Haruhito Tsuge

Electron spin resonance spectroscopic evaluation of scavenging activity of tea catechins on superoxide radicals generated by a phenazine methosulfate and NADH system

Abstract The scavenging effects of tea catechins on superoxide radicals (·O2−) generated non-enzymatically by a phenazine methosulfate (PMS) and reduced β-nicotinamide adenine dinucleotide (NADH) system, was studied using an electron spin resonance (ESR) spectrometer along with a spin-trapping agent, 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO). The presence of 3′, 4′, 5′-trihydroxyl groups attached to the B-ring of the flavan skeleton enhanced the radical scavenging efficiency displayed by the catechin family in comparison to those with 3′, 4′-dihydroxyl groups, and the insertion of a galloyl moiety into three positions of the C-ring exerted a synergistic impact on ·O2− scavenging activity. Catechin constituents accounted for 86% of the total ·O2− scavenging capacity of green tea extract, with a particularly high contribution ascribed to (−)-epigallocatechin gallate [(−)-EGCG] (48%) and (−)-epigallocatechin [(−)-EGC] (26%).

Theanine, γ-glutamylethylamide, is metabolized by renal phosphate-independent glutaminase

Abstract The distribution of theanine-degrading activity in Wistar rats was examined and this activity was detected only in the kidney. Judging from polyacrylamide gel electrophoresis, theanine-degrading enzyme from rat kidney was purified almost to homogeneity. Theanine-degrading activity was co-purified with glutaminase activity, and the relative activity for theanine was about 85% of that for l -glutamine throughout purification. Substrate specificity of purified enzyme preparation coincided well with the data of phosphate-independent glutaminase [EC 3.5.1.2], which had been previously reported. It was very curious that γ-glutamyl methyl and ethyl esters were more effectively hydrolyzed than theanine and l -glutamine, in view of relative activity and K m value. It was suggested that γ-glutamyl moiety in theanine molecule was transferred to form γ-glutamylglycylglycine with relative ease in the presence of glycylglycine. On the other hand, purified phosphate-dependent glutaminase did not show theanine-degrading activity at all. Thus, it was concluded that theanine was hydrolyzed by phosphate-independent glutaminase in kidney and suggested that, as for the metabolic fate of theanine, its glutamyl moiety might be transferred by means of γ-glutamyl transpeptidase reaction to other peptides in vivo.

Effects of Vitamin B-6 on (n-3) Polyunsaturated Fatty Acid Metabolism

To investigate interactions between vitamin B-6 and fatty acid metabolism, male Wistar rats were fed a vitamin B-6 (B-6)-deficient diet consisting of 70% vitamin-free casein and 10% perilla oil [approximately 63% alpha-linolenic acid, (n-3)] for 5 wk. The amounts of linoleic acid (n-6) and arachidonic acid (n-6) in the B-6-deficient group changed only slightly compared with those in a pair-fed control group. The amount of linoleic acid increased and arachidonic acid decreased in the plasma total lipid fraction, and the ratios of both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the B-6-deficient group were significantly lower than for the controls. The ratios of alpha-linolenic acid and EPA were higher, and DHA lower, in the B-6-deficient group than in the pair-fed control group in the total lipid as well as phospholipid fractions in liver microsomes. The activity of delta6-desaturase was significantly lower in the B-6-deficient group than in the pair-fed control group (approximately 64%), and acyl-CoA oxidase activity, an initial enzyme of the peroxisomal beta-oxidation pathway, was reduced by approximately 80% in the B-6-deficient group. These data suggest that B-6 deficiencies impair the metabolism of (n-3) PUFA from alpha-linolenic acid to EPA and DHA with the most pronounced reduction in the production of DHA.

Modulation of albumin gene expression by amino acid supply in rat liver is mediated through intracellular concentration of pyridoxal 5′-phosphate

Abstract Rats were nourished by infusion of total parenteral nutrition solutions containing 0% or 3.3% amino acids for 7 days. The level of albumin mRNA in the liver of amino acid-infused rats was found to be several fold higher than that in the liver of amino acid-depleted rats. Expression of albumin gene is known to be regulated by tissue-specific transcription factors such as HNF-1 and C EBP . We determined the binding activities of liver nuclear extracts to the HNF-1- and C EBP -binding sites by gel mobility-shift assay and found that the activities of the extract prepared from liver of amino acid-infused rats were greater than those of amino acid-depleted rats. In view of our recent finding that vitamin B6 modulates albumin gene expression through inactivation of tissue-specific transcription factors by direct interaction with pyridoxal phosphate, we determined the intracellular concentrations of vitamin B6 derivatives. We found that the concentration of pyridoxal phosphate in the liver of amino acid-infused rats was decreased to almost half of that of amino acid-depleted rats, whereas the concentration of pyridoxamine phosphate was increased in the opposite direction. These observations suggest that an increase in albumin mRNA level in the liver of amino acid-infused rats may be caused by a decrease in intracellular concentration of pyridoxal phosphate, which in turn relieves inactivation of tissue-specific transcription factors.

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.

[49] Activity staining for flavoprotein oxidases

Publisher Summary This chapter describes the activity staining of flavoprotein oxidases. The determination of the products and the enzyme activities, by staining, after electrophoresis has been successfully adopted as an analytical tool for enzymes or isoenzymes. Activity staining, in combination with usual protein staining, provides for the assessment of change of an enzyme activity as well as a criterion for homogeneity during purification. One can distinguish whether the activity comes from true isozyme(s) or artifact(s), and monitors the tendency toward dissociation or association of enzyme protein. Among the methods used to stain the oxidase activity in a gel, using appropriate substrate, the peroxidase- o -dianisidine method is especially suitable, because H 2 O 2 is generated from molecular oxygen that is the direct hydrogen acceptor from reduced flavin. The method is based on the formation of a red-colored dye, oxidized o -dianisidine, which is produced by peroxidase, acting on o -dianisidine in the presence of H 2 O 2 . Any enzyme producing H 2 O 2 and peroxidase per se could be stained, with a similar method, in which appropriate substrate and buffer solution are used. The great advantage of this method is the stability and the fact that the staining process is not affected by light. Furthermore, within the pH range between 5 and 8.5, oxidized o -dianisidine remains insoluble within the polyacrylamide gel matrix.

Popping Mechanism of Popcorn Grain

PC粒の形態,主成分である澱粉の性質,および大気条件下で, 160～200℃に加熱した時の膨化機構等,以下の事項についてDC, WCと比較検討した. i)コーン粒のいくっかの種類より単離した澱粉粒子の比較, ii)粒構造の特性, iii)加熱中の膨化における粒の走査電子顕微鏡による観察. (1) PCより単離した澱粉粒子の性質は,アミロース含量が, DCのものより高いものの(約3%),糊化特性や糊化温度において, DCと類似した値を示した. PC種の特徴はむしろ穀粒の特異な構造にあると考えられた. (2) 膨化中のPC粒の走査電子顕微鏡による観察から,角質胚乳における澱粉は, 160℃以上で糊化し,空気の膨張によるはちの巣状の構造をつくることが明らかとなった.膨化した生成物において,膜のような薄いフィルムは,糊化,焼結した澱粉によりっくられた. (3) 膨化するコーン粒において,澱粉粒子は130～170℃の範囲で糊化し,糊化の程度は65～75%であった,PCの調製において,本質的に重要なことは,加熱中に内部蒸気圧に十分耐えることを可能とする外皮の性質にある.

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。