Tokuji Shimomura

Purification and Substrate Specificity of Brewer's Yeast α-Glucosidase

Three kinds of α-glucosidase (I, II and III) were isolated from brewer’s yeast, α-Glucosidases I and II were homogeneous in disc electrophoresis, but α-glucosidase III was not. Their pH-optima were found to be in the range of 6.3 to 7.1. Each of them was a typical α-glucosidase showing a preferential activity on phenyl-α-glucoside. α-Glucosidase I was so-called isomaltase which could hydrolyze isomaltose but not maltose. α-Glucosidases II and III showed no activity on isomaltose. The ratio of velocity of hydrolysis for phenyl-α-glucoside (Km, 2.9 mm) and isomaltose (Km, 28.6 mm) of α-glucosidase I was calculated to be 100:9 in that order, and those for phenyl-α-glucoside (Km’s, 0.8 mm and 3.6 mm) and maltose (Km’s, 14.3 mm and 71.4 mm) of α-glucosidases II and III were calculated to be 100: 17 and 100: 18, respectively.

Substrate Specificity and Some Properties of Crystalline Mold Maltase

The substrate specificity of crystalline mold maltase was investigated.The enzyme acts upon various α-heteroglucosides or saccharides. Aryl-α-glucosides were hydrolyzed much faster than alkyl-α-glucosides. The enzyme acts on the maltose derivatives whose reducing groups have been masked. But among glucosylfructoses turanose, maltulose and isomaltulose were attacked with a slow rate while the enzyme was quite inert to sucrose. Malto- and isomalto-oligosaccharides were also hydrolyzed and the enzyme ceased its action at seven to eight units of hexose in both series of oligosaccharides.The opt. pH range of Takamaltase was 4.2~4.6 and opt. temp., 50~55°C. Cu++ and Hg++ strongly inhibited the enzyme activity but other metal ions tested had no effects. It is suggested that the enzyme is not a sulfhydryl enzyme because of the lack of effects of SH-reagents on the activity.

Comparative Biochemical Studies on α-Glucosidases

The properties of brewer’s yeast α-glucosidase have been investigated. The enzyme was capable of hydrolyzing various α-glucosides and was active especially on aryl-α-glucosides in comparison with other α-glucosides and sugars. The rate of hydrolysis decreased in following order: phenyl-α-glucosides, sucrose, matlose and isomaltose.The range of opt. temp, was 40~45°C and opt. pH, 6.5~7.0.Cu++ and Hg++ inhibited strongly the enzyme activity and Zn++, moderately. The enzyme was suggested to be a sulfhydryl enzyme from the inhibition experiments by SH-reagents and the effects of glutathione on the activity.The enzyme synthesized some oligosaccharides from maltose. As the transglucosidation products, nigerose, isomaltose, kojibiose and maltotriose were detected by paperchromatography.Pure nigerose was separated by splitting maltose with amyloglucosidase from the mixture of maltose and nigerose and by use of successive carbon column chromatography.

Comparative Biochemical Studies on α-Glucosidases:Part III. Trangsglucosidation Action of an α-Glucosidase of Schizosaccharomuces pombe

The transglucosidation action of Schizosaccharomyces pombe α-glucosidas e has been investigated by paperchromatography.The enzyme sythesized some oligosaccharides, consisting mainly of isomaltose, together with panose, isomaltotriose, nigerose, kojibiose and maltotriose by transferring glucosyl moiety from maltose. A small amount of isomaltose was also formed even from free glucose.The transglucosidation mechanism of the enzyme and the process of the further converison of the transglucosidation products were examined. The data indicated that the transglucosidation action of Schizosacch. pombe α-glucosidase also was similar to those of mould α-glucosidase.

Biochemical Studies on α-Glucosidase from Buckwheat (Fagopyrum esculentum Mönch): Part II. Purification of Buckwheat α-Glucosidase and Some Properties on Maltose and Soluble Starch

Buckwheat α-glucosidase was purified approximately 300-fold by CM-cellulose treatment, calcium phosphate gel treatment, fractionation with ammonium sulfate, chromatography on a column of calcium phosphate gel and CM-Sephadex treatment. The purified α-glucosidase showed one peak ultracentrifugally but was heterogeneous in moving-boundary electrophoresis.The enzyme hydrolyzed not only maltose but also soluble starch, and glucose was the sole product from both substrates. At a lower substrate concentration (0.5%), the rate of the initial reaction with maltose was about 3 times higher than that with soluble starch. At higher substrate concentrations (2,5% and 5%) the enzyme showed a trans-glucosylase action toward both substrates.The following properties were perfectly identical on maltose and soluble starch: optimum pH 5.0; optimum temperature, ca. 52°C; range of pH stability, 4~7.3; temperature of stability, 45°C for 10 min; enzymatic activity on each substrate, almost lost at 60°C for 30 min.

α, β Elimination of β-Chloro-l-alanme and Syncatalytic Inactivation of α-Aminoisobutyrate Decomposing Enzyme

β-Chloro-l-alanine was catalytically converted to pyruvate, ammonia and chloride by α-aminoisobutyrate (AIB) decomposing enzyme (α, β elimination), which was synchronously inactivated. There was a linear relationship between α, β elimination and inactivation. With apoenzyme, neither α, β elimination nor inactivation occurred. These facts suggest that α, β elimination is dependent on pyridoxal 5′-phosphate, and inactivation cooperates with α, β elimination (syncatalytic inactivation). But it seemed that d-form of β-chloroalanine was not a substrate for AIB decomposing enzyme, because just half amount of β-chloro-dl-alanine was decomposed to pyruvate by the enzyme.An identical active site for each of following three reactions were shown by the fact that AIB decomposing activity, transamination activity and α, β elimination activity were lost in parallel. From a kinetic study, the affinity of the enzyme toward β-chloro-l-alanine was shown to be higher than that toward AIB or l-alanine. The turnover number, a...

Studies on Enzymatic Synthesis of Oligosaccharides:Part II. α-D-Glucopyranosyl-L-sorboses Synthesized by the Transglucosidation Reaction of Brewer's Yeast α-Glucosidase

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.

The Inhibitory Effect of 3-Nitropropionate on the Oxidative Phosphorylation in Rat Mitochondria

The effect of 3-nitropropionate (3-NPA)on oxidative phosphorylation by using mitochondria prepared from both rat liver and brain were investigated in connection with the toxicity of this material. It was found that 3-NPA inhibited oxidative phosphorylation. In this inhibition, the uptake of inorganic phosphate was blocked but the oxygen uptake was not influenced at all. Furthermore, increase in ATPase activity of intact mitochondria was shown by the addition of 3-NPA. Results showed that 3-NPA disturbed oxidative phosphorylation as an uncoupler. However, the degree of inhibition by 3-NPA was not so high in comparison with other well-known uncouplers.Thus the toxicity of 3-NPA is not due to the inhibition of oxidative phosphorylation. 3-NPA also does not affect on cytochrome oxidase activity.

Studies on Organic Acid Metabolism by Rhizobium japonicum. Part I

17種類の有機酸について生育試験,メチレンブラウの脱色時間,酸素消費量を測定した結果, 17種類とも酸素を消費したが,一般に低級脂肪酸は酸素消費量が少く,且つ生育の為に有効なエネルギー源とはならない.これと反対にグルコースは生育に有効な炭素源となるが,酸素の消費量は極端に少なかった.これはグルコースの細胞膜への透過性及びグルコースに対する菌の適応性の問題と関連した現象と考える. TCAサイクル中間体は酢酸を除けば全て生育に利用され,且つ酸素消費量も大であった.これらのことより大豆根瘤菌中には一応TCAサイクル系の存在が推定されるけれど,グリオキシリックサイクル系の存在は考えられない. 砕ケトグルタール酸の酸化反応を触媒する酵素系を調製し,これを使用して行った2, 3の実験結果から動物或は他の微生物について報告された同酵素系と同様の性質を示すことを知った.

Biochemical Studies of Piricularia oryzae: Part V. Behavior towards dl-Norleucine and dl-AlaninePart VI. Behavior towards dl-α-Aminobutyric Acid and dl-Isoleucine: Formation of α-keto-β-Methylvaleric Acid from either Amino Acid

In the current experiment, the conversion of dl-norleucine and dl-alanine to their respective α-keto analogue by Piricularia oryzae was confirmed by the separation of 2,4-dinitrophenylhydrazone of either keto acid. Although the formation of keto acid from norleucine was distinctly recognized both in the presence and the absence of arsenite, the accumulation of pyruvic acid from alanine was only found in the presence of arsenite, as it has been observed with some microorganisms. In addition, the preferential utilization of alanine to a proper carbon source was identified, which may provide a step towards the investigation of the nutritional relationship between the blast fungus and the host plant.