Purification of Debranching Enzyme from Mature Rice Seeds

Two indole alkaloids which induce the Epstein-Barr virus early antigen of Raji cells (B lymphocyte) were found in the cultured broth of Actinomycetes NA34-17, from which teleocidin B was also obtained. The active compounds isolated were identified from their spectral data and chemical evidence as (—)-indolactam V and (—)-14-O-acetyl indolactam V.     

Purification of Oat Debranching Enzyme and Occurrence of Inactive Debranching Enzyme in Cereals

The interaction of some anthracycline antibiotics (adriamycin, daunomycin, aclacinomycin-A) with bacteriophage ?X174 was investigated. Adriamycin and daunomycin inactivated the infectivity of both free ?X174 phage and naked single-stranded ?X174 DNA without DNA strand scission, but aclacinomycin-A did not show this action. The phage inactivation reaction was reversibly inhibited by Superoxide dismutase, catalase or other oxygen radical scavengers. The inactivation of ?X174 by adriamycin and aclacinomycin-A was stimulated by the addition of Cu²⁺, while the ?X174 inactivation by daunomycin was inhibited by the addition of Cu²⁺. The ?X174 inactivation by adriamycin and aclacinomycin-A in the presence of Cu²⁺ was caused by degradation of DNA, and this inactivation reaction was inhibited irreversibly by oxygen radical scavengers. These results indicate that anthracycline antibiotics bind to ?X174 DNA in the form of free radicals and that during the auto-oxidation of these antibiotics in the presence of Cu²⁺, oxygen radicals were generated to cause the degradation of ?X174 DNA.     

双胸蚓纤溶酶的纯化及性质

 用硫酸铵分段盐析、超滤膜分级分离及DEAE-纤维素、Sephadex A-25和Sephadex G-50三种柱层析方法从双胸蚓组织的粗提取液中分离纯化出一种纤溶酶,分子量为29kD,由一条肽链组成。此晦具有强烈的溶解纤维蛋白的作用,对家兎实验性血凝块也具有明显的溶解作用。此酶的最适pH为8.0,在pH7.6～8.4之间活力相差不到2％;酶在PH4.7—11.0范围内稳定;酶作用的最适温度为57℃;此酶热稳定性较好,于25～50℃保温3小时,酶活力基本不变,60℃时,活力保留65％。金属离子Na~(+)、K~(+)、Mg~(2+)等可提高此酶的活力,而Hg~(2+)、Ca~(2+)等金属离子对此酶有不同程度的抑制作用。     

Antitumor and Antiviral Properties of Penicillic Acid

The effects of adenine and (or) guanosine concentration on the accumulation of inosine, xanthosine, adenosine and succino-adenosine were studied with various purine auxotrophs of Bacillus subtilis K strain. Genetical derepression of the common pathway enzymes resulted in increase in the accumulation of inosine, xanthosine and adenosine. Co-operative repression system of a common pathway enzyme, succino-AMP lyase with respect to adenine and guanosine, was confirmed under the condition of the accumulation test. From these and the relating other studies it was concluded that the synthesis of AMP was regulated mainly by the inhibition of PRPP amidotransferase by AMP and secondly by the repression of the common pathway enzymes by adenine and guanosine, that the synthesis of GMP was regulated mainly by the inhibition and repression of IMP dehydrogenase by guanine derivatives and that GMP was synthesized in preference to AMP at the branch point, IMP.     

Mode of Action of a Rice Debranching Enzyme on Pullulan and its Related Oligosaccharides

In the hydrolysis of pullulan by a rice debranching enzyme, oligosaccharides intermediately produced were more rapidly degraded than expected from the random cleavage (relative maximum velocities: hexaose 3.0, nonaose 1.7, oligosaccharides of higher polymerizations 1.2, pullulan 1.0), and the final product maltotriose accumulated extraordinarily even at early stages of the hydrolysis. Therefore, the hydrolytic action on pullulan was confirmed to proceed in an endo-fashion with the incomplete random cleavage.     

ATP Citrate Lyase from Germinating Castor Bean Endosperm: Localization and some Properties

ATP citrate lyase (EC 4.1.3.8) has been found in crude extracts from endosperm tissue of germinating castor bean and shows its maximum activity in 4- to 5-day-old seedlings. A strict requirement for coenzyme A and adenosine 5′-triphosphate was demonstrated. The pH optimum for the reaction is around 7.5. The unstable enzyme can be stabilized by freezing and addition of citrate and glycerol. (−)-Hydroxycitrate is a potent inhibitor. The molecular weight is about 400,000. The adenosine 5′-triphosphate citrate lyase is localized in the plastids, where it possibly plays a role in providing acetyl coenzyme A for lipid biosynthesis.     

Partial Purification and Properties of Tea Leaf Ribonuclease

Four fractions with ribonuclease activity have been isolated from tea leaves by DEAE-cellulose column chromatography and designated as RNase Tf-1, RNase Tf-2, RNase Tf-3 and RNase Tf-4. The bigger fractions of both RNase Tf-3 and RNase Tf-4 have been partially purified by Sephadex G-100 column chromatography.

RNase Tf-3 and RNase Tf-4 were respectively found to have their optimum pH at 4.75 and 4.9 and molecular weights of approximately 13,000 and 16,000, as determined by gel filtration. Both enzymes were inhibited by Cu²⁺ and Hg²⁺, and inactivated by heating at over 50°C. By addition of yeast RNA to the two enzymes, however, their thermostabilities increased. The activities of the enzymes were stable in a pH range of 4.5 to 6.5. Like other plant RNases, RNase Tf-3 and RNase Tf-4 appeared to have no preference for base in RNA.     

Purification and Some Properties of a Protease from Streptomyces limosus

Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.     

Activation of Rice Aminopeptidase by Anions and Some Properties of the Enzyme

In order to determine which proteases are responsible for the autolysis of krill, the effects of several protease inhibitors on the autolysis and protease activities of krill were investigated.

Homogenates of whole bodies, and the cephalothorax and abdomen parts of frozen krill were equilibrated at 37°C at different pHs between 2 to 10 and allowed to stand for 16 hr, following which the increase in the TCA soluble fraction was monitored. ¹⁴C-Hemoglobin (¹⁴C-Hb) hydrolyzing activity was also measured using each homogenate as a crude enzyme preparation. The degree of autolysis and the ¹⁴C-Hb hydrolyzing activity were maximum at pH 5 ~ 8 for the parts studied. The hydrolytic activity was highest in the cephalothorax, followed by that in the whole body and then the abdomen.

The effects of inhibitors on the ¹⁴C-Hb hydrolyzing activity were examined, and it was seen that soybean trypsin inhibitor (STI), diisopropyl fluorophosphate (DFP) and leupeptin significantly inhibited the activity at neutral pH, and pepstatin, monoiodoacetic acid (IAAcid) and leupeptin were effective at acidic pH for all the parts. Investigation of the effects of inhibitors on the autolysis at 20°C at pH 4 and 7 by SDS–polyacrylamide gel electrophoresis indicated that the autolysis of the cephalothorax and whole body at pH 7 was suppressed a little by STI and the autolysis of the abdomen and whole body at pH 4 was significantly inhibited by iodoacetamide (IAA) and leupeptin.

These results suggest that the main proteases responsible for the autolysis of krill are trypsin like-proteases at neutral pH and cathepsins (B, H and L types) at acidic pH.     

Partial Purification and Some Properties of a Benzhydrylamide-bond Hydrolyzing Enzyme in Pseudomonas diminuta

An enzyme in Pseudomonas diminuta showed hydrolyzing activity of a benzhydrylamide ( = diphenylmethylamide) bond in S-benzylcysteinylglycine benzhydrylamide. The enzyme was purified 225-fold by precipitation with ammonium sulfate, and column chromatography with ECTEOLA-cellulose, DEAE-cellulose and hydroxyapatite. It showed an optimum pH of 6 to 8 and it was markedly inhibited by Hg^{2 +} or p-chloromercuribenzoate. The preparation was more specific against S-benzylcysteinylglycine benzhydrylamide than other substrates tested.     

灰色链霉菌RX-17溶菌酶R1的纯化及性质研究

通过硫酸铵分级沉淀,CM-Sephadex C50、CM-Sepharose Fast Flow离子交换层析及Sephadex G-75凝胶过滤层析,从灰色链霉菌（Streptomyces griseus）RX17的发酵上清液中得到了电泳纯的溶菌酶R1,回收率6.89%。测得该酶分子量和等电点分别为16.8kD和9.10,作用于变链球菌（Streptococcus mutans）Ingbritt的最适温度和pH分别为70℃和6.6。R1酶在50℃以下及pH6～9的范围内保持稳定,60℃保温1h,残存酶活20.3％。Mg2+对酶有激活作用,而Zn2+、Cu2+、Fe2+、Cd2+、Pb2+则使酶完全丧失活性,螯合剂、盐酸羟胺、碘乙酸抑制酶活,β-巯基乙醇及表面活性剂则对溶菌有部分促进作用。R1酶溶菌谱广泛,对多种卵清溶菌酶不能作用的G+、G细菌均有溶解能力,对变链球菌、金黄色葡萄球菌（Staphylococcus aureus）、乳杆菌(Lactobacillus)等则呈现高活性。     

Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm

ADPglucose pyrophosphorylase from developing endosperm tissue of starchy maize (Zea mays) was purified 88-fold to a specific activity of 34 micromoles α-glucose-1-P produced per minute per milligram protein. Rabbit antiserum to purified spinach leaf ADPglucose pyrophosphorylase was able to inhibit pyrophosphorolysis activity of the purified enzyme by up to 90%. The final preparation yielded four major protein staining bands following sodium dodecyl sulfate polyacrylamide gel electrophoresis. When analyzed by Western blot hybridization only the fastest migrating, 54 kilodaltons, protein staining band cross-reacted with affinity purified rabbit antispinach leaf ADPglucose pyrophosphorylase immunoglobulin. The molecular mass of the native enzyme was estimated to be 230 kilodaltons. Thus, maize endosperm ADPglucose pyrophosphorylase appears to be comprised of four subunits. This is in contrast to the respective subunit and native molecular masses of 96 and 400 kilodaltons reported for a preparation of maize endosperm ADPglucose pyrophosphorylase (Fuchs RL and JO Smith 1979 Biochim Biophys Acta 556: 40-48). Proteolytic degradation of maize endosperm ADPglucose pyrophosphorylase appears to occur during incubation of crude extracts at 30°C or during the partial purification of the enzyme according to a previously reported procedure (DB Dickinson, J Preiss 1969 Arch Biochem Biophys 130: 119-128). The progressive appearance of a 53 kilodalton antigenic peptide suggested the loss of a 1 kilodalton proteolytic fragment from the 54 kilodalton subunit. The complete conservation of the 54 kilodalton subunit structure following extraction of the enzyme in the presence of phenylmethylsulfonyl fluoride and/or chymostain was observed. The allosteric and catalytic properties of the partially purified proteolytic degraded versus nondegraded enzyme were compared. The major effect of proteolysis was to enhance enzyme activity in the absence of added activator while greatly decreasing its sensitivity to the allosteric effectors 3-P-glycerate and inorganic phosphate.     

Partial Purification of Isocitrate Lyase from Candida tropicalis and Some Kinetic Properties of the Enzyme

Isocitrate lyase was purified partially from n-alkane-grown cells and glucose-grown cells of Candida tropicalis by means of ammonium sulfate fractionation and DEAE-cellulose column chromatography. The preparation from alkane-grown cells showed one peak of the enzyme activity, while that from glucose-grown cells showed two distinct peaks of the activity, on DEAE-cellulose column chromatography. These enzymes, having the similar pH optima (around 7.0) and Km values with dl-isocitrate (1.2 ~ 1.7 mm), were inhibited by various metabolic intermediates, such as 6-phosphogluconate and phosphoenolpyruvate.

Time-course changes in the activities of isocitrate lyase and isocitrate dehydrogenases of C. tropicalis during the growth indicated that the lyase would participate preferentially in alkane assimilation and NAD-linked isocitrate dehydrogenase in glucose utilization of the yeast.

Regulation of isocitrate metabolism in C. tropicalis through glyoxylate cycle and tricarboxylic acid cycle is discussed based on the kinetic properties, cellular localization and time- course changes in the levels of isocitrate lyase and NAD-linked and NADP-linked isocitrate dehydrogenases.     

N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

Purification and Some Properties of Tetanolysin

Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050-fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate-gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ-toxin of Cl. perfringens.     

N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

水稻胚乳甲壳素结合蛋白(ES-CBP)在发育种子中的组织专一性累积及其在萌发时的降解(英文)

免设印迹表明,水稻 ES—CBP(en—dosperm chitin—binding Protein)是胚乳专一性蛋白。蛋白休用NaCl提取后再用TritonX—100提取,所得的两个组份在作免疫印迹时,均能与抗ES—CBP的抗血清起反应,表明这种细胞器的衬质(matrix)和膜部分部有ES—CBP存在。免疫印迹还表明,在开花后7d(7DPA)的水稻种于胚乳中已经累积了相当多的 ES—CBP。在7～30 DPA之问.ES—CBP在胚乳总蛋白中的相对水平随着种子发育而下降。这些结果表明,ES—CBP在7DPA以前的胚乳中的累积是相对活跃的。水稻种子贮藏蛋白在7DPA以后累积最快,这时胚乳中已经累积了相当多的ES—CBP。种子萌发后2d(2DPG)时,ES—CBP已有明显的降解。到7DPG,胚乳中只剩下少量的甲壳素结合蛋白。ES—CBP这样的累积和降解模式支持了该甲壳素结合蛋白是水稻胚乳中蛋白体组织者的假说。     

圈卷产色链霉菌硝基烷类氧化酶的分离纯化及酶学性质

圈卷产色链霉菌硝基烷类氧化酶基因naoA在大肠杆菌中获得了成功表达,从含有重组质粒pNA101(pET23b∷naoA)的工程菌株BL21(DE3)中分离纯化了硝基烷类氧化酶,SDSPAGE检测为均一。对纯酶进行了酶学性质及动力学研究。底物为1硝基丙烷、2硝基丙烷和硝基乙烷时,在04mol/L的磷酸缓冲液中,酶的最适反应pH值为7～8,最适反应温度为48℃～56℃。室温保存6d后,酶的活性保持了43.3%,但对60℃以上的高温敏感。硫醇化合物如巯基乙醇、还原型谷胱甘肽不同程度地抑制酶活性,特别是NADH,其浓度为1mmol/L时,酶活性几乎全部丧失。以1硝基丙烷为底物时,NaoA的Km为357mmol/L,Vmax为0199μmol/(μg.min)。     

Purification and Properties of a Thermophilic Bacteriophage Lytic Enzyme

A phage lytic enzyme was isolated from lysates of Bacillus stearothermophilus (NCA 1503-4R). The enzyme was purified 1,998-fold with a 27% recovery of enzyme activity. By use of polyacrylamide gel electrophoresis and sucrose gradient centrifugation the enzyme was judged free from protein contaminants. The lytic enzyme was active over a pH range of 6.0 to 7.0, with a maximum at 6.3, and it was stable between pH 7.0 and 8.0 and at 5.0 and unstable between pH 5.5 and 6.5. The temperature coefficient (Q(10)) was 2.27 between 35 and 45 C, 2.01 between 45 and 55 C, and 2.00 between 50 and 60 C. Lytic enzyme in 0.1 m sodium phosphate was not inactivated after a 1-hr exposure to temperatures below 65.5 C, whereas a 1% inactivation was observed at 70.6 C. A 2-hr exposure at 60.1, 65.5, and 70.6 C resulted in an inactivation of 1.2, 9.6, and 12.0%, respectively. A sodium phosphate concentration of at least 0.1 m was necessary for the prolonged exposure of lytic enzyme at 55 C (pH 6.3), whereas 0.005 m was required for maximal lytic activity. Lytic activity was stimulated 169, 165, and 160% by 10(-4)m Mg(++), Ca(++), and Mn(++), respectively. Lytic activity was inhibited 75% by 10(-4)m ethylenediaminetetraacetic acid (EDTA). The EDTA inhibition could be reversed by the addition of excess Mg(++), Ca(++), or Mn(++). Lytic activity was not affected by NaCl, KCl, or NH(4)Cl. Lytic activity was inhibited 100, 91, 25, 61, and 56% by 10(-4)m Hg(++), Cu(++), Zn(++), p-chloromercuribenzoate, and p-hydroxymercuribenzoate, respectively. Cysteine or 2-mercaptoethanol did not stimulate lytic activity, nor were these sulfhydryl compounds required for maintenance of enzyme activity during handling or storage. Cell walls were rapidly solubilized when incubated with lytic enzyme. Lytic action was complete after 1.5 min, with a 70% reduction in optical density (OD). Cell walls without lytic enzyme showed no reduction in OD during this period. The solubilization of N-terminal amino groups paralleled the reduction in OD and reached a level of 0.3 mumole/mg of cell wall after 4 min of incubation. Cell walls with and without lytic enzyme treatment showed a 3- and a 1.3-fold increase, respectively, in N-terminal amino groups after 3 hr of incubation. There was no release of reducing power in either the untreated cell wall suspensions or those treated with lytic enzyme. Electron micrographs of treated and untreated cell walls showed that the enzyme partially degrades the cell wall with the release of small wall fragments.