Production and properties of a cellobiose-oxidizing enzyme from a newly isolatedcellulolytic bacterium Cytophaga sp. LX-7

A cell-bound cellobiose-oxidizing enzyme was produced by cellulolytic Cytophaga sp. LX-7. It was found that both the cellulosic substrates and the soluble carbohydrate substrates tested promoted the production of the cellobiose-oxidizing enzyme, and the highest specific activities were obtained with cellulose powder MN300, carboxy- methylcellulose CM22, maltose and cellobiose. Among the nitrogen sources examined, peptone gave the best cellobiose-oxidizing enzyme production, whereas inorganic nitrogen sources gave very poor growth. The medium buffered with Tris/HCl, pH 7.1, yielded the highest levels of cellobiose-oxidizing enzyme activity and the temperature optimum for crude enzyme activity was 40°C.     

Cellobiose-oxidizing enzymes from the lignocellulose-degrading basidiomycete Phanerochaete chrysosporium: interaction with microcrystalline cellulose

Summary Cellulose-degrading cultures of the white-rot fungus Phanerochaete chrysosporium produce two extracellular cellobiose-oxidizing enzymes, cellobiose oxidase and cellobiose: quinone oxidoreductase. These two enzymes bind strongly to microcrystalline cellulose (MCC) in the pH range 4–7; above neutral pH their affinity for MCC decreases. Cellulose-bound enzymes could not be eluted with phosphate buffer (20 mM, pH 6) containing polyols (10%), KCl (1 M), urea (1 M) or 1% ionic or non-ionic detergent. TRIS or borate buffer at pH 9 eluted 30%–35% of the cellobiose-oxidizing enzyme activity. The cellulose-immobilized enzymes oxidized cellobiose actively, suggesting that the catalytic sites are not involved in cellulose binding. These results suggest that the cellobiose-oxidizing enzymes of P. chrysosporium may be organized into two domains: a cellulose-binding domain and a catalytic domain.Offprint requests to: V. Renganathan     

垃圾填埋场土壤酶活性与化学性质和微生物数量的关系研究

通过气相色谱／质谱定性分析表明,垃圾填埋场土壤的有机物污染成分复杂,共检出有机物50种。研究表明,随着填埋深度的加深,土壤理化性质,微生物数量和土壤酶活性有很大不同。运用典型相关分析对垃圾填埋场的土壤酶活性与土壤化学性质和微生物数量的关系进行了研究。结果表明。土壤酶活性与化学性质总体上密切相关;土壤酶活性与微生物数量总体上密切相关;但在土壤酶中过氧化氢酶活性与化学性质和微生物数量不相关。     

云斑尖塘鳢消化酶活力的研究

本文采用酶学分析方法研究了云斑尖塘鳢在正常摄食状态与饥饿的状态下胃、肠及肝胰脏组织中蛋白酶、淀粉酶和脂肪酶的活性。结果显示,在30℃的条件下,正常摄食组样本在酸性条件下的蛋白酶活力表现为:胃后肠肝胰脏前肠,中性和碱性条件下:后肠肝胰脏前肠及胃;饥饿组样本仅有胃表现出较高的酸性蛋白酶活性,其他器官的蛋白酶活性均很低。在正常和饥饿实验组中肝胰脏的淀粉酶活性均高于其他器官,胃肠的淀粉酶活性均较低。正常摄食组中脂肪酶活力后肠肝胰脏;而在饥饿组中仅有肝胰脏检测到脂肪酶活性。结果表明,云斑尖塘鳢适度饥饿组较正常摄食组消化酶活性大幅降低;其高蛋白酶活力及中等脂肪酶活力与其肉食性相一致;此外云斑尖塘鳢也具备少量的淀粉消化能力。     

Cell cycle and 5'-Nucleotide phosphodiesterase activities in rat liver

The postnatal developments of liver and serum 5'-nucleotide phosphodiesterase (5'-NPD) in Fischer 344 rats were studied. The liver enzyme activities were found to correlate well with the growth of the liver as measured by the wet weight and percentages of liver cells in the G2/M phases of the cell cycle as determined by flow cytofluorometry. Therefore, this enzyme may be intimately related to the growth of the liver. Although the specific activities of the serum enzyme are three orders of magnitude less than that of the liver enzyme, very good correlation was obtained between the total serum and liver enzyme activities. This finding, therefore, suggest that the main origin of the serum enzyme is from liver. Multiple isozymes were detected in both liver and serum 5'-nucleotide phosphodiesterase after polyacrylamide gel electrophoresis.     

Effect of dietary protein on purine nucleoside phosphorylase and xanthine dehydrogenase activities of liver and kidney in chicken and pigeon

Comparative studies were made on the effects of diets of different protein contents on the activities of purine nucleoside phosphorylase and xanthine dehydrogenase of avian livers and kidneys. In chicken liver and kidney, both enzyme activities were increased with high protein diet, confirming the previous results. In pigeon liver, only purine nucleoside phosphorylase was increased but xanthine dehydrogenase activity was not detected after feeding a high protein diet, while both enzyme activities were increased in the pigeon kidney. The increase in the levels of plasma oxypurines in pigeon serum was consistent with the result that the xanthine dehydrogenase activity of pigeon was not detected in the liver but in the kidney.     

Biosynthesis and Metabolism of NAD in Lemna paucicostata 151

We have already reported that NiA and related compounds had plant-growth-promoting and flower-inducing activities in duckweed, Lemna paucicostata 151. These effects may be concerned with the biosynthesis and metabolism of NAD. So, for the first step, the various enzyme activities related to them were investigated in this study to obtain some fundamental information on the action mechanism of these compounds and metabolites. Extremely high enzyme activity of nicotinamidase and very high enzyme activity of N AD glycohydrolase were found. The enzyme activities of nicotinate phosphoribo- syltransferase, quinolinate phosphoribosyltransferase, and nicotinate methyltransferase were easily detected. In contrast, nicotinamide phosphoribosyltransferase and ADP-ribosyltransferase activities were very low and nicotinamide methyltransferase activity was not detectable. NiA and NAm administered to the plant were rapidly incorporated into N AD and metabolized to several compounds. Postulation of the action mechanism is discussed.     

Developmental variation in effects of the second and third chromosomes on the activities of the glucose 6-phosphate and 6-phosphogluconate dehydrogenases in Drosophila melanogaster

Developmental profiles of the second- and third-chromosome modifiers of the activities of glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) in Drosophila melanogaster were investigated. Third-chromosome modifiers showed very strong effects on both enzyme activities at larval, pupal, and adult stages, whereas second-chromosome effects were detected mainly at larval and adult stages. For both enzyme activities and both chromosomes, the correlation over line means between larval and pupal stages was significantly positive, but the correlation between larval or pupal stage and adult stage was not significant. This result suggests that the actions of modifiers on G6PD and 6PGD activities are influenced by the change of developmental stages. Correlation between G6PD and 6PGD activities was positive and highly significant throughout the developmental stages for both sets of chromosomes, although third-chromosome correlations were slightly higher than second-chromosome correlations. The magnitude of the correlation between G6PD and 6PGD activities does not seem to be influenced by the change of development. Diallel crosses for both sets of chromosomes indicate that the action of activity modifiers is mainly additive for both sets of chromosomes, but dominance effects were detected in some cases in adult males. Significant maternal effects were detected for the third chromosome for both enzyme activities until the pupal stage. The change of the activity modifier action after emergence of the imago and the significant correlation between G6PD and 6PGD activities were also detected for diallel progeny.This work was supported by Public Health Service Grant NIH-GM11546.Paper No. 10211 of the journal series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695.     

Unusual Growth Phase and Oxygen Tension Regulation of Oxidative Stress Protection Enzymes, Catalase and Superoxide Dismutase, in the Phytopathogen Xanthomonas oryzae pv. oryzae

The enzymes catalase and superoxide dismutase play major roles in protecting phytopathogenic bacteria from oxidative stress. In Xanthomonas species, these enzymes are regulated by both growth phase and oxygen tension. The highest enzyme levels were detected within 1 h of growth. Continued growth resulted in a decline of both enzyme activities. High oxygen tension was an inducing signal for both enzyme activities. An 80,000-Da monofunctional catalase and a manganese superoxide dismutase were the major forms of the enzymes detected at different stages of growth. The unusual regulatory patterns are common among several Xanthomonas strains tested and may be advantageous to Xanthomonas species during the initial stage of plant-microorganism interactions.     

Transmural distribution of glucose metabolizing enzymes across the left and the right ventricle heart walls in three different mammalian species

The transmural distribution of five glucose metabolizing enzymes (hexokinase; glucose-6-phosphate dehydrogenase; phosphofructokinase; aldolase; and lactate dehydrogenase) were explored in the left and in the right ventricle wall of rat, ox and pig hearts. The levels of most of these enzyme activities were different in the different animal species and (within the same species) in the two ventricles. Most of these enzyme activities were found to be non-uniformly distributed across the left (but not across the right) ventricle wall. Differences in the transmural distribution of enzyme activities were detected among the three examined mammalian species.     

水稻籽粒淀粉分支酶活性的遗传分析

用由247个株系组成的珍汕97B/密阳46重组自交系群体及其含207个分子标记的连锁图谱,在2002年和2003年分别测定亲本和重组自交系群体开花后10 d和20 d籽粒的淀粉分支酶的活性,检测到3个控制开花后10 d Q酶活性的主效应QTL(qnantitative trait loci),联合贡献率为10%,其中qQ10-6与环境发生显著的互作;分别检测到5对和2对染色体区间对开花后10 d、20 d Q酶活性的影响具有加性×加性上位性作用,其中开花后10 d的3对染色体区间具有显著的上位性×环境互作效应.由此可见,水稻籽粒Q酶活性相关基因的表达,受到环境因子的极大影响.     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Differences in tissue expressions of enzyme activities in interspecific sunfish (Centrarchidae) hybrids and their backcross progeny

The extent of naturally occurring variations of enzyme locus expression was determined for three tissues (liver, muscle, and eye) in two species of sunfish (Centrarchidae), the green sunfish (Lepomis cyanellus) and the redear sunfish (L. microlophus). The genetic basis for species differences in tissue enzyme specific activities of malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), phosphoglucomutase (EC 2.7.5.1), and glucosephosphate isomerase (EC 5.3.1.9) was investigated by determining enzyme specific activities in the tissues of the reciprocal F1 hybrids and of their backcross progenies. The specific activities for most enzymes in hybrids were intermediate between those of the parental species. Significant differences in enzyme specific activity were detected among the F1 progeny as well as those of backcrosses. Variations in specific activity levels in one tissue were often independent of variations in specific activities in a different tissue. However, the changes in the specific activities of different enzymes within the same tissue were often positively correlated. The tissue glucosephosphate isomerase activity differences appear not to be due to different functional contributions of the glucosephosphate isomerase allelic isozymes. Cluster analysis of distributions of specific activities revealed no simple Mendelian pattern of inheritance for control of tissue enzyme activity. Our results suggest a polygenic control of tissue enzyme specific activity levels.     

Distribution of Tetrahydromethanopterin-Dependent Enzymes in Methylotrophic Bacteria and Phylogeny of Methenyl Tetrahydromethanopterin Cyclohydrolases

The methylotrophic proteobacterium Methylobacterium extorquens AM1 possesses tetrahydromethanopterin (H(4)MPT)-dependent enzymes, which are otherwise specific to methanogenic and sulfate-reducing archaea and which have been suggested to be involved in formaldehyde oxidation to CO(2) in M. extorquens AM1. The distribution of H(4)MPT-dependent enzyme activities in cell extracts of methylotrophic bacteria from 13 different genera are reported. H(4)MPT-dependent activities were detected in all of the methylotrophic and methanotrophic proteobacteria tested that assimilate formaldehyde by the serine or ribulose monophosphate pathway. H(4)MPT-dependent activities were also found in autotrophic Xanthobacter strains. However, no H(4)MPT-dependent enzyme activities could be detected in other autotrophic alpha-proteobacteria or in gram-positive methylotrophic bacteria. Genes encoding methenyl H(4)MPT cyclohydrolase (mch genes) were cloned and sequenced from several proteobacteria. Bacterial and archaeal Mch sequences have roughly 35% amino acid identity and form distinct groups in phylogenetic analysis.     

Variation among Extracted Lines of Drosophila Melanogaster in Triacylglycerol and Carbohydrate Storage

Whole larvae and whole adult extracts from 26 second chromosome replacement lines of Drosophila melanogaster were analyzed to determine the amounts of stored triacylglycerols and carbohydrates as well as the activities of 13 enzymes in relevant biochemical pathways. Analysis of covariance revealed significant differences among lines in stored lipids and carbohydrates, as well as in activities of most of the enzymes. Significant broad-sense genetic correlations (among adjusted line means) were detected for a number of enzyme pairs. Multiple regression techniques were applied to assess the extent to which the amounts of stored triacylglycerols and carbohydrates could be predicted from the enzyme activities. Significant regressions were found in both adults and larvae, suggesting that modulation of enzyme activities is reflected in different sizes of storage pools. The population genetic consequences of natural selection acting on a phenotype such as energy storage is considered in light of models of metabolic flux in biochemical pathways.     

The influence of the parasite invasion on antioxidant enzyme activity in the liver and muscles of a host, the Black Sea flounder Psetta maxima maeotica

The influence of the intestine cestode Botriocephalus gregarius on the antioxidant enzyme system in the liver and muscle of its host, the Black Sea flounder Psetta maxima maeotica, was studied. The significant increase of superoxidedismutase activity in examined tissues and changes of catalase and peroxidase activities were detected. High correlation (r > or = 0.6) between enzyme activities and the parasite number was noted. The possibility to use antioxidant enzyme activity as biomarkers of the parasite invasion in fish is discussed.     

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.     

Characterization, Biosynthesis, and Regulation of Granulose in Clostridium acetobutylicum

Synthesis of granulose was investigated in 15 solvent-producing Clostridium strains. Only one of the strains did not produce granulose. The structure of granulose in Clostridium acetobutylicum P262 consisted of a high-molecular-weight polyglucan containing only (1-->4) linked d-glucopyranose units. Biosynthesis of granulose in C. acetobutylicum P262 was dependent on ADPglucose pyrophosphorylase, and granulose synthase and mutants defective in granulose accumulation lacked either one or both enzyme activities. Granulose-positive revertants exhibited both enzyme activities. ADPglucose pyrophosphorylase and granulose synthase were not subject to allosteric control by metabolites. Granulose accumulation and the biosynthetic enzyme activities were initiated immediately before the pH breakpoint and were detected in cells only at the end of the exponential growth phase. Granulose accumulation did not occur under conditions of nitrogen limitation, excess carbon, or excess energy.