ability to bind to the high-affinity cell surface B/E receptor but both bound to mouse peritoneal macrophages with saturable high-affinity kinetics. At 4°C, the macrophages bound ¹²⁵I-labelled citraconyl-LDL more avidly (K = 21 · 10⁻³ ml/ng) than they bound labelled acetyl-LDL(K = 2 · 10⁻³ ml/ng). Competitive inhibition studies indicated that acetyl-LDL and citraconyl-LDL were bound to non-identical sites on the macrophage monolayer surface and that the binding site for citraconyl-LDL was also different from that recognized by hypercholesterolaemic rabbit plasma VLDL (βVLDL).     

Binding of calcium by parvalbumin fragments

Parvalbumin fragments from carp pI 4.47 parvalbumin corresponding to its residues 1–75 and 76–108 bind Ca²⁺ with affinities corresponding to K_d 0.9 · 10⁻⁴ M and K_d 3 · 10⁻³ M, respectively.     

Sugar-metal ion interaction. Synthesis,spectroscopic and structural analysis of Zn(II), Cd(II), and Hg(II) sugar complexes containing l-arabinose

Interaction between l-arabinose and the zinc group metal-ion salts has been studied in aqueous solution and solid complexes of the type M(l-arabinose)X₂·nH₂O, where M = Zn(II), Cd(II), and Hg(II) ions, X = Cl⁻ or Br⁻, and n = 0–2 have been isolated and characterized. On comparison with the structurally known Ca(l-arabinose) Cl₂·4H₂O and the corresponding magnesium compounds, it is concluded that the Zn(II) and Cd(II) ions are six-coordinated, binding to two arabinose moieties via 03, 04 of the first and 01, 05 of the second sugar molecule as well as to two H₂O molecules. The Hg(II) ion binds only to two sugar molecules in a similar fashion to zinc and cadmium ions, resulting in a four coordination around the mercury ion. The strong intermolecular hydrogen bonding network of the free arabinose is rearranged to that of the sugar OH...H₂O...halide system upon metalation. The β-anomer sugar conformation is predominant in the free sugar, while the α-anomer conformation is preferred by the alkaline earth and Zn(II), Cd(II), and Hg(II) cations.     

Kinetics of nucleotide binding to chloroplast coupling factor (CF1)

Studies of the kinetics of association and dissociation of the formycin nucleotides FTP and FDP with CF₁ were carried out using the enhancement of formycin fluorescence. The protein used, derived from lettuce chloroplasts by chloroform induced release, contains only 4 types of subunit and has a molecular weight of 280 000.In the presence of 1.25 mM MgCl₂, 1 mol of ATP or FTP is bound to the latent enzyme, with K_d = 10^?7 or 2 · 10^?7, respectively. The fluorescence emission (λ_max 340 nm) of FTP is enhanced 3-fold upon binding, and polarization of fluorescence is markedly increased. The fluorescence changes have been used to follow FTP binding, which behaves as a bimolecular process with K₁ = 2.4 · 10⁴ M^?1 · s^?1. FTP is displaced by ATP in a process apparently involving unimolecular dissociation of FTP with k_?1 = 3 · 10^?3 s^?1. The ratio of rates is comparable to the equilibrium constant and no additional steps have been observed.The protein has 3 sites for ADP binding. Rates of ADP binding are similar in magnitude to those for FTP. ADP and ATP sites are at least partly competitive with one another.The kinetics of nucleotide binding are strikingly altered upon activation of the protein as an ATPase. The rate of FTP binding increases to at least 10⁶ M^?1 · s^?1. This suggests that activation involves lowering of the kinetic barriers to substrate and product binding-dissociation and has implications for the mechanism of energy transduction in photophosphorylation.     

Tissue kallikrein processes small proenkephalin peptides

Tissue kallikrein may play a role in processing precursor polypeptide hormones. We investigated whether hydrolysis of natural enkephalin precursors, peptide F and bovine adrenal medulla docosapeptide (BAM-22P), by hog pancreatic kallikrein is consistent with this concept. Incubation of peptide F with this tissue kallikrein resulted in the release of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin. Met⁵-Lys⁶-enkephalin was the main peptide released, indicating that the major cleavage site was between two lysine residues. At 37°C and pH 8.5, the K_M values for formation of Met⁵-enkephalin and Met⁵-Lys⁶-enkephalin were 129 and 191 μM, respectively. Corresponding k_cat values were 0.001 and 0.03 s⁻¹ and k_cat/K_M ratios were 8 and 1.6·10² M⁻¹ · s⁻¹, respectively. Cleavage of peptide F at acidic pH (5.5) was negligible. When BAM-22P was used as a substrate, Met⁵-Arg⁶-enkephalin was released, thus indicating cleavage between two arginine residues. At pH 8.5, K_M was 64 μM, k_cat was 4.5 s⁻¹, and the k_cat/K_M ratio was 7 · 10⁴ M⁻¹ · s⁻¹. At 5.5, the pH of the secretory granules, K_M, k_cat and k_cat/K_M were 184 μM, 1.9 s⁻¹ and 10⁴ M⁻¹ · s⁻¹, respectively. It is unlikely that peptide F could be a substrate for kallikrein in vivo; however, tissue kallikrein could aid in processing proenkephalin precursors such as BAM-22P by cleaving Arg-Arg peptide bonds.     

Sugar interaction with metal ions: synthesis,spectroscopic, and structural analysis of Zn(II), Cd(II), and Hg(II) complexes,containing d-glucuronate anion

Interaction between D-glucuronic acid and Zn(II), Cd(II), and Hg(II) metal ion salts has been studied in solution and solid complexes of the type M(D-glucuronate)X · nH₂O and M(D-glucuronate)₂·nH₂O, where M = Zn(II), Cd(II), and Hg(II), X = Cl⁻ or Br⁻, and n = 0–2 were isolated and characterized. Spectroscopic and other evidence indicated that in the metal-halide-sugar complexes the Zn(II) and Cd(II) ions bind to two D-glucuronate moieties via 06, 05 of the carboxyl oxygen atoms of the first and 04, 06' of hydroxyl and carbonyl groups of the second as well as to two H₂O molecules, whereas in the corresponding M(D-glucuronate)₂ · nH₂O salts, the metal ions are bonded to two sugar anions through 06 and 06' of the ionized carboxyl groups and two water molecules, resulting in a six-coordination around each metal cation. The Hg(II) ion binds to 06 and 05 oxygen atoms of a sugar anion and to a halide anion or water molecule, in the Hg(D-glucuronate)X·nH₂O compounds, while in the corresponding metal-glucuronate salt mercury is bonded to 06 and 06' of the two glucuronate anions with four-coordination around the Hg(II) ion. The β-anomer sugar conformation is predominant in the free acid and in these series of metal-sugar complexes.     

Thermodynamics of hexokinase-catalyzed reactions.

The enthalpies of the hexokinase-catalyzed phosphorylation or glucose, mannose, and fructose by ATP to the respective hexose 6-phosphates have been measured calorimetrically in TRIS/TRIS HCl buffer at 25.0, 28.5, and 32.0°C. The effects on the measured enthalpy of the glucose/hexokinase reaction due to variation of pH (over the range 6.7 to 9.0) and ionic strength (over the range 0.02 to 0.25) have been examined. Correction for enthalpy of buffer protonation leads to δH^o and δC_p^o values for the processes: eq-D-hexose + ATP⁴⁻ = eq-D-hexose 6-phosphate²⁻ + ADP³⁻+ H⁺. Results are δH^o = −23.8 ± 0.7 kJ · mol⁻¹ and δC_p^o = −156 ± 280 J·mol⁻¹·K⁻¹ for glucose. δH^o = −21.9 ± 0.7 kJ·mol⁻¹ and δC_p^o = 10 ± 140 J·mol⁻¹·K⁻¹ for mannose, and δH^o = −15.0 ± 0.9 kJ·mol⁻¹ and δC_p^o = −41 ± 160 J·mol⁻¹·K⁻¹ for fructose. Combination of these measured enthalpies with Gibbs energy data for hydrolysis of ATP⁴⁻ and that for the hexose 6-phosphates lead to δS^o values for the above hexokinase-catalyzed reactions.     

Uptake and binding of β-ecdysone in imaginal discs of Drosophila melanogaster

The kinetics of uptake and retention of β-ecdysone by imaginal discs from late third instar larvae of Drosophila melanogaster correspond well with those of the first synthetic response of discs to hormone, an increase in RNA synthesis.Competition studies indicate the presence of two types of hormone binding sites, specific and non-specific. The specific sites are saturated at hormone concentrations which fully induce morphogenesis. Results are consistent with the hypothesis that analogs which induce morphogenesis at differing concentrations bind to the same sites. Experiments with the inhibitors N-ethylmaleimide, actinomycin d, and cycloheximide suggest that the binding sites are pre-existing in the cell and require functional sulfhydryl groups for binding.Specific binding, binding that is competed by excess unlabeled β-ecdysone, is saturable (70–80 nM). Kinetic rate constants for this specific binding were estimated to be k_a = 1.5 × 10⁵M^?1 min^?1, k_d = 3 × 10^?2 min^?1. The equilibrium dissociation constant calculated from the kinetic rate constants was K_eq = 2 × 10^?7M compared to 1.7 × 10^?7M β-ecdysone required to induce morphogenesis in vitro and 2.5 × 10^?7M determined to be the in vivo concentration at the time of induction of morphogenesis.