Equilibrium and rapid kinetic studies on nocodazole-tubulin interaction

The interaction between nocodazole and calf brain tubulin in 10(-2) M sodium phosphate, 10(-4) M GTP, and 12% (v/v) dimethyl sulfoxide at pH 7.0 was studied. The number of binding sites for nocodazole was shown to be one per tubulin monomer of 50,000 as a result of equilibrium binding studies by gel filtration and spectroscopic techniques. The presence of microtubule-associated proteins did not significantly affect the binding of nocodazole to tubulin. The apparent equilibrium constant measured at 25 degrees C was (4 +/- 1) X 10(5) M-1. Temperature does not significantly affect the apparent equilibrium constant; hence, the binding of nocodazole to tubulin is apparently entropy driven. Stopped flow spectroscopy was employed to monitor the rate of nocodazole binding under pseudo first order conditions. The effects of temperature and nocodazole concentration were studied. The apparent rate constants were dependent on the concentration of nocodazole in a nonlinear manner. In conjunction with results from structural and thermodynamic studies the kinetic results were interpreted to suggest a mechanism of T + N in equilibrium with TN in equilibrium with T* N, where T and N are tubulin and nocodazole, respectively. T and T* represent two conformational states of tubulin. Furthermore, the kinetic data are consistent with the thermodynamic data only if a model of two parallel similar reactions were considered, one rapid and the other slow. The initial binding step for both the rapid and slow phases was characterized by identical binding constants; however, there was a significant difference in the rates of isomerization. Hence, nocodazole is potentially a useful probe for amplifying differences in solution properties of tubulin subspecies.     

Equilibrium, kinetic, and footprinting studies of the Tus-Ter protein-DNA interaction.

Arrest of DNA replication in the terminus region of the Escherichia coli chromosome is mediated by protein-DNA complexes composed of the Tus protein and 23 base pair sequences generically called Ter sites. We have characterized the in vitro binding of purified Tus protein to a 37-base pair oligodeoxyribonucleotide containing the TerB sequence. The measured equilibrium binding constant (KD) for the chromosomal TerB site in KG buffer (50 mM Tris-Cl, 150 mM potassium glutamate, 25 degrees C, pH 7.5, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 100 micrograms/ml bovine serum albumin) was 3.4 x 10(-13) M. Kinetic measurements in the same buffer revealed that the Tus-TerB complex was very stable, with a half-life of 550 min, a dissociation rate constant of 2.1 x 10(-5) s-1, and an association rate constant of 1.4 x 10(8) M-1 s-1. Similar measurements of Tus protein binding to the TerR2 site of the plasmid R6K showed an affinity 30-fold lower than the Tus-TerB interaction. This difference was due primarily to a more rapid dissociation of the Tus-TerR2 complex. Using standard chemical modification techniques, we also examined the DNA-protein contacts of the Tus-TerB interaction. Extensive contacts between the Tus protein and the TerB sequence were observed in the highly conserved 11 base-pair "core" sequence common to all identified Ter sites. In addition, protein-DNA contact sites were observed in the region of the Ter site where DNA replication is arrested. Projection of the footprinting data onto B-form DNA indicated that the majority of the alkylation interference and hydroxyl radical-protected sites were arranged on one face of the DNA helix. We also observed dimethyl sulfate protection of 2 guanine residues on the opposite side of the helix, suggesting that part of the Tus protein extends around the double helix. The distribution of contacts along the TerB sequence was consistent with the functional polarity of the Tus-Ter complex and suggested possible mechanisms for the impediment of protein translocation along DNA.     

Equilibrium and kinetic studies of the interactions of a porphyrin with low-density lipoproteins

Low-density lipoproteins (LDL) play a key role in the delivery of photosensitizers to tumor cells in photodynamic therapy. The interaction of deuteroporphyrin, an amphiphilic porphyrin, with LDL is examined at equilibrium and the kinetics of association/dissociation are determined by stopped-flow. Changes in apoprotein and porphyrin fluorescence suggest two classes of bound porphyrins. The first class, characterized by tryptophan fluorescence quenching, involves four well-defined sites. The affinity constant per site is 8.75 x 10(7) M(-1) (cumulative affinity 3.5 x 10(8) M(-1)). The second class corresponds to the incorporation of up to 50 molecules into the outer lipidic layer of LDL with an affinity constant of 2 x 10(8) M(-1). Stopped-flow experiments involving direct LDL porphyrin mixing or porphyrin transfer from preloaded LDL to albumin provide kinetic characterization of the two classes. The rate constants for dissociation of the first and second classes are 5.8 and 15 s(-1); the association rate constants are 5 x 10(8) M(-1) s(-1) per site and 3 x 10(9) M(-1) s(-1), respectively. Both fluorescence and kinetic analysis indicate that the first class involves regions at the boundary between lipids and the apoprotein. The kinetics of porphyrin-LDL interactions indicates that changes in the distribution of photosensitizers among various carriers could be very sensitive to the specific tumor microenvironment.     

The interaction of calcium and magnesium ions with deoxyribonucleic acid.

Conductance and equilibrium dialysis studies are reported for the aqueous systems (native calf thymus) DNA-CaCl₂ and DNA-MgCl₂ at various pH values and ionic strengths at 25 °C. Discontinuities occur in the conductance curves at mole ratios of Ca²⁺ and Mg²⁺ to nucleate phosphorus of 0.125, 0.30, and 0.50. The dialysis results show the formation of complexes of stoichiometry 0.50 and 1.00 mol Ca²⁺ or Mg²⁺/mol nucleate phosphorus (2:1 and 1:1 complexes), the latter only in neutral or alkaline solutions, in agreement with the conductance discontinuity at 0.50. The other discontinuities may be due to preferential binding in the formation of the 2:1 complex. Binding constants for the 2:1 complexes are evaluated. Absorption-temperature profiles have been determined for “native” and dialysed DNA in the presence of NaCl, CaCl₂, and MgCl₂. For dialysed DNA at 26 ° C and 260 nm the decrease of absorbance with increased salt concentration was halted for MgCl₂ and CaCl₂ at a concentration corresponding to the formation of the 2:1 complex. The absorbance of “native” DNA did not decrease. T_m and the reciprocal of the hypochromic rise ($\text{1}\text{h}$) increased linearly with log (salt concn). Values of T_m were the same at 230, 260, and 280 nm, but h was greater at 230 and 280 than at 260 nm, which may be due to the existence of alternating blocks of (A + T) and (G ? C) pairs. The entropy of transition was in the order Ca > Mg ? Na.     

Formation constants for interaction of citrate with calcium and magnesium ions

Formation constants for the interaction of citrate ion with calcium and magnesium ions in solution at 37°C and a constant ionic strength of 0.15 were determined by potentiometric titration. Values for the formation of CaL^? and CaHL⁰ complexes were 1.88 × 10³ and 67, respectively. Corresponding constants for MgL^? and MgHL⁰ were 2.19 × 10³ and 42, respectively. The existence of other complexes was not confirmed. Protonation constants were also determined under the same conditions.     

Equilibrium and kinetic studies on reversible and irreversible denaturation of micrococcal nuclease

The effect of pH and temperature on the thermal denaturation of micrococcal nuclease wer4e investigated. The ranges employed were between pH3.30 and pH9.70 and between 10 degrees C and 85 degrees C, respectively. The reversible denaturation involved in the whole process was clearly discriminated from the irreversible one. The former took place with a large enthalpy change of 384 kJ mol(-1) at pH 9.70, where the enzyme exhibited it s maximum activity. The latter probably led to aggregation because the successive long incubation after complete deactivation caused precipitation. A reasonable scheme explaining the process involving both denaturations was proposed and the kinetic on the irreversible deactivation was performed. It was revealed that the irreversible deactivation involved two types of reactions whose activation energies were relatively small: 22.2 kJ mol(-1) and 18.8kJ mol(-1). The presence of sucrose suppressed the reversible denaturation without significant influence on enthalpy change, whereas it affected little the irreversible deactivation kinetically. The effects of pH change and addition of sucrose on the denaturation were discussed thermodynamically, especially in terms of the entropy change. (c) 1994 John Wiley & Sons, Inc.     

Blood magnesium, and the interaction with calcium, on the risk of high-grade prostate cancer

Background

Ionized calcium (Ca) and magnesium (Mg) compete as essential messengers to regulate cell proliferation and inflammation. We hypothesized that inadequate Mg levels, perhaps relative to Ca levels (e.g. a high Ca/Mg ratio) are associated with greater prostate cancer risk.

Study Design

In this biomarker sub-study of the Nashville Men''s Health Study (NMHS), we included 494 NMHS participants, consisting of 98 high-grade (Gleason≥7) and 100 low-grade cancer cases, 133 prostate intraepithelial neoplasia (PIN) cases, and 163 controls without cancer or PIN at biopsy. Linear and logistic regression were used to determine associations between blood Ca, Mg, and the Ca/Mg ratio across controls and case groups while adjusting for potential confounding factors.

Results

Serum Mg levels were significantly lower, while the Ca/Mg ratio was significantly higher, among high-grade cases vs. controls (p = 0.04, p = 0.01, respectively). Elevated Mg was significantly associated with a lower risk of high-grade prostate cancer (OR = 0.26 (0.09, 0.85)). An elevated Ca/Mg ratio was also associated with an increased risk of high-grade prostate cancer (OR = 2.81 (1.24, 6.36) adjusted for serum Ca and Mg). In contrast, blood Ca levels were not significantly associated with prostate cancer or PIN.Mg, Ca, or Ca/Mg levels were not associated with low-grade cancer, PIN, PSA levels, prostate volume, or BPH treatment.

Conclusion

Low blood Mg levels and a high Ca/Mg ratio were significantly associated with high-grade prostate cancer. These findings suggest Mg affects prostate cancer risk perhaps through interacting with Ca.     

Rapid kinetic studies on calcium interactions with native and fluorescently labeled calmodulin

Stopped-flow studies on calcium binding to calmodulin showed that under pseudo first order conditions the reaction was complete within 2.5 milliseconds. The time course for calcium dissociation from the native protein showed a single kinetic phase (τ1^?1 = 10S^?1) while that from the dansylated derivative revealed a second slower kinetic phase (τ1^?1 = 10S^?1, τ2^?1 = 0.31S^?1) that accounted for about one-half of the total fluorescence decrease. Therefore the dansyl derivative of calmodulin may provide a useful tool for studying conformational changes in the protein not reflected by the active site tyrosines.     

Equilibrium partition studies of the interaction between aldolase and myofibrils

The adsorption of aldolase to myofibrils derived from rabbit skeletal muscle has been investigated by partition equilibrium studies at pH 6.8, I = 0.158 M, and the results interpreted in terms of an intrinsic association constant of 410,000 m^?1 for the interaction of four sites on aldolase with myofibrillar sites, there being one such site for every 10–12 heptameric repeat units of F-actin-tropomyosin-troponin thin filament. Involvement of the active site of the enzyme in the adsorption process is indicated by the fact that competitive inhibition of the phenomenon by phosphate may be accounted for by an intrinsic association constant of 400 m^?1 for the aldolase-phosphate interaction, a value in good agreement with that describing phosphate inhibition of the enzymatic hydrolysis of fructose-1,6-bisphosphate under similar conditions. On the basis of these equilibrium constants plus the aldolase and thin filament contents of muscle, resting muscle is indicated as containing a significant proportion (25–30%) of aldolase in the bound form, with changes in the subcellular distribution of the enzyme being likely during exercise due to the increased concentrations of Ca²⁺ and fructose-1,6-bisphosphate that then prevail.     

Equilibrium and kinetic studies on the binding of des-N-tetramethyltriostin A to DNA

The interaction between TANDEM (a des-methyl analogue of triostin A) and poly(dA-dT) results in extension of the helix by 6.8 Å for each ligand molecule bound, exactly as predicted for a bis-intercalation reaction. Cooperativity is evident in Scatchard plots for the interaction at ionic strengths of 0.2 and 1.0, where the binding constant is diminished compared to that which pertains at low salt concentration. Binding to a natural DNA (calf thymus), already considerably weaker than binding to poly(dA-dT), is also sensitive to increased ionic strength. With a self-complementary octanucleotide d(G-G-T-A-T-A-C-C) the binding curve indicates the presence of a single des-N-tetramethyltriostin A binding site per helical fragment with a non-cooperative association constant about 6·10⁶ M^?1. Detergent-induced dissociation of des-N-tetramethyltriostin A-poly(dA-dT) complexes results in a simple exponential decay at all levels of binding, but the time constant of decay is dependent upon the initial binding ratio. This behaviour cannot directly explain the cooperativity of equilibrium binding isotherms but suggests the occurrence of relatively long-lived perturbations of the helical structure by binding of the ligand. [Ala³, Ala⁷]des-N-tetramethyltriostin A, which has a more flexible octapeptide ring lacking the disulphide cross-bridge, dissociates from poly(dA-dT) much faster than des-N-tetramethyltriostin A. Dissociation of des-N-tetramethyltriostin A from calf thymus DNA is more rapid than dissociation of triostin A or other quinoxaline antibiotics, which may account for its low antimicrobial activity.     

The interaction of magnesium ions with the calcium pump of sarcoplasmic reticulum.

1. In the presence of Ca2+, ATP phosphorylates the Ca2+ pump of sarcoplasmic reticulum at the same site and to the same extent regardless of whether Mg2+ is added or not to the incubation media, the main effect of added Mg2+ being to increase the rate of phosphorylation. 2. When phosphoenzyme is made in Mg2+-containing media it dephosphorylates about 30-times faster than when it is made in the absence of added Mg2+. Addition of Mg2+ after phosphorylation is uneffective in accelerating the hydrolysis of phosphoenzyme even in solubilized enzyme, suggesting that phosphorylation of the Ca2+ pump results in occlusion of the site at which Mg2+ combines to accelerate the release of phosphate. 3. Occlusion of the site for Mg2+ can be partially reversed by trans-1,2-diaminocyclohexonetetraacetic acid (CDTA). Use was made of this property to demonstrate that for the rapid release of phosphate to occur Mg2+ has to be bound to the enzyme. 4. Results seem to indicate that Mg2+ combines with the Ca2+ pump prior to phosphorylation.     

Equilibrium studies of the cyclic AMP receptor protein-DNA interaction

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6.3 X 10(8) M-1 at 0.2 microM-cAMP, and increases to 8.4 X 10(10) M-1 at 5.0 microM-cAMP. The latter is about 10(5) times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0.2 microM to 10 microM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6 X 10(6) M-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.     

Equilibrium and kinetic studies of unfolding of homologous cytochromes c.

Extensive investigations of the unfolding equilibria and kinetics of oxidized and reduced cytochromes c are reported. It is found that all cytochromes c have similar unfolding free energies (deltaGD = 7 +/- 1 kcal/mol). Differences among species do not correlate in any way with the metabolic differences among species. The stabilization of cytochrome c on reduction is estimated at 1.1 kcal/mol. Stability differences among species are mirrored in their denaturation kinetics. For cytochrome c (III), the unfolding exhibits multiple phases. The rate constants for the two observable phases both change by a factor of 3 between horse cytochrome c (III) and cow cytochrome c (III). On reduction, all unfolding appears to occur in a single step. The rate of this unfolding still varies between species, however, the results can be accommodated to a sequential model, with some assumptions. The observations are consistent with chain reversal occurring at an early stage in the reaction and suggest that previously observed rapid processes may be ligand exchange processes.     

Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution.

An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.     

Equilibrium and kinetic studies of ligand BMXD complexation with copper(II) and glycylglycine

Equilibrium studies of BMXD complexation with copper(II) and glycylglycine were performed by potentiometric and UV-visible titration. The equilibrium constants determined by potentiometry for the ternary system are: [LCu₂P³⁺]/[LCu₂⁴⁺][P⁻]=8.11(3), [LCu₂HP⁴⁺]/[LCu₂P³⁺][H⁺]=5.39(2), [LCu₂H₋₁P²⁺][H⁺]/[LCu₂P³⁺]=−6.33(2), [LCu₂(OH)H₋₁P⁺][H⁺]/[LCu₂H₋₁P²⁺]=−8.20(1) and [LCu₂(OH)₂HH₋₁P][H⁺]/[LCu₂(OH)H₋₁P⁻¹]=−10.33(3), which are in good agreement with the values determined by UV-visible titration. The species formed were confirmed by electrospray mass spectrometry. The association constants, determined by kinetic studies, for the association of the second molecule of glycylglycine with the LCu₂P complex, for two ionic media, are 1.29 (KCl) and 1.27 (KNO₃). These are in good agreement with the values determined by thermodynamic measurements 1.41 (KCl) and 1.26 (KNO₃).