Polyamines secreted by cancer cells possibly account for the impairment of the human erythrocyte sodium pump activity.

Using an original microcalorimetric method, we previously showed that in erythrocytes from cancer patients, the sodium pump activity was decreased and returned to normal in patient in remission. In addition we suggested that a plasma-borne factor probably secreted by cancer cells accounted for this impairment of the sodium transporter. In the present study we sought to identify this factor as well as its mechanism of action. First we determined the effect of culture media from undifferentiated and differentiated colon cancer cell lines (Caco-2 and HT29-D4) on the sodium pump activity of normal human erythrocytes. The inhibitory powers of culture media from undifferentiated cells were higher than those of differentiated cells (38.6 +/- 3.5% vs 6.9 +/- 4.6%, p<0.05 for Caco-2 and 45.8 +/- 6.2% vs 9.0 +/- 5.0%, <0.05 for HT29-D4). The use of alpha difluoro-methylomithine (2 mM) to inhibit ornithine decarboxylase, the rate-limiting enzyme for polyamine biosynthesis, dramatically reduced the sodium pump inhibition induced by the two undifferentiated cell lines (75% for Caco-2 and 89% for HT29-D4). Polyamines secreted by undifferentiated cells and then taken up by human erythrocytes thus appeared as inhibitors of sodium pump of these red blood cells. Putrescine, spermidine, and spermine (the main polyamines) exerted a similar inhibitory effect (33 +/- 2%). Tested in vitro on Na,KATPase, these polyamines (3 mM) were inhibitors (putrescine = 23 +/- 2%; spermidine= 48 +/- 3%; spermine= 55 +/- 2%) when assay condition for the ATPase reaction was suboptimal (Na+ = 10 mM; K+ = 1 mM). The inhibitory effect appeared to be related to their charge and their aliphatic chain length. The effect of spermidine and spermine on the ionic substrates and ATP-Mg showed that molecules decreased the affinity (Km) of the Na,K-ATPase for Na+ (11.24 +/- 0.49 mM for control vs 23.51 +/- 1.53 mM for spermine and 18.86 +/- 0.98 mM for spermidine), indicating that polyamines exerted their inhibitory effect in a competitive manner.

Dissociation constants and thermal stability of complexes of Bacillus intermedius RNase and the protein inhibitor of Bacillus amyloliquefaciens RNase.

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40,82Ala mutant are about 10(-12) M, only 5 to 43 times higher than those of the barnase-barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase-barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

Thermostability of the barnase-barstar complex.

Scanning microcalorimetry was used to study heat denaturation of barnase in complex with its intracellular inhibitor barstar. The heat denaturation of the barnase-barstar complex is well approximately by two two-state transitions with the lower temperature transition corresponding to barstar denaturation and the higher temperature one to barnase denaturation. The temperature of barnase melting in its complex with barstar is 20 degrees C higher than that of the free enzyme. The barstar melting temperature is almost the same in the complex or alone (71 degrees C at pH 6.2 and 68 degrees C at pH 8.0). It seems possible that when barstar unfolds it can remain bound to barnase, while the latter unfolds only on dissociation of the denatured barstar.

Thermodynamic study of dihydrofolate reductase inhibitor selectivity.

The thermodynamic parameters of the binding of some folate analogues (methotrexate, trimetrexate and trimethoprim) to dihydrofolate reductases from different species have been measured with a flow microcalorimetric method at 37 degrees C. In the absence of NADPH, the three inhibitors exhibited a higher affinity for E. coli DHFR than for vertebrate DHFRs. This selectivity in favor of bacterial DHFR is entropy driven and is correlated with a weaker conformational change for bacterial DHFR than for vertebrate DHFRs, and with additional hydrophobic contacts, provided by this enzyme to the ligands. In presence of NADPH, as reported in the literature, trimetoprim shows a high selectivity in favor of bacterial DHFR, contrarily to methotrexate and trimetrexate, whose affinities are elevated and highly similar for mammalian and bacterial enzymes. The positive cooperative effect of NADPH, which has an enthalpic origin, fluctuates widely with inhibitor structure and with enzyme species. For trimethoprim, the cooperative effect is much more pronounced for bacterial DHFR than for vertebrate DHFRs. But the role of NADPH is not to induce a selectivity: it only increases the selectivity that trimethoprim already presented in absence of NADPH. Inversely, for methotrexate and trimetrexate, the cooperative effect is stronger for vertebrate enzymes than for the bacterial enzyme, and thus, NADPH cancels the selectivity the two antifolic compounds had, in the absence of NADPH, for the bacterial enzyme.

Comparative thermodynamic study of the interaction of some antifolates with dihydrofolate reductase.

The thermodynamic parameters of the binding of antifolate drugs to bovine liver dihydrofolate reductase (EC 1.5.1.3., 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase) have been measured with a flow microcalorimetric method. These parameters are greatly influenced by the structure of the inhibitor and/or by the presence of NADPH and above all by temperature. For all the compounds studied, binding at 37 degrees C is driven by favourable enthalpy variations, whereas entropy variations are unfavourable. At 10 degrees C, reactions are both enthalpically and entropically driven. These effects can be explained by a partial thermal denaturation of dihydrofolate reductase at 37 degrees C, which is restructured by NADPH and/or the antifolate. The refolding induced by the antifolate trimetrexate may explain its high association constant in the binary system (without NADPH), and the weaker cooperative effect of NADPH in the ternary system, as compared to methotrexate. In contrast, the poor affinity of trimethoprim for mammalian dihydrofolate reductase in binary and ternary systems at 37 degrees C is the result of a weaker stabilizing effect of this compound as regards temperature increase. Heat capacity variation linked to the complex formation reaction showed that this conformational transition is more pronounced between 25 and 37 degrees C than between 10 and 25 degrees C. Thus, the ability of the inhibitors to give to dihydrofolate reductase a more stable thermal behaviour at 37 degrees C is determinant in their binding.

Thermodynamic study of the influence of NADPH on the binding of methotrexate and its metabolites to a mammalian dihydrofolate reductase.

Interaction of methotrexate and some of its metabolites with a mammalian dihydrofolate reductase was studied using two complementary methods, potentiometry and microcalorimetry. The major plasma metabolite of this anticancer agent, 7-hydroxymethotrexate, was found to have a different binding behavior from that of polyglutamyl derivatives and of methotrexate itself. Indeed, 7-hydroxymethotrexate binds without a pK shift to dihydrofolate reductase, whereas polyglutamyl derivatives bind to the enzyme with a proton uptake, as the parent drug does. NADPH increases the association constant of the 7-hydroxy metabolite by a factor of 10-20, while for methotrexate and for polyglutamates this increase is about 100-fold. It was demonstrated that the enhancement of the binding by NADPH had an enthalpic origin. Finally, the binding behavior of dihydrofolate reductase seemed to be independent of its enzymatic activity.