Catechol oxidase from green olives: Properties and partial purification

Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.     

Chemical modification of electric eel acetylcholinesterase by tetranitromethane

The reaction of acetylcholinesterase (acetylcholinehydrolase, EC 3.1.1.7) with tetranitromethane has been studied. The reaction caused a decrease in enzyme activity as measured with the substrate acetylthiocholine under conditions where hydrolysis of the neutral substrate indophenyl acetate was unaltered. The inactivation of acetylcholinesterase by tetranitromethane was greatly accelerated by the quaternary oximes pyridine-2-aldoxime methyl nitrate or toxogonin, though not by other quaternary inhibitors tested and not by an aliphatic oxime. The enhanced inactivation by tetranitromethane in the presence of pyridine-2-aldoxime methyl nitrate was blocked by the enzyme inhibitor decamethonium.The oxime-induced inactivation of acetylcholinesterase by tetranitromethane was accompanied by significant changes in the immunological properties of the enzyme as demonstrated by complement fixation. The reaction also resulted in the disappearance of tyrosine and appearance of nitrotyrosine.     

Inhibition of phagocytotic metabolic changes of leukocytes by an intracellular calcium-antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate.

The role of calcium in regulating the activity of leukocytes to generate and release superoxide was studied by using an intracellular calcium-antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate. The antagonist inhibited the release of superoxide anions induced by a calcium-ionophore A23187 and the inhibition was relieved by the addition of calcium ions. The release induced by cytochalasin D or by the ingestion of bacteria was similarly inhibited by the calcium-antagonist. The result supports the hypothesis that an intracellular translocation of calcium is regulating the phagocytotic metabolic activity of leukocytes. The release of granule enzymes induced by the ionophore was also inhibited by the calcium antagonist.     

小蜡果皮水溶性色素的提取及光敏感性

以去离子水和不同体积分数乙醇从小蜡果皮中浸提红色素,用光谱扫描法检测该红色素的光吸收特性,比较不同浸提时间及不同体积分数乙醇对浸提效果的影响,并用暴露方式进行光敏感性测定。结果显示,小蜡果皮色素在纯水中的溶解性最好,属水溶性色素,延长浸提时间可提高色素的浸出率,但杂质质量分数也随之提高,紫外线对色素的色度影响最大,直接照射可使色素的色度大幅度下降。     

The acetates of p-nitrophenyl alpha-L-arabinofuranoside--regioselective preparation by action of lipases

All possible di-O-acetates and mono-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were prepared by chemoenzymatic way using lipases. The 2,3-di-O-acetate was obtained in 90% yield by deacetylation of the primary acetyl group of per-O-acetylated p-nitrophenyl alpha-L-arabinofuranoside by Candida cylindracea lipase (CCL) or Candida rugosa lipase (LAY). The 2,5- and 3,5-di-O-acetates were obtained by acetylation of p-nitrophenyl alpha-L-arabinofuranoside by Pseudomonas cepacia lipase (LPS-30) in organic solvents. The 5-O-acetate was regioselectively synthesised in 95% yield by acetylation of p-nitrophenyl alpha-L-arabinofuranoside catalysed by porcine pancreas lipase. Finally, the 2- and 3-O-acetates of p-nitrophenyl alpha-L-arabinofuranoside were obtained in two steps. The enzymatic di-O-acetylation of p-nitrophenyl alpha-L-arabinofuranoside by LPS-30 was followed by enzymatic hydrolysis of the primary acetyl group by CCL or LAY.     

Purification of membrane proteins from Acholeplasma laidlawii by agarose suspension electrophoresis in Tween 20 and polyacrylamide and dextran gel electrophoresis in detergent-free media.

Four of the membrane proteins from Acholeplasma laidlawii that are soluble in the nonionic detergent Tween 20 have been purified by preparative electrophoretic techniques utilizing different supporting media. The last purification step for two of the major proteins was a preparative polyacrylamide gel electrophoresis performed in the absence of any detergent. The proteins were recovered by continuous elution. The purity of the fractions was examined by analytical polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Two of the minor proteins were purified by dextran gel electrophoresis as the final step, which was also performed in a detergent-free buffer. The separation was followed by scanning the dextran gel in ultraviolet light. The proteins were recovered by slicing the gel and degrading the gel slices with dextranase. The homogeneity of the fractions was checked by electroimmunoassay.     

长臂光敏生物素标记葡萄扇叶病毒外壳蛋白基因cDNA探针的制备及其在检测上的应用

由葡萄扇叶病毒（Ｇｒａｐｅｖｉｎｅｆａｎｌｅａｆｖｉｒｕｓ,ＧＦＬＶ）导致的葡萄扇叶病在世界各地葡萄园内均有发生,是最为普遍和严重的病毒病害之一,感病葡萄可减产２０％～８０％。近年来,许多国家开展了葡萄脱毒苗木的培育和推广工作,同时进行了葡萄扇叶病毒...     

Enzymes of agmatine degradation and the control of their synthesis in Klebsiella aerogenes.

The degradation of agmatine to succinate by Klebsiella aerogenes occurs in five steps. The enzyme catalyzing the first step, agmatinase, is induced by agmatine. The enzymes catalyzing the second and third steps, putrescine aminotransferase and 4-aminobutyraldehyde dehydrogenase, are induced by putrescine and also by their product, 4-aminobutyrate. The enzymes catalyzing the fourth and fifth steps, 4-aminobutyrate aminotransferase and succinate semialdehyde dehydrogenase, are induced by 4-aminobutyrate. This compound also serves as gratuitous inducer of the catabolic acetylornithine aminotransferase. The formation of the enzymes responsible for agmatine degradation is regulated not only by induction, but also by catabolite repression and activation by glutamine synthetase.     

重组小鼠血管内皮生长因子在毕赤酵母菌中的表达和纯化

目的：建立毕赤酵母重组小鼠血管内皮生长因子（mVEGF）的制备方法,为研究mVEGF的生物活性、抗原性等提供基础。方法：通过全基因合成方法获得编码mVEGF的基因片段,将其克隆至表达载体pPICZaA上,电转化整合到毕赤酵母GS115基因组中,用甲醇诱导表达目的蛋白,表达上清经硫酸铵沉淀、SephadexG25柱脱盐、阳离子交换层析三步纯化获得目的蛋白;用还原型和非还原型SDS-PAGE检测目的蛋白的聚体状态,用Westelqq印迹验证纯化蛋白;通过PNGaseF酶切分析目的蛋白的N-糖基化修饰;通过人脐静脉内皮细胞（HUVEC）增殖实验检测目的蛋白的生物活性。结果：获得mVEGF的重组毕赤酵母表达菌株,SDS-PAGE分析可见GSll5表达的重组mVEGF在还原状态下表观相对分子质量约为20×10^3,在非还原状态下约为40×10^3;经Western印迹检测,这些条带均为目的蛋白条带,能被兔抗mVEGF抗体特异性结合,PNGase F酶切后相对分子质量降至18×10^3左右,证明目的蛋白发生了Ⅳ-糖基化修饰;细胞测活实验表明,mVEGF具有刺激HUVEC增殖的生物活性。结论：利用毕赤酵母菌制备了具有生物活性的重组mVEGF。     

Studies on the relationship between the oxygen uptake and the release of amylase and sialic acid

The relationship between the oxygen uptake and the release of amylase and sialic acid induced by pilocarpine was investigated in dog submandibular glands. Pilocarpine dose-dependently stimulated the oxygen uptake. The dose required for the maximal response was 10 microM. The release of amylase and sialic acid induced by pilocarpine was inhibited by the addition of iodoacetic acid, malonic acid, 2, 4-dinitrophenol, antimycin A or sodium azide. The oxygen uptake induced by pilocarpine was significantly inhibited by iodoacetic acid, malonic acid, antimycin A or sodium azide. On the other hand, 2, 4-dinitrophenol further stimulated the oxygen uptake by pilocarpine. The increase in the oxygen uptake or the release of amylase and sialic acid induced by pilocarpine was significantly inhibited by ouabain. The Na+, K+-ATPase activity ratio in the microsomal fraction of dog submandibular glands was dose-dependently increased by pilocarpine. The Na+, K+-ATPase activity ratio induced by pilocarpine was significantly inhibited by ouabain, antimycin A, oligomycin or 2, 4-dinitrophenol. The pilocarpine-induced Na+, K+-ATPase activity ratio was significantly inhibited by the removal Ca2+ from the medium or the addition of 2 mM EGTA. These results suggest that the increase in the oxygen uptake by pilocarpine is profoundly involved in the energy supply for the process of amylase and sialic acid release. In particular, the energy supply demanded for the activation of Na+ pump may play a role in the mechanism by which pilocarpine induces the oxygen uptake.     

Anatomical variations of the arterial pattern in the right hemiliver

The arterial supply to the right hemiliver was studied in 80 liver casts. The arteries were divided into 10 groups according to their origin and branching pattern. The right hemiliver was supplied by one artery in 96% of cases and by two arteries in 4%. When there was only one artery it originated from the proper hepatic artery in 73/77 cases and from the superior mesenteric artery in 4/77 cases. The replacing right hepatic artery which originated from the superior mesenteric vessel supplied the whole right hemiliver in 5% of cases. The incomplete replacing right hepatic artery which supplied only a part of the right hemiliver was found in 4% of cases. The anterior section (segments 5 and 8) was supplied by one artery in 61%, by two arteries in 30% and by three arteries in 9% of cases. The posterior section (segments 6 and 7) was supplied by one artery in 66%, by two arteries in 31% and by three arteries in 3% of cases. Segments 5 and 7 were predominantly supplied by one artery, whereas segments 6 and 8 by two arteries.     

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.     

Mechanisms underlying burst generation of the pyloric muscle in the mantis,shrimp, Squilla oratoria

The pyloric constrictor muscles of the stomach in Squilla can generate spikes by synaptic activation via the motor nerve from the stomatogastric ganglion. Spikes are followed by slow depolarizing afterpotentials (DAPs) which lead to sustained depolarization during a burst of spikes. 1. The frequency of rhythmic bursts induced by continuous depolarization is membrane voltage-dependent. A brief depolarizing or hyperpolarizing pulse can trigger or terminate bursts, respectively, in a threshold-dependent manner. 2. The conductance increases during the DAP response. The amplitude of DAP decreases by imposed depolarization, whereas it increases by hyperpolarization. DAPs from successive spikes sum to produce a sustained depolarizing potential capable of firing a burst. 3. The spike and DAP are reduced in amplitude by decreasing [Ca]o, enhanced by Sr2+ or Ba2+ substituted for Ca2+, and blocked by Co2+ or Mn2+. DAPs are selectively blocked by Ni2+, and the spike is followed by a hyperpolarizing afterpotential. 4. The spike and DAP are prolonged by intracellular injection of the Ca2+ chelator EGTA. A hyperpolarizing afterpotential is abolished by EGTA and enhanced by increasing [Ca]o. The DAP is diminished in Na(+)-free saline and reduced by tetrodotoxin. 5. It is concluded that the muscle fiber is endowed with endogenous oscillatory properties and that the oscillatory membrane events result from changes of a voltage- and time-dependent conductance to Ca2+ and Na+ and a Ca2+ activated conductance to K+.     

The identification of the membrane proteins of human and animal viruses]

The review deals with the methods of identification of virus-specific proteins on virion and infected cell surface. The isotopic labeling of membrane proteins, their extraction by proteolytic enzymes and selective solubilization by detergents are considered. The plasmatic membrane isolation by centrifugation and by means of microcarriers is described. The methods of membrane protein localization using monoclonal antibodies and radioimmunoprecipitation are presented.     

The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.     

Photosynthesis-related infrared light transmission changes in spinach leaf segments

The time courses of infrared light transmission changes and fluorescence induced by light in spinach leaf segments were measured. The illumination by red light exhibited a complex wave pattern. The transmission approached the baseline after repeating decreases and increases. Illumination by far-red light decreased the transmission. One of the differences between the two responses was the difference between the two amplitudes of the first increasing component. The component in the red light response was larger than the component in the far-red light response. The transmission decrease by far-red light is supposed to correspond to "red drop." The transmission decrease by far-red light was suppressed by red light. This is due to an activation of a transmission-increasing component. This probably corresponds to "enhancement." A proportional correlation existed between the intensity of far-red light and the minimum intensity of red light that suppressed the transmission decrease induced by far-red light. The component which made Peak D in the time course of fluorescence yield and the first increasing component in the transmission changes were suppressed by intense light.     

Correlation between transglutaminase activity and polyamine levels in human neuroblastoma cells. Effect of retinoic acid and alpha-difluoromethylornithine

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.     

Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon. 1. The mRNA of PEPCK was induced maximally 2-3 h after addition of 10 nM glucagon, as detected by Northern-blot analysis after hybridization with a biotinylated antisense RNA of PEPCK. 2. The synthesis rate of PEPCK increased maximally 2-3 h after application of glucagon as revealed by pansorbin-linked immunoprecipitation of [35S]methionine-labelled PEPCK. 3. The enzyme amount and activity was maximally induced 4 h after glucagon application. 4. The mRNA of PEPCK was half-maximally induced by 0.1 nM and maximally by 1 nM and 10 nM glucagon. The half-maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin. The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon-dependent increase in mRNA and enzyme-synthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1-1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range, with glucagon being the dominant hormone.     

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Control Mechanisms at the Level of FDP-Aldolases in Algae

Abstract

The FDP-aldolase I from Euglena gracilis is a four-chain enzyme, as shown by the amino acid analysis of the C and N terminals. The protein is dissociated by acid pH to a monomer. The reassociation-dissociation process goes through a dimer stage. The enzyme, inhibited either by mercurials or by ATP, is dissociated to a dimer. This process is readly reversible either by mercaptoethanol and by AMP.