Disruption of functions of wild-type p53 by hetero-oligomerization.

We report that a p53 segment (p53 del 1-293) containing the oligomerization domain interferes with the functions of wild-type p53. Wild-type p53 inhibits transcription mediated by human cytomegalovirus (CMV) immediate-early promoter significantly; however, co-expression of p53 del 1-293 drastically reduces this repression. We show that wild-type p53 forms hetero-oligomers with p53 del 1-293 suggesting that the hetero-oligomers are defective in repressing the CMV promoter. A synthetic promoter with p53-binding sites is transactivated significantly by wild-type p53. However, co-expression of p53 del 1-293 drastically reduces this activation. At a high concentration, a deletion mutant of wild-type p53 (del 393-327) defective in oligomerization transactivates efficiently a promoter with synthetic p53-binding sites. This transactivation remains unaffected by co-expression of p53 del 1-293. p53 del 393-327 also fails to hetero-oligomerize with p53 del 1-293 indicating that hetero-oligomerization is necessary for disruption of wild-type p53-mediated transactivation. Immunostaining experiments show that hetero-oligomerization does not lead to changes in localization of nuclear p53 demonstrating that delocalization of p53 is not the reason for inactivation. We also show that co-expression of p53 del 1-293 significantly reduces the G1/S arrest by wild-type p53 suggesting that a proper oligomeric form is necessary for wild-type p53-mediated cell cycle arrest. Thus, our work shows that hetero-oligomerization disrupts wild-type p53's biological functions and suggests a mechanism by which p53 mutants may disrupt functions of wild-type p53.

Release of iron from haemoglobin--a possible source of free radicals in diabetes mellitus.

Increased blood glucose in diabetes mellitus stimulates nonenzymatic glycosylation of several proteins, including haemoglobin. Although iron is tightly bound to haemoglobin, it is liberated under specific circumstances yielding free reactive iron. Studies with purified haemoglobin from normal individuals and diabetic patients revealed that concentration of free iron was significantly higher in the latter cases and increased progressively with extent of the disease. In vitro glycosylation of haemoglobin also led to increase in release of iron from protein. This increase in free iron, acting as a Fenton reagent, might produce free radicals, which, in turn might be causing oxidative stress in diabetes.

Structural organisations of hemoglobin and myoglobin influence their binding behaviour with phenothiazines.

Binding modalities of chlorpromazine and trifluoperazine, two widely used antipsychotic phenothiazine drugs with hemoglobin and myoglobin have been studied to understand how the quaternary, tertiary and secondary structural organisations of the proteins regulate the binding process. NaCl-induced alteration in the quaternary structure of hemoglobin influences its binding modality with phenothiazines. Minor alterations in the tertiary structure of thermally denatured myoglobin (denaturation temperature ranging between 30-70 degrees C) do not affect its affinity and the modality of binding with the drugs, but alterations in the secondary structure of the protein denatured at temperatures between 70-80 degrees C influence its binding.

Protoporphyrin IX potentiates horseradish peroxidase-catalyzed oxidation of NADH:Involvement of enzyme-porphyrin interaction.

Protoporphyrin IX potentiates horseradish peroxidase-catalyzed hydrogen peroxide-mediated NADH oxidation, but the porphyrin cannot change the enzyme-catalyzed o-dianisidine oxidation. Spectrofluorimetric studies reveal that an interaction occurs between horseradish peroxidase and protoporphyrin IX. The interaction is predominantly hydrophobic and entropy-driven endothermic process. This interaction may influence the potentiation effect of the protoporphyrin IX on horseradish peroxidase-catalyzed NADH oxidation because the latter has a positive correlation with the extent of binding of the protein with the porphyrin.

Studies on the interaction of hematoporphyrin with hemoglobin.

Spectrophotometric and spectrofluorimetric studies reveal that an interaction occurs between hemoglobin and hematoporphyrin, a photosensitizing drug used in photodynamic therapy. Two concentration ranges of hematoporphyrin, 0.4-0.9 microM and 1.8-3.6 microM, representing significantly monomeric and aggregated (dimeric) state, respectively, have been used in the binding studies. The binding affinity constant (K) decreases, while the possible number of binding sites (p) increases as the concentration range of the porphyrin is increased. The nature of interaction has been studied by fluorescence quenching titration method under different ionic strengths and temperature conditions. It appears to be predominantly electrostatic and enthalpy-driven in the lower range of porphyrin concentration. However, the interaction follows mostly hydrophobic and entropy-driven modality in the higher concentration range of the ligand. The porphyrin-hemoglobin interaction results in release of oxygen from the protein. The extent of oxygen release depends on the stoichiometric ratio of hematoporphyrin:hemoglobin.

Trifluoperazine is more effective than chlorpromazine in releasing oxygen from haemoglobin and myoglobin.

The extent of oxygen release from two heme proteins, haemoglobin and myoglobin have been studied in the presence of trifluoperazine and chlorpromazine (5-1000 microM). At a molar ratio (drug:protein) of 1.5, the release of oxygen from haemoglobin was 4 and 15% in the presence of chlorpromazine and trifluoperazine respectively, while from myoglobin the corresponding values were 20 and 40%. The findings were attributed to the greater extent of local conformational change around tryptophan moieties of each of the proteins induced by trifluoperazine.

Comparative studies on the interaction of protoporphyrin with hemoglobin and myoglobin.

The binding parameters of protoporphyrin IX (PPIX) with hemoglobin (Hb) were studied spectrofluorimetrically and the results were compared with those of PPIX interacting with myoglobin (Mb). Two concentration ranges of PPIX (0.3 microM-1.5 microM and 1.5 microM-3.0 microM) were used. For both hemoglobin and myoglobin, the binding affinity constant (K) decreased while the number of binding sites (p) increased as the concentration range of PPIX increased. The interactions occurred in non-cooperative mode. Over a particular PPIX range, the interaction of PPIX with hemoglobin decreased significantly with increasing NaCl molarity indicating a trend in electrostatic interaction, whereas PPIX binding with myoglobin did not change significantly indicating mostly non-electrostatic mode of interaction. Total bound charge (z psi) decreased significantly with increased PPIX concentration range in case of hemoglobin-PPIX interaction, but remained almost same in case of myoglobin-PPIX interactions. Thermodynamic analysis revealed that binding of PPIX to hemoglobin was mostly electrostatic at lower concentration range of PPIX but became less electrostatic at higher concentration range and myoglobin-PPIX interaction, predominantly hydrophobic in nature, became more hydrophobic with increased range of PPIX concentration. The difference in binding modality between PPIX-Hb and PPIX-Mb has been discussed in relation to the state of aggregation of porphyrin as well as the subunit interaction property present and absent in hemoglobin and myoglobin, respectively.

Accumulation of a murein-membrane attachment site fraction when cell division is blocked in lkyD and cha mutants of Salmonella typhimurium and Escherichia coli.

Membrane fractionation studies were performed on Salmonella typhimurium lkyD(Ts) and E. coli cha(Ts) mutants that appeared to be blocked at a late stage of the cell division cycle. In both cases growth of the mutant strains at nonpermissive temperatures was associated with accumulation of a characteristic cell envelope fraction (fraction OML) that contained inner membrane, murein, and outer membrane components. The isolated fraction corresponded in composition and bouyant density to a fraction from wild-type strains that had previously been suggested (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-767, 1982; K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, B. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) to contain adhesion sites between inner membrane, murein, and outer membrane. The accumulation of OML in LkyD- and Cha- cells was prevented by treatments that blocked DNA synthesis. The effects of interference with DNA synthesis did not appear to involve the SOS response.

Characterization of microsomal ATPases from developing human placenta.

Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na+ + K+ + Mg2+, Mg2+, and Ca2+ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg2+-dependent and Na+ + K+ + Mg2+-dependent ATPases possess nearly the same Km (apparent) for ATP, while the Ca2+-dependent enzyme shows a different one. Mg2+-dependent ATPase shows higher substrate affinity than Ca2+-dependent ATPase, although the Vmax of the Mg2+-dependent enzyme is lower than that of the latter. However, for each enzyme, the Km remains almost constant and Vmax varies during ontogenic development. Vmax of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, L-phenylalanine, L-leucine, and L-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.

Rate of formation of hydrogen peroxide (H2O2) in IUD-fitted human endometrium--a preliminary report.

Endometrial biopsy samples of (i) women (20-40 yr) using no contraceptive methods and (ii) women (25-45 yr) fitted with intrauterine contraceptive devices (CuT/Lippes loop) were analysed for the rate of H2O2 formation. The mean value for a normal proliferative endometrium was 10.56 +/- 1.45 nmoles H2O2 per milligram protein per 2 min, whereas the rate observed for samples of the same phase of the IUCD-fitted group without any bleeding episodes was 14.76 +/- 1.28 and that for members of the same group but who experienced excessive menstrual blood loss (menorrhagia) was 17.26 +/- 2.00, which was significantly higher than that of the control group.

Relationship between mitochondrial ATPase and membrane lipids from developing human placenta.

Mitochondrial lipids and ATPase activity have been studied in developing human placenta. The enzyme activity in the placenta increases steadily with gestational age until the 18th to 21st week, and decreases in the second half of pregnancy. Both Mg2+ and Ca2+ ions have been found to activate the enzyme system. The solubilization of membrane lipids by deoxycholate inhibits the enzyme activity. Total lipid and total phospholipid contents of the mitochondrial membranes also increase during the early development of the placenta with peaks in the 14th to 17th week, after which the levels decrease. However, the percentage composition of individual phospholipids remains almost the same throughout pregnancy. The profile of the ATPase activity exhibits a correlation with that of membrane lipids in human placenta during intrauterine development of the tissue.