Physicochemical characterization of the alkaline denaturation of cytochrome c peroxidase.

The alkaline denaturation of cytochrome c peroxidase and apocytochrome c peroxidase was investigated by analytical ultracentrifugation, gel-filtration chromatography, and circular dichroism. The results indicate that both cytochrome c peroxidase and the apoenzyme undergo extensive structural modifications upon exposure to alkaline pH, including dimer formation. The midpoint of the transition for dimer formation in the native enzyme occurs at pH 9.6 +/- 0.1, while loss of tertiary and secondary structure occurs with transition midpoints at pH 10.3 +/- 0.1 and pH 11.3 +/- 0.1, respectively. Studies performed in the presence of dithiothreitol and with carboxymethylated cytochrome c peroxidase indicate that dimer formation occurs via a disulfide crosslink involving the single cysteine residue in the enzyme. Denaturation of cytochrome c peroxidase in the presence of guanidine hydrochloride gave results similar to those obtained for the alkaline denaturation.

Errors from using 3H-labeled ribonucleoside triphosphates to monitor nuclear RNA synthesis in vitro.

The [3H]XTPs are used widely to monitor RNA synthesis in vitro. Recently, we discovered that they reflected only 40-45% of the true rate of nuclear RNA synthesis. Thus, when [8-14C]GTP was used, 1466 pmol [8-14C]GMP was incorporated per mg DNA/10 min. On the other hand, when [8-3H]GTP was used, only 564 pmol [8-3H]GMP was incorporated per mg DNA/10 min. There are three obvious factors that could have contributed to this greater than 2-fold difference in the apparent incorporation rate: commercial [8-3H]GTP sample was contaminated with substances causing the assay medium to be less efficient in RNA synthesis; 3H exchange occurred during acid washing of the [3H]RNA; and there was a greater quenching effect on [3H]RNA. Experiments were designed to test each of these alternatives. We are able to conclude that none of the above three are contributing factors. Our data also show that the 3H label was removed after it was incorporated into RNA. Similar differences were observed when 3H and 14C labeled pairs of ATP, UTP and CTP were compared. Furthermore, when nuclei were fractionated into nucleolar and nucleoplasmic fractions and carried out RNA synthesis, the loss of 3H label was observed mainly from the nucleoplasmic fraction.

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase.

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.

Sedimentation equilibrium studies on the interaction between cytochrome c and cytochrome c peroxidase.

The interaction between cytochrome c and cytochrome c peroxidase was investigated using sedimentation equilibrium at pH 6,20 degrees C, in a number of buffer systems varying in ionic strength between 1 and 100 mM. Between 10 and 100 mM ionic strengths, the sedimentation of the individual proteins was essentially ideal, and sedimentation equilibrium experiments on mixtures of the two proteins were analyzed assuming ideal solution behavior. Analysis of the distribution of mixtures of cytochrome c and cytochrome c peroxidase in the ultracentrifuge cell based on a model involving the formation of a 1:1 cytochrome c-cytochrome c peroxidase complex gave values of the equilibrium dissociation constant ranging from 2.3 +/- 2.7 microM at 10 mM ionic strength to infinity (no detectable interaction) at 100 mM ionic strength. Attempts to determine the presence of complexes involving two cytochrome c molecules bound to cytochrome c peroxidase were inconclusive.

Evidence for an indirect mechanism of aflatoxin B1 inhibition of rat liver nuclear RNA polymerase II activity in vivo.

Previous studies suggested multiple sites of action of aflatoxin B1 (AFB1) in vivo to inhibit rat liver nuclear RNA synthesis--it impairs nucleolar DNA template function and inhibits RNA polymerase II activity. We have previously shown that AFB1 activated in vitro inhibits nucleolar RNA synthesis. The question is whether AFB1 can inhibit RNA polymerase II under these in vitro conditions. Male Sprague-Dawley rats, 200 g, were injected i.p. with 0.6 mg AFB1 and liver nuclei were isolated 2 h later. When the total nuclear free RNA polymerases were extracted and assayed in the absence and presence of alpha-amanitin (3.2 micrograms/ml), we found that only alpha-amanitin-sensitive (i.e., RNA polymerase II) activity was inhibited (97%). DEAE-Sephadex chromatography confirmed this result. When total nuclear free RNA polymerases were incubated with AFB1 activated in vitro under conditions producing 70% inhibition of nucleolar RNA synthesis, no inhibition was observed for either alpha-amanitin-sensitive or -resistant activities. Similar results were obtained with low and high (28 and 167 micrograms/ml) concentrations of AFB1. This was further confirmed using highly purified RNA polymerase II. We conclude that AFB1 inhibition of RNA polymerase II activity in vivo is not a result of direct interaction of AFB1 to the enzyme.