Kazuji Ohashi

Change of choline metabolism in rat liver on chronic ethionine-feeding.

On chronic ethionine-feeding (0.1% DL-ethionine in 0.5% sucrose solution) for 2, 4 or 6 months, choline metabolism in rat hepatic cells was altered considerably, although RNA contents were virtually unchanged. Choline dehydrogenase activity in the hepatic mitochondrial fraction was suppressed by about 1/2 or 1/3, compared to its normal level, whereas choline kinase activity, which existed in the cytoplasmic fraction, was elevated more than 1.5-fold. The normal value for choline-metabolizing enzymes in terms of the choline dehydrogenase/choline kinase activity ratio was estimated to be about 70 under the defined conditions, while the average value of the activity ratio drastically changed to about 10-20 on chronic ethionine-feeding. The present results suggest that an alteration of hepatic choline-flux occurred, due both to an increase in choline kinase activity and to a counteractive decrease in choline dehydrogenase activity.

Partial purification and property of pyridoxine (pyridoxamine)-5'-phosphate oxidase isozymes from wheat seedlings.

Abstract Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.

Immobilization of yeast pyridoxamine (pyridoxine) 5'-phosphate oxidase by organomercurial agarose.

Pyridoxamine (pyridoxine) ?-phosphate oxidase (EC 1.4.3.5) catalyzes the formation of pyridoxal 5’-phosphate (PLP) from pyridoxamine 5’-phosphate (PMP) or pyridoxine 5’-phosphate (PNP) [l-4] . The enzyme has been highly purified from rabbit liver [4,5] and one bacterium [3] . Baker’s yeast contains a high amount of this enzyme activity [6] , however, trials for the purification have not been done since Pogell reported the existence of the enzyme in 1958 [l] . Recently, we have reported the results on the partial purification of the enzyme from yeast [6] . In the advances of the purification, we have now found that the enzyme covalently attached to an organomercurial agarose, Aff-Gel501, is active in situ, and the enzyme column can be applicable to not only kinetic studies but also the production of PLP from PMP or PNP in relatively high yield. The preparation and some properties of the immobilized enzyme are described here.

Rat Liver Choline Dehydrogenase

コリン代謝上の問題点を解明する目的で, 酸化分解系の初発酵素, コリン脱水素酵素 (EC 1. 1. 99. 1) の3ヵ月齢ラットにおける臓器分布, 細胞内顆粒局在性, および, その可溶化と可溶化酵素の安定化法を検討した。1) コリン脱水素酵素は肝および腎臓にのみ存在し, ミトコンドリアに結合し, 通常の細胞破砕の条件では容易に漏出しないことを確かめた。また, 両臓器における比活性は同程度であるが, 総活性の80%は肝臓に存在した。2) 可溶化の方法として, 超音波処理後, 2% TritonX-100でかくはん抽出する方法を採用し, 可溶化率約50%の結果を得た。3) 可溶化酵素はTriton X-100を含まない溶液中で容易に失活する。失活を防止する目的で, 総たん白質量に対し1/15のジチオビスニトロ安息香酸 (DTNB) を添加し, 化学修飾した酵素標品は溶液中で安定で, かつ, 通常の酸素電極法で活性測定できることを明らかにした。