The effect of pH and temperature on the reaction of fully reduced and mixed-valence cytochrome c oxidase with dioxygen

The reaction of fully reduced and mixed-valence cytochrome oxidase with O2 has been followed in flow-flash experiments, starting from the CO complexes, at 428, 445, 605 and 830 nm between pH 5.8b and 9.0 in the temperature range of 2-40 degrees C. With the fully reduced enzyme, four kinetic phase with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 2.5 x 10(4), 1.0 x 10(4) and 800 s(-1), respectively, are observed. The rates of the three last phases display a very small temperature dependence, corresponding to activation energies in the range 13-54 kJ x mol(-1). The rates of the third and fourth phases decrease at high pH due to the deprotonation of groups with pKa values of 8.3 and 8.8, respectively, but also the second phase appears to have a small pH dependence. In the reaction of the mixed-valence enzyme, three kinetic phases with rate constants at pH 7.4 and 25 degrees C of 9 x 10(4), 6000 and 150 s(-1), respectively, are observed. The third phase only has a small temperature dependence, corresponding to an activation energy of 20 kJ x mol(-1). No pH dependence could be detected for any phase. Reaction schemes consistent with the experimental observations are presented. The pH dependencies of the rates of the two final phase in the reaction of the fully reduced enzyme are proposed to be related to the involvement of protons in the reduction of a peroxide intermediate. The temperature dependence data suggest that the reorganization energies and driving forces are closely matched in all electron transfer steps with both enzyme forms. It is suggested that the slowest step in the reaction of the mixed-valence enzyme is a conformation change involved in the reaction cycle of cytochrome oxidase as a proton pump.     

Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex.

Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

The influence of temperature on antiproliferative effects, cellular uptake and DNA platination of the clinically employed Pt(II)-drugs

Cellular uptake of a drug is one of the most important factors influencing its pharmacodynamics and pharmacokinetics. Our laboratory has previously studied platinum uptake following cisplatin, carboplatin and oxaliplatin treatment at sub-lethal doses of selected tumour cell lines. Here we report on the influence of temperature on dose-dependent antiproliferative effects, cellular uptake and DNA platination of these platinum-based drugs tested on MCF-7 human mammary carcinoma cell line. Inductively coupled plasma-mass spectrometry (ICP-MS) technique has been chosen to perform Pt determinations on cells treated with drug concentrations similar with those usually found in vivo in human plasma. The high sensitivity and analytical rapidity of this technique made possible to carry out a very large amount of Pt determinations (about 300) necessary for this study. Hyperthermia (43 degrees C) proved a synergistic effect with cisplatin on cell growth inhibition, while only an additive effect was demonstrated for carboplatin and oxaliplatin. This behaviour might be explained by the higher DNA platination ratio between data at 43 and 37 degrees C of cisplatin with respect to those of carboplatin and oxaliplatin.     

Pharmacokinetics and tissue distribution of novel traditional Chinese medicine-platinum anticancer agents in rats

The pharmacokinetics and tissue distribution profiles of a novel series of traditional Chinese medicine-platinum (TCM-Pt) compounds [Pt(C(8)H(8)O(5))(NH(2)R)(2)]: 1 (where R=H), 3 (R=CH(3)) and 5 (R=C(6)H(10)), were studied in Sprague-Dawley rats following a single bolus intravenous (i.v.) injection. Platinum concentrations in total plasma, plasma ultrafiltrate, urine and tissues were measured by flameless atomic absorption spectroscopy. Pharmacokinetic studies showed that plasma concentrations of total and free platinum for the novel TCM-Pt compounds as well as cisplatin and carboplatin declined in a biexponential manner with a short distribution half-life (t(1/2alpha): 0.12-0.34h). Compared with cisplatin, the novel TCM-Pt compounds had a longer elimination half-life (t(1/2beta)), larger dose normalized area under the curve (AUC/D), larger volume of distribution at steady-state (V(ss)), slower clearance (CL) of free platinum and higher percentage of cumulative urinary excretion (CUE), which can be attributed to their lower chemical reactivities. In tissues, the highest Pt concentrations were found in the kidney, followed by the liver and the lowest in the heart; no Pt was detected in the brain. Twenty-four hours after drug administration, platinum concentrations in tissues were significantly lower for the novel TCM-Pt compounds. These findings suggest that the novel compounds might afford higher clinical efficacy and reduced systemic side effects, when compared with cisplatin.     

Remarkably stereoselective photoinduced electron-transfer reaction between zinc myoglobin and optically active binaphthyl bisviologen

We have designed and synthesized new optically active bisviologens ([BNMV](4+)) containing a binaphthyl moiety to examine the stereoselective photoinduced electron-transfer (ET) reactions with zinc-substituted myoglobin (ZnMb) by flash photolysis. The photoexcited triplet state of ZnMb, (3)(ZnMb)*, was successfully quenched by [BNMV](4+) ions to form the radical pair of a ZnMb cation (ZnMb(.+)) and a reduced viologen ([BNMV](.3+)), followed by a thermal ET reaction to the ground state. The rate constants ( k(q)) for the ET quenching at 25 degrees C were obtained as k(q)( R)=(2.9+/-0.2)x10(7) M(-1) s(-1) and k(q)( S)=(2.2+/-0.2)x10(7) M(-1) s(-1), respectively. The ratio of k(q)( R)/ k(q)( S)=1.3 indicates that the ( R)-isomer of the chiral viologen preferentially quenches (3)(ZnMb)*. On the other hand, the rate constants ( k) for the thermal ET reaction from [BNMV](.3+) to ZnMb(-+) at 25 degrees C were k( R)=(1.2+/-0.1)x10(8) M(-1) s(-1) and k( S)=(0.47+/-0.03)x10(8) M(-1) s(-1), respectively, and the ratio remarkably increased to k( R)/ k( S)=2.6. The activation parameters, Delta H(not equal) and Delta S(not equal), were determined from the kinetic measurements at various temperatures (10-30 degrees C) to understand the ET mechanisms. In the quenching reaction, the energy differences of Delta Delta H*(R- S) and T Delta Delta S*( R- S) at 25 degrees C were calculated to be -3.9+/-1.6 and -3.3+/-0.2 kJ mol(-1), respectively, whereas Delta Delta H*( R-S)=7.7+/-1.9 kJ mol(-1 )and T Delta Delta S*( R-S)=9.9+/-0.5 kJ mol(-1 )were found for the thermal ET reaction. Therefore, the thermal ET reaction to the ground state was proved to be dominated by the entropy term, and the large stereoselectivity may arise from the decrease in charge repulsion between donor and acceptor.     

Binding of branched-chain 2-oxo acids to bovine serum albumin.

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 x 10(3) M-1, k2 = 0.09 x 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 x 10(3) M-1, k2 = 0.08 x 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 x 10(3) M-1, k2 = less than 0.05 x 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.     

Structure and properties of the 5a,6-anhydrotetracycline-platinum(II) dichloride complex: a theoretical ab initio study

Ab initio structural parameters, relative energies and spectroscopic properties are reported for the 5a,6-anhydrotetracycline-platinum(II) dichloride complex. Distinct coordination modes were analyzed in gas phase and aqueous medium indicating the tricarbonylmethane moiety (O3-Oam-O1) as the most probable Pt(II) complexation sites, supporting experimental proposals. The theoretical (1)H NMR analysis in conjunction with the observed data suggests that the Pt(II) might be bound to the O3-Oam preferentially, even though this site was found slightly unfavorable, based on energetic grounds, relative to O1-Oam. The aquation reaction, which is an important activation step in the action mechanism of cisplatin like molecules, was also investigated showing rate constants (0.5-1 x 10(-5) M(-1) s(-1)) of the same order of magnitude as those for the parent cisplatin compound. This is an important result stressing the importance of tetracyclines-Pt(II) complexes as lead compounds for the development of new platinum based antitumor drugs.     

Reactivity of platinum-based antitumor drugs towards a Met- and His-rich 20mer peptide corresponding to the N-terminal domain of human copper transporter 1

Cellular uptake of platinum-based antitumor drugs is a critical step in the mechanism of the drug action and associated resistance, and deeper understanding of this step may inspire development of novel methods for new drugs with reduced resistance. Human copper transporter 1 (hCtr1), a copper influx protein, was recently found to facilitate the cellular entry of several platinum drugs. In the work reported here, we constructed a Met- and His-rich 20mer peptide (hCtr1-N20) corresponding to the N-terminal domain of hCtr1, which is the essential domain of hCtr1 for transporting platinum drugs. The interactions of the peptide with cisplatin and its analogues, including transplatin, carboplatin, oxaliplatin, and [Pt(l-Met)Cl₂], were explored at the molecular level. Electrospray ionization (ESI) mass spectrometry (MS) data revealed that all of the platinum(II) complexes used in present study can bind to hCtr1-N20 in 1:1 and 2:1 stoichiometry. Four Met residues should be involved in binding to cis-platinum complexes on the basis of the tandem MS spectrometry and previously reported data. Time-dependent 2D [¹H,¹⁵N] heteronuclear single quantum coherence NMR spectra indicate the reaction of cisplatin with hCtr1-N20 is a stepwise process. The intermediate, however, is transient, which is consistent with the ESI-MS results. Time-dependent ESI-MS data revealed that the geometry and the properties of both the leaving and the nonleaving groups of platinum(II) complexes play essential roles in controlling the reactivity and formation of the final products with hCtr1-N20.     

Interaction of anticancer drug cisplatin with guanine: density functional theory and surface-enhanced Raman spectroscopy study

Surface-enhanced Raman spectroscopy (SERS) in a silver sol assisted by density functional theory (DFT) calculations is shown to be a promising tool in the characterization of platinum complexes and their interaction with nucleic acid bases. This is demonstrated using cisplatin and guanine as a model. The energies and geometric parameters of cisplatin, guanine, and their reaction products are calculated at Becke's nonlocal three parameter exchange and correlation functional and the Lee-Yang-Parr correlation functional level using the 6-31++G(d,p) basis set on the light elements and the effective core potential by Hay and Wadt on platinum. Available X-ray crystallography data are mostly in agreement with predictions within the experimental precision level, although Pt-N bond lengths tend to be systematically overestimated. The normal Raman spectrum of cisplatin is assigned. The SERS spectra of cisplatin and its reaction product with guanine are measured from 10(-6) M aqueous solution. The observed spectral changes in the SERS spectrum of guanine upon cisplatin binding are modeled by DFT calculations. The best agreement between theory and experiment is achieved when the adsorbed reaction product is assumed to be the 1:1 adduct cis-Pt(NH3)2ClG in which Pt is bound to N7 and guanine is deprotonated at N9.     

Quantitative measurement of the reduction of platinum(IV) complexes using X-ray absorption near-edge spectroscopy (XANES)

The platinum(II) drugs cisplatin, carboplatin and oxaliplatin are usefully employed against a range of malignancies, but toxicities and resistance have spurred the search for improved analogs. This has included investigation of the platinum(IV) oxidation state, which provides greater kinetic inertness. It is generally accepted that Pt(IV) complexes must be reduced to Pt(II) for activation. As such, the ability to monitor reduction of Pt(IV) complexes is critical to guiding the design of candidates, and providing mechanistic understanding. Here we report in full that the white line height of X-ray absorption near-edge spectra (XANES) of Pt complexes, normalized to the post-edge minima, can be used to quantitatively determine the proportion of each oxidation state in a mixture. A series of Pt(IV) complexes based on the Pt(II) complexes cisplatin and transplatin were prepared with chlorido, acetato or hydroxido axial ligands, and studies into their reduction potential and cytotoxicity against A2780 human ovarian cancer cells were performed, demonstrating the relationship between reduction potential and cytotoxicity. Analysis of white line height demonstrated a clear and consistent difference between Pt(II) (1.52 ± 0.05) and Pt(IV) (2.43 ± 0.19) complexes. Reduction of Pt(IV) complexes over time in cell growth media and A2780 cells was observed by XANES, and shown to correspond with their reduction potentials and cytotoxicities. We propose that this method is useful for monitoring reduction of metal-based drug candidates in complex biological systems.     

Enthalpic barriers to the hydrophobic binding of oligosaccharides to phage P22 tailspike protein

The structural thermodynamics of the recognition of complex carbohydrates by proteins are not well understood. The recognition of O-antigen polysaccharide by phage P22 tailspike protein is a highly suitable model for advancing knowledge in this field. The binding to octa- and dodecasaccharides derived from Salmonella enteritidis O-antigen was studied by isothermal titration calorimetry and stopped-flow spectrofluorimetry. At room temperature, the binding reaction is enthalpically driven with an unfavorable change in entropy. A large change of -1.8 +/- 0.2 kJ mol(-1) K(-1) in heat capacity suggests that the hydrophobic effect and water reorganization contribute substantially to complex formation. As expected from the large heat-capacity change, we found enthalpy-entropy compensation. The calorimetrically measured binding enthalpies were identical within error to van't Hoff enthalpies determined from fluorescence titrations. Binding kinetics were determined at temperatures ranging from 10 to 30 degrees C. The second-order association rate constant varied from 1 x 10(5) M(-1) s(-1) for dodecasaccharide at 10 degrees C to 7 x 10(5) M(-1) s(-1) for octasaccharide at 30 degrees C. The first-order dissociation rate constants ranged from 0.2 to 3.8 s(-1). The Arrhenius activation energies were close to 50 and 100 kJ mol(-1) for the association and dissociation reactions, respectively, indicating mainly enthalpic barriers. Despite the fact that this system is quite complex due to the flexibility of the saccharide, both the thermodynamic and kinetic data are compatible with a simple one-step binding model.     

Kinetics and mechanisms of the recombination of Zn2+, Co2+, and Ni2+ with the metal-depleted catalytic site of horse liver alcohol dehydrogenase

The kinetics of the recombination of the metal-depleted active site of horse liver alcohol dehydrogenase (LADH) with metal ions have been studied over a range of pH and temperature. The formation rates were determined optically, by activity measurements, or by using the pH change during metal incorporation with a pH-indicator as monitor. The binding of Zn2+, Co2+, and Ni2+ ions occurs in a two-step process. The first step is a fast equilibrium reaction, characterized by an equilibrium constant K1. The spectroscopic and catalytic properties of the native or metal-substituted protein are recovered in a slow, monomolecular process with the rate constant k2. The rate constants k2 5.2 X 10(-2) sec-1 (Zn2+), 1.1 X 10(-3) sec-1 (Co2+), and 2 X 10(-4) sec-1 (Ni2+). The rate constants increase with increasing pH. Using temperature dependence, the activation parameters for the reaction with Co2+ and Ni2+ were determined. Activation energies of 51 +/- 2.5 kJ/mol (0.033 M N-Tris-(hydroxymethyl)methyl-2-aminomethane sulfonic acid (TES), pH 6, 9) for Co2+ and 48.5 +/- 4 kJ/mol (0.033 M TES, pH 7, 2) for Ni2+ at 23 degrees C were found. The correspondent activation entropies are - 146 +/- 10 kJ/mol K for Co2+ and - 163 +/- 9 kJ/mol K for Ni2+. Two protons are released during the binding of Zn2+ to H4Zn(n)2 LADH in the pH range 6.8-8.1. The binding of coenzyme, either reduced or oxidized, prevents completely the incorporation of metal ions, suggesting that the metal ions enter the catalytic site via the coenzyme binding domain and not through the hydrophobic substrate channel.     

Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs

The sulfur-containing tripeptide glutathione (GSH) is one of the most abundant molecules in cells. Elevated levels of GSH render some types of cancer cells resistant against well-known platinum anti-cancer drugs such as cisplatin and carboplatin. Platinum complexes are often very reactive towards the cysteine residue of GSH, which detoxifies these compounds by a rapid binding mechanism. Clearly, this resistance mechanism poses a severe obstacle to any new platinum drugs designed to overcome cisplatin resistance. In the present study the cytotoxicity of dinuclear platinum compounds of the 1,1/t,t type, as developed by Farrell, is determined in human ovarium A2780 cells and in the cisplatin-resistant cell line A2780cisR, which possesses elevated levels of GSH. Further, the effect of depletion of GSH levels by L-buthionine-S,R-sulfoximine (L-BSO) in A2780cisR was investigated. The experiments show that detoxification by GSH is an effective resistance mechanism against dinuclear platinum compounds. However, the dinuclear complexes are less sensitive towards detoxification compared to cisplatin. This is probably because of the rapid binding of dinuclear cationic complexes to DNA. Compared to cisplatin, the rapid binding to DNA reduces the time during which the drug molecules are exposed to GSH in the cytosol. The reaction of a representative dinuclear compound with glutathione (pH 7, 37 degrees C) was studied in detail by 195Pt NMR. The dinuclear complex BBR3005 ([trans-PtCl(2)(NH(3))(2)(mu-H(2)N(CH(2))(6)NH(2))](2+), abbreviated as 1,1/t,t n=6), follows different pathways in the reaction with GSH, depending on the molar ratio of the reactants. When reacted in stoichiometric amounts (1:1), first a chloride on each platinum is replaced by a sulfur, forming a PtN(3)S product at -2977 ppm. After 2-3 h, this intermediate reacts further to form a sulfur-bridged N(3)Pt-S-PtN(3) species as the main product at -2811 ppm. When BBR3005 is reacted with GSH in a ratio of 1:4, the sulfur-bridged species is not observed. Instead, the final product is trans-Pt(GS)(2)(NH(3))(2) (at -3215 ppm); the same product appears if GSH is reacted with trans-PtCl(2)(NH(3))(2). Apparently, GSH first replaces the chlorides and subsequently degrades the dinuclear compound by replacement of the diaminealkyl linker.     

Thermoregulatory responses to exercise in dehydrated dogs

Measurements of rectal temperature (Tre), water lost by evaporation (Eresp) and drooling, cardiac output (CO), and common carotid blood flow (CCBF) were made in dogs (mean hydrated wt 31.0 +/- 1.5 kg) running for 1 h on a level treadmill at 7.5 km/h at an ambient temperature of 25 degrees C. Each animal was studied when it was hydrated ad libitum and when it had been dehydrated by removal of drinking water until 9-10% of the initial body weight had been lost. Dehydrated exercising animals had significantly higher Tre and lower rates of Eresp, CO, and CCBF. Tre and Eresp were measured in seven animals. Average Tre during running was 39.11 +/- 0.10 degrees C in hydrated and 39.80 +/- 0.25 degrees C in dehydrated animals (P less than 0.01). Average Eresp during running was 3.9 +/- 0.3 g/min in hydrated animals and 2.3 +/- 0.3 g/min in dehydrated animals (P less than 0.01). Average CO during exercise, measured in five animals, was 11.1 +/- 0.7 1/min in the hydrated state and 8.6 +/- 0.5 1/min in the dehydrated state (P less than 0.01). Unilateral CCBF during exercise, measured in four animals, was 602 +/- 40 ml/min in the hydrated state and 418 +/- 22 ml/min in the dehydrated state (P less than 0.01). Water lost by drooling in seven exercising animals was 41.5 +/- 11 g/h when they were hydrated and 0.6 +/- 0.4 g/h when they were dehydrated. It is concluded that dehydrated dogs doing mild exercise can save water by reducing Eresp and regulating body temperature above hydrated levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein phosphatase 2A inhibition and circumvention of cisplatin cross-resistance by novel TCM-platinum anticancer agents containing demethylcantharidin

Novel TCM-platinum compounds [Pt(C(8)H(8)O(5))(NH(2)R)(2)] 1-5, derived from integrating demethylcantharidin, a modified component from a traditional Chinese medicine (TCM) with a platinum moiety, possess anticancer and protein phosphatase 2A inhibition properties. The compounds are able to circumvent cisplatin resistance by apparently targeting the DNA repair mechanism. Novel isosteric analogues [Pt(C(9)H(10)O(4))(NH(2)R)(2)] A and B, devoid of PP2A-inhibitory activity, were found to suffer from an enhanced DNA repair and were cross-resistant to cisplatin. The results advocate a well-defined structure-activity requirement associating the PP2A-inhibiting demethylcantharidin with the circumvention of cisplatin cross-resistance demonstrated by TCM-Pt compounds 1-5.     

Purification and characterization of protein kinase A from liver of the freeze-tolerant wood frog: role in glycogenolysis during freezing

Freeze tolerance by various amphibians includes cryoprotectant production in the form of glucose. Activation of the catalytic subunit of liver cAMP-dependent protein kinase (PKAc) facilitates activation of glycogenolysis, a critical biochemical process necessary for production of glucose. Here, we purified PKAc from Rana sylvatica liver to determine the extent to which cold temperature, which stimulates cryoprotectant production, affected PKAc activity and function. PKAc was purified to greater than 95% homogeneity, with a final specific activity of 71 nmol phosphate transferred/min/mg protein. The molecular weight of frog liver PKAc was 47.6 +/- 1.1 kDa and K(m) values for the phosphate acceptor kemptide and Mg-ATP were 9.0 +/- 0.1 and 51.8 +/- 1.0 microM at 22 degrees C, respectively. K(m) values for both substrates dropped significantly at 5 degrees C. The enzyme was sensitive to specific inhibitors of mammalian PKAc (PKA(i), H89) but was only moderately inhibited by high salt concentrations. Furthermore, salt inhibition was reduced at low temperature. The effect of temperature on enzyme activity indicated a conformational change in PKAc at 10 +/- 2 degrees C, with calculated activation energies of 51 +/- 4 kJ/mol at temperatures above 10 degrees C and 110 +/- 9 kJ/mol below 10 degrees C. PKAc in wood frog liver plays a crucial role in mediating the freeze-induced glycogenolysis that is responsible for the production of 200-300 mM levels of glucose as a cryoprotectant. Differential effects of low temperature on enzyme function, increased substrate affinity and reduced ion inhibition, appear to be central to this role.     

Influence of treatment temperature on the genotoxic effects of cisplatin in CHO cells: cytotoxicity, mutagenicity and induction of lesions in DNA

In cells exposed in vitro to the cytotoxic and mutagenic antitumor drug cisplatin (cis-Pt(NH3)2Cl2), various adducts with nuclear DNA are formed. A comparative study was made of the influence of temperature variation during treatment of cultured Chinese hamster ovary (CHO) cells with cisplatin on cytotoxicity, mutation induction and Pt-DNA adduct formation. Before and after treatment (1 h at 32, 37 or 40 degrees C) cells were kept at 37 degrees C. Cytotoxicity increased with temperature; D0 values were 29.6 +/- 1.6, 21.1 +/- 1.2 and 11.4 +/- 0.6 microM at 32, 37 and 40 degrees C, respectively. Pt-DNA binding to DNA at 40 degrees C was 2.0 (+/- 0.3) times as high as at 32 degrees C. This factor remained practically constant over a 24-h post-treatment incubation of the cells, during which about 60% of DNA-bound Pt were removed. As the increase in cytotoxicity between 32 and 40 degrees C was roughly in proportion to that in Pt binding, no substantial changes in the spectrum of adducts appeared to occur. The induction of DNA interstrand cross-links, studied at 32 and 40 degrees C, varied linearly with dose. Influence of temperature on cross-link formation was comparable to that on total Pt binding. Amounts of cross-links highly increased during 24 h after treatment. Plots of cross-links against survival after treatments at 32 and 40 degrees C almost coincided. Induction of 6-thioguanine-resistant (HGPRT) mutants at various cisplatin concentrations did not show a clear temperature dependency. Consequently, equitoxic treatments were significantly more mutagenic at 32 degrees C than at 40 degrees C, the opposite of what has been reported for E. coli.