Nobuko Ohishi

Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

The reaction of lipid peroxides in animal tissues with thiobarbituric acid was dependent on pH of the reaction mixture as was the case for linoleic acid hydroperoxide. The optimum pH was found to be 3.5. Taking this fact into consideration, a standard procedure for the assay of lipid peroxide level in animal tissues by their reaction with thiobarbituric acid was developed as follows. Ten percent (wv tissue homogenate was mixed with sodium dodecyl sulfate, acetate buffer (pH 3.5), and aqueous solution of thiobarbituric acid. After heating at 95°C for 60 min, the red pigment produced was extracted with n-butanol-pyridine mixture and estimated by the absorbance at 532nm. As an external standard, tetramethoxy-propane was used, and lipid peroxide level was expressed in terms of nmol malondialdehyde. Using this method, the liped peroxide level in the liver of rats suffering from carbon tetrachloride intoxication was investigated. The results were in good agreement with previously reported data obtained by measuring diene content.

Lipid peroxide level in plasma of diabetic patients.

Abstract Recently, one of the authors (1) devised a reliable method for the microdetermination of lipid peroxide in plasma. This has enabled us to study the plasma lipid peroxide level of patients suffering from diabetes. This paper deals with the data obtained with a large sample of patients, and the relation between the lipid peroxide level and the different features of the disease.

Proteins with covalently-bound flavin in rat liver mitochondria.

Abstract By SDS-polyacrylamide gel electrophoresis, mitochondrial proteins having covalently-bound flavin were analyzed. Mitochondria were prepared from the liver of rat injected with radioactive riboflavin. Radioactivity was found to be associated with four protein components. Their subunit molecular weights were 91,000, 72,000, 60,000 and 44,000. The first two components exhibited yellowish fluorescence on a gel under ultraviolet illumination. The component of the highest molecular weight seems to be a new protein containing covalently-bound flavin.

lnfrared spectra and molecular association of lumiflavin and riboflavin derivatives

Abstract Infrared spectra of lumiflavin (Lf), [3-ND]Lf, [3-NMe]Lf, [2- 13 C]Lf, [4a- 13 C]Lf, [1,3- 15 N]Lf, [1,3,5- 15 N]Lf, riboflavin (Rf), [3-NMe]Rf, riboflavin-2′,3′,4′,5′-tetraacetate (RfT) and [3-NMe]RfT were measured in their solid and solution states. The vibrational assignments of lumiflavin have been made on the basis of the observed isotope frequency shifts in the i.r. spectra. The differences between the skeletal vibrations of Lf and those of Rf are discussed. The modes of molecular association of Lf and Rf are also proposed based on the i.r. spectra of the carbonyl stretching region.

Covalently bound flavin as prosthetic group of choline oxidase.

Abstract Highly purified choline oxidase of Arthrobacter globiformis fluoresced as a yellow band on SDS gel in 7% acetic acid. The absorption spectrum of the enzyme showed marked hypsochromic shift of the second absorption band. Aminoacyl flavin obtained from this enzyme was identified with 8α-[N(3)-histidyl]FAD.

Kinetic isotope effect on the reaction of D-Amino-acid oxidase

Upon anaerobic mixing of D-alanine and D-aminoacid oxidase (EC 1.4.3.3), by rapid reaction technique, transitory appearance of long-wavelength absorbing intermediate was observed [ 1, 21, which was found to be a single species and was identified spectrophotometrically with the purple complex observed in an equilibrium state [2]. The absorbance at 550 nm increases rapidly and subsequently decreases slowly, indicating the rapid formation of the purple intermediate and its slow conversion to the fully reduced enzyme. The reaction sequence in anaerobic reduction of the enzyme with the substrate is expressed as follows [l, 21:

Release of α-imino acid as primary product in d-amino-acid oxidase reaction

Abstract Due to the formation of oxidation products from noncyclic amino acids, such as d -alanine and d -leucine, on catalysis with d -amino-acid oxidase ( d -amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) in slightly alkaline solution the pH of the solution decreased and subsequently increased towards the initial level. In the case of cyclic amino acids such as d -proline, on the other hand, only a decrease in pH was observed. These results canbe interpreted to mean that an imino acid, of which the imine group is unprotonated, is released as the primary oxidation product from the enzyme before its hydrolysis to keto acid and NH4+.

Solvent effect on fluorescence of fat-soluble riboflavin derivatives

Abstract In aqueous solution, riboflavin 2′,3′,4′,5′-tetranicotinate showed a fluorescence spectrum having a peak around 520 mμ, which is similar to that of riboflavin. In solvents of low polarity, however, the fluorescence was intensified with the appearance of fine structure, and the emission peak shifted to the blue. Using dioxane-water system, a linear relation was found between the polarity of the solvent and the fluorescence intensity. Similar effect was also found when the fluorescence spectra of riboflavin 2′,3′,4′,5′-tetrabutyrate were measured in the various media of different polarities. On the basis of these findings, binding aspect of d -amino-acid oxidase with benzoate was interpreted.

Plasma flavin levels of patients receiving long-term hemodialysis

Abstract When hemodialysis was monitored by blood urea nitrogen (BUN) or serum creatinine, the total amounts of flavins in plasma of patients were elevated. This means that the amount of flavins removed through the dialyser was less than that absorbed through the intestine. It was also noticed that the dialysance of flavins was lower than that of urea or creatinine. Accordingly, dietary flavins appear to be enough to replace losses; further supplement of riboflavin is not necessary.

Identification of a covalently bound flavoprotein in rat liver mitochondria with sarcosine dehydrogenase

Abstract A covalently bound flavoprotein having highest molecular weight among four covalently bound flavoproteins found in rat liver mitochondria was partially purified and characterized. Its subunit molecular weight was estimated to be 94,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its absorption maxima were observed at 353 and 460 nm. Since this flavoprotein was reduced by either sarcosine or dimethylglycine and oxidized by phenazine methosulfate, it was identified with sarcosine dehydrogenase.