Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases

Resonance Raman scattering from cow milk lactoperoxidase (LPO) and its complexes with various electron donors and inhibitors was investigated. The Raman spectrum of LPO is strikingly close to that of hog intestinal peroxidase but distinctly dissimilar to that of horseradish peroxidase (HRP). The v10 frequency suggested the six-coordinate high-spin structure of heme for native LPO in contrast with the five-coordinate high-spin structure for HRP. For the v10 band, benzohydroxamic acid caused a frequency shift with HRP but not with LPO. Guaiacol, o-toluidine, and histidine brought about a frequency shift of the v4 mode for LPO but not for HRP. The frequency shift was restored upon removal of the substrate or inhibitor by dialysis. The down shift of the v4 frequency is considered to represent an appreciable donation of electrons from the substrate or inhibitor to the porphyrin LUMO and thus their direct interaction with the heme group. From the relative intensity of the shifted and unshifted v4 lines, the dissociation constant was determined to be Kd = 52 mM for guaiacol and Kd = 87 mM for histidine at pH 7.4. The binding of histidine was relatively retarded in the presence of sulfate anion (Kd = 150 mM for 0.53 M sulfate present), and imidazole alone yielded no frequency shift, indicating the binding of the carboxyl group of histidine to the protein cationic site on one hand and a weak charge-transfer interaction between the imidazole group and the heme group on the other.     

Glutathione depletion by 2-nitroimidazole-1-acetohydroxamic acid as a radiosensitizer in hypoxic rat hepatocytes

The effects of nitroimidazoles as radiosensitizers on intracellular glutathione (GSH) level were investigated in rat isolated hepatocytes. Dinitroimidazoles have lowered almost completely GSH level during the incubation for 30 min under oxic (95% O2+5% CO2) condition, while mononitroimidazoles had scarcely affected. In the case of hypoxic (95% N2+5% CO2) condition, however, 2-nitroimidazoles, not 4-nitroimidazoles, as well as 2,4- and 4,5-dinitroimidazoles have caused the significant depletion of GSH. This suggests that nitro group in the 2-position of imidazoles may be responsible for the GSH depletion under hypoxia. Especially, 2-nitroimidazole-1-acetohydroxamic acid (KIH-801) was found to be the most potent GSH depletor only under hypoxic, not oxic conditions, and might be useful for the new hypoxic cell radiosensitizer instead of misonidazole.     

Purification and some properties of NADPH-cytochrome P-450 reductase from crab-eating monkey liver microsomes

NADPH-cytochrome P-450 reductase was purified to 30.8 units/mg from monkey liver microsomes. The purified reductase showed one major protein band (78,000) and two minor ones (58,000 and 20,000) on analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Monkey, rat, and guinea pig reductases were not immunochemically identical to each other judged from Ouchterlony double diffusion analysis and immunotitration with regard to NADPH-cytochrome c reductase activity.     

Hormonal regulation of carbamoyl-phosphate synthetase I synthesis in primary cultured hepatocytes and Reuber hepatoma H-35. Defective regulation in hepatoma cells

Regulation of carbamoyl-phosphate synthetase I (CPS) synthesis by various hormones was compared in primary cultured hepatocytes from adult rat and in Reuber hepatoma H-35 by pulse labeling of the cells with [35S]methionine. CPS synthesis in hepatocytes was stimulated 8-fold and 5-fold by dexamethasone and glucagon respectively. CPS synthesis in hepatocytes was synergically (about 50-fold) stimulated by a combination of dexamethasone and glucagon. Less synergic stimulation was observed by combining dexamethasone with N6, O2'-dibutyryladenosine 3',5'-monophosphate (dibutyryl-cAMP) or with isoproterenol. The basal level of CPS synthesis in hepatoma cells was higher than that in hepatocytes. CPS synthesis in hepatoma cells was stimulated by dexamethasone and dibutyryl-cAMP but the extent was only 3-fold and 1.8-fold respectively. The synergic effect of combination of dexamethasone and dibutyryl-cAMP was not observed in hepatoma cells. Neither glucagon nor isoproterenol exhibited an appreciable effect on CPS synthesis in hepatoma cells. Insulin and epinephrine suppressed CPS synthesis both in hepatocytes and hepatoma cells. The effect of epinephrine was indicated to be through alpha-adrenergic receptors. The effects of insulin and epinephrine were additive on CPS synthesis both in hepatocytes and hepatoma cells.     

Expression of the insulinoma gene rig during liver regeneration and in primary cultured hepatocytes

We have isolated a novel gene, rig (rat insulinoma gene) from rat insulinomas. In the present study, rig was found to be expressed in rat regenerating liver and in primary cultured rat hepatocytes. The level of rig mRNA was increased at the proliferative phase of liver regeneration. In synchronously cultured hepatocytes, the rig mRNA level was elevated at the G1 phase of the cell cycle and the rig-protein was accumulated in the nuclei during the S phase. These results indicated that rig could be involved in a more general way in growth or cell replication.     

Circulating interleukin 6 as a useful marker for predicting postoperative complications.

We examined postoperative serial changes in the levels of serum interleukin 6 (IL-6), serum acute phase reactants (APRs) and plasma neutrophil elastase (NE) in patients with various cancers and reviewed these changes in patients who did, and did not, show postoperative complications. Serum IL-6 level was elevated after surgery, peaking on the first postoperative day. Elevation of serum APRs and plasma NE levels also followed. There was a significant correlation between the serum peak level of IL-6 and those of APRs and NE (P less than 0.01). Moreover, there was a significant difference in the serum IL-6 level in patients with and without complications. The relationship between the serum IL-6 greater than 400 pg/ml and the incidence of postoperative complications was also marked. These results suggest that circulating IL-6 is a clinically useful marker for the earliest detection and prediction of postoperative complications.     

Aqueous solubility and acidity constants of cholic, deoxycholic, chenodeoxycholic, and ursodeoxycholic acids.

Cholic acid, deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid were purified by a foam fractionation method. Using thermogravimetric analysis, the attached water molecule was found to be completely removed from solids of the latter three at 100 degrees C, while cholic acid still had one water molecule of crystallization per two cholic acid molecules at that temperature. The acidity constants of the acids were accurately determined from aqueous solubility measurements at different pH values and at 15, 25, 35, and 45 degrees C. The enthalpy change of dissolution from temperature dependence of solubility as undissociated acid monomer was much less than those of common ionic surfactants. This results from a smaller entropy increase on dissolution due to the hardly flexible hydrophobic structure of these bile acids.