Kei Yamanaka

Binding of copper to albumin and participation of cysteine in vivo and in vitro

Albumin, the major copper-binding protein in blood serum, was shown to form different albumin-copper complexes in in vivo and in vitro. Cupric ions added in vitro to control rat serum bound preferentially to mercaptalbumin and the mercaptalbumin-copper complex remained unchanged with time. Cupric ions injected intravenously into the rat first formed the mercaptalbumin-copper complex; this binary complex changed gradually with time to form an albumin-copper-cysteine complex. The participation of cysteine in the formation of this complex was demonstrated in vitro and further suggested that its conversion was an oxidative reaction. Glutathione also participated in forming the complex, but it was not as effective as cysteine. Albumin-copper complexes were separated on a gel filtration column and detected simultaneously by high-performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry.

[98] d-Arabinose (l-fucose) isomerase from Aerobacter aerogenes

Publisher Summary D-arabinose (L-fucose) isomerase catalyzes principally two reactions: the interconversion of D-arabinose and D-ribulose and of L-fucose and L-fuculose. The chapter describes the assay method, purification procedure, and properties of D-arabinose (L-fucose) isomerase from Aerobacter Aerogenes . For routine assay, the colorimetric method can be employed but the reaction mixture is incubated for 10 min rather than 20 min. The reaction mixture (1.0 ml) contains 0.5 ml of glycine buffer (pH 9.3), 0.05 ml of MnCl 2 , and 0.01–0.20 ml of appropriately diluted enzyme solution. After equilibrium for 5 min at 35°, the reaction is initiated by addition of 0.05 ml of D-arabinose or L-fucose. The mixture is incubated at 35° for 10 min, and the reaction is terminated by addition of 0.05 ml of 50% trichloroacetic acid. Ketopentose is determined by the cysteine-carbazole test. Color is developed for 20 min at 20° for ribulose, or at 35° for fuculose, and read at 540 nm or 550 nm, respectively. The reading is corrected for the blank with heated enzyme or with no enzyme. The purification procedure involves the steps, such as growth of cells, extraction, second PEG precipitation, and crystallization. The crystalline enzyme is stable at 2° as sediment in PEG solution for at least 1 month. Enzyme is stable for more than 1 month in 50 m M Tris HC1 buffer at pH 7.5 containing 1 m M of MnCI, and mercaptoethanol. Crystalline enzyme is active on two pentoses and the apparent Michaelis constants are 51 m M for L-fucose and 160 m M for D-arabinose. Manganese ion is required specifically for both activities. Other properties are also discussed.

Sugar Isomerases:Part I. Production of D-Glucose Isomerase from Heterolactic Acid Bacteria

Enzymatic activity for isomerizing d-glucose to d-fructose was demonstrated in the extracts from the d-xylose-grown cells of heterolactic acid bacteria; the enzyme was called the d-glucose isomeras...

New PQQ-Enzyme: Aromatic Alcohol and Aldehyde Dehydrogenases in Rhodopseudomonas Acidophila M402

Two NAD+-independent, dye-linked aromatic alcohol and aldehyde dehydrogenases of Rhodopseudomonas acidophila M402 were purified. PQQ was demonstrated in both enzymes: the characteristic absorption spectra and activation by PQQ on apo-dehydrogenase preparations. Anaerobically-grown cells do not show the dehydrogenation activity on any alcohols, but the activities were appeared when the bacterium grew on the medium supplemented PQQ under anaerobic conditions. Absorption spectrum of the purified aromatic alcohol dehydrogenase showed absorption maxima at 277, 416, 523 and 552 nm with a shoulder at 290 nm. This indicates the presence of PQQ and also cytochrome(s). Therefore, this enzyme should be classified as quinohemoprotein aromatic alcohol dehydrogenase.

Studies on the Pentose Isomerases of Lactic Acid Bacteria: Part VI. Effect of Metals on Isomerase ProductionPart VII. Chromatography of Pentose IsomerasesPart VIII. Separation of D-Xylose and l-Arabinose Isomerases

Production of d-xylose and l-arabinose isomerases by lactic acid bacteria was greatly promoted by the addition of manganese ions in cultural medium. Effective concentration of the ions was 5 × 1O-3 m. Ferrous ions were also effective for the production of d-xylose isomerase and cobaltous ions were somewhat effective for the production of l-arabinose isomerase. Zinc and cadmium ions inhibited bacterial growth. It was possible to increase the production of isomerase by changing MnSO4 concentration to 5× 10-3 m (0.l1 %) in place of 0.001 per cent in the normal medium.Column chromatographic procedures for the purification of pentose isomerases were carried out. Cation and anion exchange resins were not suitable because of their low exchange capacities and instability of the enzyme at acidic pH range. But the isomerases were successfully purified by DEAE-cellulose column chromatography with high recovery (85~90%). Using a Tris buffer, KCl concentration was increased in gradient. d-Xylose isomerase was eluted a...