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In a previous paper it has been described that α-amylase formation in Aspergillus oryzae is stimulated by soluble starch, glycogen and maltose, whereas it is inhibited by glucose, which is added into a growing medium or a secondary incubation medium as the carbon source. The present paper reports that isomaltose and panose are the most effective inducers among a large number of sugars examined here, and suggests the importance of transglucosidase action demonstrated in view of α-amylase formation. The initial action of inducers in this system is also discussed.

α-Amylase in Aspergillus oryzae is generally known to be present in mycelium as well as in medium, whether it is grown with or without shaking. The interesting fact is reported in this paper, when A. oryzae was grown in a phosphate deficient medium, however, α-amylase accumulation occurred only within the cell. From the results of pH-activity curve, starch gel zone electrophoresis and differential centrifugation, there seemed to be no distinctions between exo- and endocellular α-amylase. The liberation of α-amylase from the cell was observed to be dependent on pH and the concentrations of anions. The significance of the α-amylase accumulation and liberation in this system is discussed.

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl2 and AgNO3 caused complete inhibition of the enzyme activity at 2 × 10‒5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.

(1) 麹菌のα-アミラーゼ生成過程に於ける,菌体内遊離アミノ酸の検出を行なった. (2) α-アミラーゼの生成はエチオニン,グルタミン酸-γ-ヒドラチド及びノルロイシンにより阻害されたが,それぞれのホモローグアミノ酸により阻害は回復された. (3) 菌体内のグルタミン酸を,アイソトープ稀釈分析法で定量した結果,洗滌菌体によるα-アミラーゼの生成に於いては, lag phaseに於いてグルタミン酸量は増加し,α-アミラーゼの生成に伴って見かけ上減少したが,エチオニンを添加した時は初めから増加した. (4) Ageの古い菌体ではエチオニン阻害が少なく,又inducerの効果も少いことがわかった. (5) エチオニン添加により,核酸区分へのP32のincorporationが小さくなることを認めた. (6) 以上の結果から,少くとも若い菌体によるα-アミラーゼの生成には,遊離アミノ酸が何等かのかたちで関係しているように推定された.しかしながら,古いageの菌体の場合は,これらの関係に尚不明の点が残されている.

(1) 酵母のRNAとspermのDNAとのAsp. oryzae 557号菌によるα-アミラーゼの生成に対する影響を検討した結果,その有効性がプリン ピリ 塩基にあることが判明した. (2) 2, 6-diaminopurineや2-thiouracilの如きプリン・ピリミジン塩基のアナローグといわれる物質が,本麹菌の場合にはα-アミラーゼの生成に阻害的に作用しなかつた. (3) 酵母のRNAの加水分解物を添加すると,麹菌のRNAが増加することがわかつたので,更に核酸代謝に関係の深い物質を添加して, RNAが増加する条件を求めた. (4) 菌体の核酸含量を高めるとα-アミラーゼ生成力も高くなり, α-アミラーゼの生成に核酸が関係しているように考えられる間接的なデータが得られた. (5) 菌体のageによる核酸含量の変化を観察し, α-アミラーゼ生成能との関係を考察した.

(1)土壌から分離したPen. acueatum var. apiculatum No. 2282の生産する粘質物の沈降定数,極限粘度,偏比容などを測定し, Mandelkern式から分子量を求め,分子量約200万の値をえた. (2) この粘質物およびそれからえられた多糖類をそれぞれhigher molecular weight luteic acidおよびhigher molecular weight luteoseと呼ぶことを提案した.

It was previously reported that α-amylase accumulation is caused within the mycelium grown in a phosphate deficient medium and the concentration of anions or pH in a surrounding medium is responsible for its liberation. As it was subsequently found that α-amylase liberation from the mycelium of Aspergillus oryzae is stimulated by peptone, an attempt was made on purification of effective substances from it. The present paper describes on purification and properties of phosphopeptides found as an effective substance for α-amylase liberation, and discusses on the stimulation effect, comparing with the effects on pH and concentration of anions which were previously observed.

新しく分離した菌株S-48のD-マンノースイソメラーゼの酵素的性質について調べた結果は,次のようである. (1) 最適pHは7.8に,また最適温度は40°付近に認められた. (2) 熱には弱く50°, 5分間の加熱で90%以上失活するが,この熱失活はCaCl2, NaClの存在によりかなり保護される. (3) 本酵素反応は可逆反応であり,平衡はD-マンノースに作用させたとき約75%前後D-フラクトースに異性化され,またD-フラクトースに作用させたときには約25%前後のD-マンノースが生成した. (4) 本酵素の基質について調べた結果は, D-マンノースのみに作用し, D-グルコース, D-ガラクトース, D-キシロース, L-アラビノースからはいずれも相当するケトースは生成しなかった.またD-ソルビトール, D-マンニトールからもD-フラクトースの生成は見られなかった. (5) 阻害剤について試験した結果, p-クロロマーキュリベンゾエートにより強く阻害され,またモノヨード酢酸,アザイドによっても阻害された. p-クロロマーキュリベンゾーエートによる阻害は,システインやグルタチオンの添加により回復するが,システインやグルタチオンを単独に添加したときには賦活効果は見られなかった. (6) 金属イオン添加の効果について試験した結果,いずれも効果なく, Cu++, Zn++, Ni++などにより強く阻害され, Mg++, Mn++, Co++,によってもわずかに阻害された. (7) 金属キレート剤の効果について試験した結果,いずれも阻害効果なく,また賦活効果もないので本酵素反応には金属イオンを要求しないものと推定される. (8) 本酵素は培地炭素源としてD-マンノース, D-グルコースによって強く酵素が生成されたが,このほかD-ガラクトース, D-フラクトース, D-マンニトール, D-キシロース,蔗糖,澱粉などによっても生成された. (9) S-48菌の培養経過と酵素活性について試験した結果,単位菌体中のD-マンノースイソメラーゼ含有量は対数生育期に比較的多く存在した.

In the previous paper, it was reported that the higher molecular weight luteose which consisted of β-1,6-linked d-glucose units was made by Pen. aculeatutn var. apiculatum. The isolation of the microorganism which is able to produce β-d-1,6-glucanase and some properties of this glucanase were described in this paper. The optimum reaction temperature for this enzyme was at about 40°C, and the optimum pH was at about 6.7. At the maximum degree of the hydrolysis, only d-glucose and gentiobiose were detected, and at this point, the hydrolysis degree of higher molecular weight luteose was about 55% as d-glucose. The formation of β-d-1,6-glucanase was induced by the addition of higher molecular weight luteose. From the property of this enzyme, the authors proposed the name “lutease” for this enzyme.

(1) Bacillus megaterium A-1のグルコース異性化酵素についてその酵素的性質を調べた. (2) グルコース異性化酵素の最適pHは7.7附近に,また最適温度は35°附近に認められた. (3) 熱に対してはかなり不安定で, 50°, 2分間加熱することにより残存活性は約28%, 70°, 2分間加熱すると活性は殆んど認められない. (4) この酵素の基質特異性について試験した結果,各種のペントース,ヘキソース,糖アルコールからはいずれも相当するケトースの生成は認められず,グルコースのみに作用することがわかった. (5) SH試薬として,グルタチオン,システイン,シスチンにつきその効果を調べたが,いずれも添加の効果もなく,また阻害作用も殆んど認められなかった. (6) 各種試験した阻害剤のうち,砒酸,亜砒酸,アザイド,ヒドロキシルアミン,ヒドラジン, p-ニトロフェノール, KCN,尿素,チオ尿素等により阻害作用を認めた. p-クロロマーキュリベンゾェート,モノヨード酢酸, 2, 4-ジニトロフェノール,フッ化ソーダ等による阻害は認められなかった. (7) 各種金属イオンの効果について試験した結果, Fe++, Fe+++, Hg++により強く阻害をうけ, Mg++, Mn++,Co++によってもわずかに阻害をうけた. (8) キレート試薬および金属結合剤について試験を行なった結果,活性の低下は認められなかった.

An enzyme, which catalyzes the isomerization of D-glucose to D-fructose, has been found in a newly isolated bacterium which tentatively identified as Pacacolobacterum aerogenoides. The enzyme converts not only D-glucose but also D-mannose to D-fructose, and NAD and Mg++ are required as cofactor for this isomerization. The properties of this enzyme were sum-marized as follows: (1) As a cofactor for the isomerization by this enzyme, NAD was absolutely necessary, whereas NADP, FMN and FAD were not. (2) The optimum pH was found to be at 7.5 and optinum temperature was at about 40°C. (3) The enzyme activity was markedly reduced by EDTA treatment and the reduced activity by EDTA was restored by the addition of Mg++, Mn++ or Co++. (4) The enzyme activity was strongly inhibited by monoiodoacetate, p-chloromercuribenzoate, and Cu++, however, the activity was recovered by adding cysteine or glutathione.

It has been found that exocellular α-amylase could be fixed to mycelia of Aspergillus oryzae at an acidic pH region and fixed α-amylase was released reversely at an alkaline pH region. The fixation has more remarkably been observed in mycelia obtained from a phosphate deficient medium where endocellular accumulation of a-amylase occurs more easily than in an ordinary mycelia which secrete a large amount of α-amylase into medium. Bound form of α-amylase was more resistant to low pH and less active than the free form. The results appear to support the previous suggestion that a large quantity of endocellular α-amylase might be located on mycelial surface of the mold.

A newly isolated bacterium, strain S-48, having mannose isomerase which converts d-mannose to d-fructose was identified as Xanthomonas rubrilineans. From the results of the investigation on the isomerase activities of bacteria belonging to Xanthomonas examined, it was found that the isomerase was found in all species of Xanthomonas examined. The isomerase was produced not only when the bacteria were grown on mannose, but also when on glucose, fructose, galactose, xylose, sucrose or starch, and even on bouillon medium without carbohydrate. Therefore, the formation of mannose isomerase of these bacteria seemed to be constitutive.

A newly isolated bacterium, strain S-48, having mannose isomerase which converts d-mannose to d-fructose was identified as Xanthomonas rubrilineans. From the results of the investigation on the isomerase activities of bacteria belonging to Xanthomonas examined, it was found that the isomerase was found in all species of Xanthomonas examined. The isomerase was produced not only when the bacteria were grown on mannose, but also when on glucose, fructose, galactose, xylose, sucrose or starch, and even on bouillon medium without carbohydrate. Therefore, the formation of mannose isomerase of these bacteria seemed to be constitutive.

Monofluoroacetate (FA) is well known as a toxic substance and has been used as an insecticide: C-F linkage of which is very stable. Bacteria, capable of being grown in a defined salt medium containing FA as a sole source of carbon, have been isolated from soil. It has been found that the defluorination of FA involved an enzyme which catalyzes the conversion of FA to glycolate and fluoride ions and that the enzyme was induced when bacteria were grown in a defined salt medium containing FA as a sole source of carbon. The activity of defluorination was fairly stable and strong in this organism.

A Mn2+-dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C′ subunit and a 63 kDa regulatory A′ subunit, was purified from human erythrocyte cytosol. C′ and A′ produced V8- and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+-independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C′A and CA′ revealed that the metal dependency resided in C′ and not in A′. In CA, 0.87±0.12 mol zinc and 0.35±0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C′A′. Pre-incubation of C′ with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+-independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+- and Fe2+-metalloenzyme and that C′ is the apoenzyme.

(1) グルコース異性化酵素はグルゴースの酸化物であるグルコン酸, 2-ケトグルコン酸により阻害されるのを認めた.しかし5-ケトグルコン酸による阻害は認められなかった.グルコン酸による阻害度は5×10-3Mで約50%である. (2) Bacillus megaterium A-1のグルコース脱水素酵素について試験を行い, DPN, FMNにlinkする脱水素酵素の存在を認めた. (3) グルコース脱水素酵素はMg++を要求する. PCMB,ヒドラジン, Zn++, Cu++, Hg++, Co++, Ni++等により強く阻害される.砒酸ソーダ,亜砒酸ソーダ,シアン化カリ,ナトリウムアザイドによる阻害は認められなかった. (4) 砒酸ソーダあるいは亜砒酸ソーダのグルコース異性化酵素に対する阻害性とグルコース脱水素酵素に対する非阻害性の性質を利用し,グルコース異性化酵素反応(グルコースーフラクトース)は可逆性であることを認めた. (5) Glucostatを用いPCMBの存在下でフラクトースから生成するグルコースを定量した.

Human B cell stimulatory factor 2 (BSF-2) was originally characterized and isolated as a T cell-derived factor that caused the terminal maturation of activated B cells to immunoglobulin-producing cells. Molecular cloning of the complementary DNA predicts that BSF-2 is a protein of relative molecular mass (Mr) 26,000 similar or identical to interferon beta 2, hybridoma plasmacytoma growth factor and hepatocyte stimulating factor. IL-6 has been proposed as a name for this molecule. It is now known that BSF-2 has a wide variety of biological functions and that its target cells are not restricted to normal B cells. Responses are also seen in T cells, plasmacytomas, hepatocytes, haematopoietic stem cells, fibroblasts and rat phoeochromocytoma, PC12 (Satoh, T. et al., manuscript in preparation). Of particular interest to this report is that human BSF-2 is a potent growth factor for murine plasmacytomas and hybridomas. This observation suggested to us that constitutive expression of BSF-2 or its receptor could be responsible for the generation of human myelomas. In this study we report that myeloma cells freshly isolated from patients produce BSF-2 and express its receptors. Moreover, anti-BSF-2 antibody inhibits the in vitro growth of myeloma cells. This is direct evidence that an autocrine loop is operating in oncogenesis of human myelomas.

The content of peripheral blood B cells (B1+) was reduced in patients of multiple myeloma (MM) and not in those with benign monoclonal gammopathy (BMG) compared to normal donors (P less than 0.01). This observation correlated with the suppression of synthesis of normal immunoglobulin (Ig) in MM. Thus, cytokine activities regulating the proliferation of normal mature B cells, such as B cell stimulatory factor 1 (BSF-1)/interleukin 4 (IL-4), B cell growth inhibitory factor (BIF) and IL-2 in peripheral blood T cells, and IL-1 in peripheral blood adherent cells, were investigated in patients with BMG (n = 7) and MM (n = 28). All patients of MM having a marked suppression of synthesis of all other normal Ig, had significantly lower levels of BSF-1 activity and inversely higher levels of BIF activity than those of normal donors. However, patients with BMG having no suppression of synthesis of normal Ig had BSF-1 and BIF activities similar to normal donors. There was no significant difference in IL-1 and IL-2 activities between both normal donors and BMG versus MM patients. These data show that in MM altered cytokine activities correlate with suppression of synthesis of normal Ig.

Acid carboxypeptidase (EC 3.4.12.-) crystallized from culture filtrate of Penicillium janthinellum has been investigated for its use in carboxy-terminal sequence determination of Z-Gly-Pro-Leu-Gly, Z-Gly-Pro-Leu-Gly-Pro, angiotensin, I, native lysozyme, native ribonuclease T1, and reduced S-carboxymethyl-lysozyme. The examination indicated that proline and glycine were liberated from Z-Gly-Pro-Leu-Gly-Pro. At high enzyme concentration, the enzyme catalyzed complete sequential release of amino acids from the carboxy-terminal leucine to the amino-terminal aspartic acid of angiotensin I. The enzyme released the carboxy-terminal leucine from native lysozyme, however, no release of the carboxy-terminal threonine from native ribonuclease T1 was observed after a prolonged period of incubation with the enzyme. The sequence of the first nine carboxy-terminal residues of denatured lysozyme, leucine, arginine, S-carboxymethyl-cysteine, glycine, arginine, isoleucine, tryptophane, alanine, and glutamine, could be deduced unequivocally from a time release plot of an incubation mixture with the enzyme.

Cultured broths were screened by measuring the inhibition of monoamine oxidase. Using serotonin as the substrate, two active agents were isolated from Actinomycetes and shown to be pimprinine and trans-cinnamic acid amide, and a compound isolated from a mushroom was identified to be harman. Using benzylamine as the substrate, an inhibitor was isolated from a Streptomyces and shown to be phenethylamine.

ACCORDING to Reese et al.1, β-D-1,6-glucan rarely occurs in Nature, yet many fungi are able to produce an enzyme capable of hydrolysing it. Production of β-D-1,6-glucanase by Streptomyces has not been reported.

A dopamine β-hydroxylase inhibitor produced by Gloeophyllum striatum was isolated and found to be identical with oosponol. The inhibition is competitive with tyramine and noncompetitve with ascorbic acid. This inhibitor showed a hypotensive effect.

1. The influences upon amylase forming ability of Asp. oryzae by addition of several organic acids to the CZAPEK medium (surfure culture) were studied. 2. Aconitic acid, α-ketoglutaric acid, maleic acid, malic acid, oxalacetic acid and malonic acid were effective and pyruvic acid, citric acid, fumaric acid and succinic acid were ineffective upon amylase formation.

IN the course of work on metabolic products of newly isolated moulds from soil, Penicillium aculeatumRaper and Fennell var. apiculatumAbe strain No. 2282 was found to produce a luteic acid-like substance of high viscosity1. The substance consisted of glucose polymerized by glucosidic linkage and malonic acid. As a result of periodate oxidation, almost all the glucosidic linkages of polysaccharide were of the β-1,6 type. Since the molecular weight of the polysaccharide calculated from a result of ultra-centrifugal method (1.6 × 106) was greater than that of luteose, which was first reported by H. Raistrick and M. L. Rintoul2, and consisted of 84 glucose units3, it was found that the polysaccharide preparation is lutease of higher molecular weight.

Background The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear. Results We report the first genome-wide identification and characterization of TR4 in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors. Conclusions Our data suggest that TR4 plays a pivotal role in fundamental biological processes across different cell types. In addition, the identification of cell type specific TR4 binding sites enables future studies of the pathways underlying TR4 action and its possible role in metabolic diseases.