Decreased activity of inducible nitric oxide synthase type 2 and modulation of the expression of glutathione S-transferase alpha, bcl-2, and metallothioneins during the differentiation of CaCo-2 cells.

Reactive oxygen species modulate the cell growth of a wide variety of mammalian cells. To determine whether oxidative metabolism is altered during the differentiation process, we studied the expression of pro- and antioxidant proteins in proliferating and differentiated CaCo-2 cells, a human colon adenocarcinoma cell line. Nitric oxide synthase type 2 (iNOS) produces nitric oxide (NO). Depending on its rate of synthesis, NO may either promote cellular and DNA damage or reduce the ability of other free radicals to induce cell injury. Using Western and Northern blot analysis and arginine conversion assay, we demonstrate that the expression of iNOS decreases when cells undergo differentiation. This biological event entails a diminished production of NO metabolites and correlates with the loss of activation of soluble guanylate cyclase activity. In differentiated cells, a 2-fold down-regulation of the nuclear factor kappa B activity was observed, suggesting that nuclear factor kappa B could be one of the iNOS gene regulatory factors in the CaCo-2 model. In parallel, we studied the expression of other antioxidant proteins including glutathione S-transferase alpha (GST alpha), bcl-2, and the metallothioneins (MTs). We show that the protein levels of GST alpha and MT increase during the differentiation of CaCo-2 cells, whereas bcl-2 levels decrease. Our investigation indicates that the expression of iNOS, GST alpha, bcl-2, and MT is associated with the enterocytic differentiation. The shift in the expression of specific antioxidant genes during CaCo-2 cell differentiation may occur to avoid alterations in the cell redox potential

Anion-linked polymerization of the tetrameric hemoglobin from Scapharca inaequivalvis. Characterization and functional relevance.

The anion-linked polymerization of the tetrameric hemoglobin from Scapharca inaequivalvis has been characterized by means of sedimentation velocity experiments in terms of its dependence on the binding of other protein ligands, oxygen, and protons. The linkage with oxygen manifests itself at any given anion concentration in the markedly different sedimentation behavior of the oxygenated and deoxygenated protein; whereas the former sediments always as a single peak (congruent to 4.3 S), the latter exhibits bimodal Schlieren patterns with a fast peak that attains congruent to 10 S. A comparison of experimental and computer-simulated (Cox, D. J. (1971) Arch. Biochem. Biophys. 146, 181-195) boundaries shows that the behavior of oxyhemoglobin can be represented by a rapidly reversible dimerization of the native 4.3 S molecule, whereas the behavior of the deoxygenated protein can be described adequately in terms of the polymerization of the native structure into tetramers and octamers. The interplay between the binding of protons and anions is brought out by the different dependence of polymer formation on anion concentration at different pH values. In the case of chloride, polymerization goes through a maximum at around 20-50 mM Cl- at pH 6.3 but decreases monotonically above 5 mM at pH 5.5. On the basis of these data and of the effect of other anions such as phosphate and perchlorate, a tentative picture of the high affinity anion-binding sites has been proposed. From a functional point of view, the hemoglobin polymers are characterized by a lower oxygen affinity and a higher cooperativity than the tetrameric structure; hence, polymerization results in a shift of the lower asymptote of the Hill plots while the upper asymptote is unaltered. The effect of polymerization on oxygen binding has been analyzed in terms of the polysteric linkage scheme (Colosimo, A., Brunori, M., and Wyman, J. (1974) Biophys. Chem. 2, 338-343). The data obtained at pH 6.3 as a function of chloride concentration could be fitted satisfactorily by taking the self-association behavior of the protein into account