Iron-sulphur clusters in fumarate reductase from Vibrio succinogenes

(1) The fumarate reductase complex from Vibrio succinogenes contains one FAD molecule, one [4Fe-4S]3+(3+,2+) and one [2Fe-2S]2+(2+,1+) cluster per enzyme molecule. Both clusters can be partly reduced by succinate. In the presence of excess Na2S2O4 and fumarate, the [2Fe-2S] cluster is completely oxidized, whereas the other cluster is largely reduced. (2) The [2Fe-2S] cluster is localized in the Mr, 31,000 subunit. The EPR spectrum of the reduced cluster in the isolated subunit differs slightly in line width, but not in g-value, from the spectrum of reduced, intact enzyme complex. The demonstrates that the immediate environment of th cluster is little perturbed by dissociating this subunit from the FAD-containing Mr 79,000 subunit. The temperature dependence of the power-saturation behaviour has, however, greatly decreased in the isolated subunit, the saturation at 11 K of the paramagnetic cluster being much less than in the enzyme complex. Moreover, the temperature dependence of th power-saturation behaviour of this cluster in the enzyme is greater with succinate as reducing agent, than with dithionite. (3) The [4Fe-4S] cluster is located on the Mr 79,000 subunit. This cluster is unstable in air when the subunit has been dissociated from the enzyme complex.     

Cell yields of Vibrio succinogenes growing with formate and fumarate as sole carbon and energy sources in chemostat culture

Vibrio succinogenes which gains all the ATP by anaerobic electron transport phosphorylation, was grown in continuous culture on a defined medium with formate and fumarate as sole energy sources. The growth yield at infinite dilution rate (Y ^max) was obtained by extrapolation from the growth yields measured at various dilution rates. With formate as the growth limiting substrate, Y ^max was found as 14 g dry cells/mol formate. Under these conditions growth was limited by the rate of energy supply, because formate is used only as a catabolic substrate (Bronder et al. 1982). The Y _ATP ^max calculated from the ATP requirement for cell synthesis was 18 g dry cells/mol ATP. This gives an ATP/2e ratio of 0.8. The ATP/2e ratio in vitro had been measured as 1 (Kröger and Winkler 1981). It is concluded that growing V. succinogenes gain at least 80% the stoichiometrically possible amount of ATP, when growth is limited by energy supply.     

Preliminary crystallographic study of an L-asparaginase from Vibrio succinogenes

Crystals of an L-asparaginase from Vibrio succinogenes were obtained with the hanging drop method from ammonium sulphate-containing solutions. The crystals belong to the orthorhombic space group P22(1)2(1) with unit cell dimensions of a = 71.3 A, b = 85.8 A, c = 114.0 A, and contain two tetrameric enzyme molecules per unit cell. There are two subunits in the asymmetric unit; a molecular dyad is coincident with the crystallographic dyad. The crystal lattice is similar to that reported for an Escherichia coli asparaginase. Rotation function calculations have revealed that the V. succinogenes enzyme has 222 point group symmetry in the crystal. The second and third molecular dyads differ, however, from the corresponding E. coli asparaginase dyads by approximately 40 degrees. The crystals diffract to at least 2.2 A resolution and are suitable for X-ray crystallographic structure determination.     

Requirement of succinate for the growth of Vibrio succinogenes

Vibrio succinogenes required relatively small amounts of succinate for growth when formate plus nitrate was supplied as the energy source. The requirement for succinate was not apparent when formate plus fumarate was the energy source because fumarate is reduced to succinate. l-Asparagine, fumarate, and malate replaced succinate, and it appears likely that they do so by being converted to succinate. Formate plus l-aspartate or l-asparagine served as energy sources for growth. The stoichiometry of the reduction of aspartate with H(2) by resting cells suggests an aspartase reaction followed by reduction of fumarate to succinate. Oxalacetate or pyruvate plus bicarbonate did not substitute for succinate, nor did many other compounds that were tested. (14)C-succinate was mainly incorporated into the alcohol-soluble fraction of cells, although there was significant incorporation into the hot trichloroacetic acid-soluble and -insoluble fractions.     

Asparaginase production by human clinical isolates of Vibrio succinogenes

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

云南美登木悬浮培养细胞的化学成分

利用组织培养方法以云南美登木(Maytenus hookeri Lose.)未木质化的茎和嫩叶为材料诱导出愈伤组织,经过继代培养建立了悬浮细胞培养系.培养物的乙酸乙酯提取物显示抗橙色青霉(Penicillium avellaneum UC-4376)生长的生物活性.对培养物进行化学成分研究,分离鉴定了9个化合物:2,3-diacetoxyl maytenusone (1)、角鲨烯 (squalene,2)、β-谷甾醇 (β-sitosterol,3)、2′,3′,4′-triacetylsitoindoside Ⅰ (4)、salaspermic acid (5)、美登酮酸 (maytenonic acid,6)、2α-羟基美登酮酸(2α-hydroxy-maytenonic acid,7)、6,11,12-trihydroxy-8,11,13-abietrien-7-one (8)和11,12-dihydroxy-8,11,13-abietrien-7-one (9),其中化合物1为新化合物.通过2D NMR对化合物5-7的NMR数据进行了全指定,并修正了化合物5和6的部分碳谱数据指定.     

云南美登木悬浮培养细胞的化学成分(英)

利用组织培养方法以云南美登木 (MaytenushookeriLose .)未木质化的茎和嫩叶为材料诱导出愈伤组织 ,经过继代培养建立了悬浮细胞培养系。培养物的乙酸乙酯提取物显示抗橙色青霉 (PenicilliumavellaneumUC_4 376 )生长的生物活性。对培养物进行化学成分研究 ,分离鉴定了 9个化合物 :2 ,3_diacetoxylmaytenusone (1)、角鲨烯 (squa lene ,2 )、β_谷甾醇 (β_sitosterol,3)、2′ ,3′,4′_triacetylsitoindosideⅠ (4)、salaspermicacid (5 )、美登酮酸 (maytenonicacid ,6 )、2α_羟基美登酮酸 (2α_hydroxy_maytenonicacid ,7)、6 ,11,12_trihydroxy_8,11,13_abietrien_7_one (8)和 11,12_dihydroxy_8,11,13_abietrien_7_one (9) ,其中化合物 1为新化合物。通过 2DNMR对化合物 5 - 7的NMR数据进行了全指定 ,并修正了化合物 5和 6的部分碳谱数据指定。     

Lysis of Vibrio succinogenes by ethylenediamine-tetraacetic acid or lysozyme

Wolin, M. J. (University of Illinois, Urbana). Lysis of Vibrio succinogenes by ethylenediaminetetraacetic acid or lysozyme. J. Bacteriol. 91:1781-1786. 1966.-Cell suspensions of Vibrio succinogenes are lysed by ethylenediaminetetraacetic acid (EDTA) or lysozyme. Lysis occurs at alkaline pH and is prevented by 0.15 m NaCl or KCl or 0.3 m sucrose. The addition of 10(-3)m Mg(++), 10(-3)m spermine, or 10(-2)m Ca(++) prevents lysozyme lysis, and 10(-4)m spermine prevents EDTA lysis. EDTA lysis leads to the formation of a cell ghost, and lysozyme lysis leads to the formation of an empty round body. Freezing and thawing of cells permits lysozyme attack which is not prevented by the protective agents mentioned above. Much of the cell protein, and almost all of the nucleic acids, are released from the cells during EDTA lysis. Treatment of frozen-thawed cells with lysozyme at neutral pH does not cause release of more than 50% of the cell protein and 60% of the nucleic acids of the cells.     

Asparaginase synthesis by semi-continuous fermentor cultures of Vibrio succinogenes

Summary Vibrio succinogenes produces an asparaginase that does not hydrolyze glutamine, is not immunosuppressive, and has antitumor activity. Fermentor cultures initiated by small inocula exhibit a pattern of increasing enzyme activity consistent with induction during exponential phase. Semi-continuous cultures permit the harvesting of fully induced cells.     

Phosphorylative fumarate reduction in Vibrio succinogenes: Stoichiometry of ATP synthesis

1. Cells of Vibrio succinogenes, treated with EDTA at pH 8, catalyze the phosphorylation of their endogenous ADP and AMP as a function of the electron transport from formate to fumarate. The P/fumarate ratio obtained from the initial velocity of the phosphorylation on initiation of the electron transport and from the activity of fumarate reduction in the steady state was 0.90. The phosphorylation was prevented by 10μmol/g protein carbonylcyanide-3-chlorophenylhydrazone.
2. The esterification of external phosphate in the presence of ADP, hexokinase and glucose is catalysed by a membrane preparation of V. succinogenes in the steady state of fumarate reduction by H₂. The phosphorylation was fully abolished by either 5μmol/g protein carbonylcyanide-4-trifluoromethoxyphenylhydrazone or 30μmol/g protein carbonylcyanide-3-chlorphenylhydrazone. Phosphorylation was blocked also by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent membrane bound ATP synthase, and by low concentrations of the inhibitors of electron transport 2-(n-nonyl)-4-hydroxyquinoline-N-oxide or 4-chloromercuriphenylsulfonate.
3. The P/fumarate ratios, measured with the membrane preparation, were found to increase with progressive inhibition of the electron transport from hydrogen to fumarate by means of 4-chloromercuriphenylsulfonate. The extrapolated ratio at vanishing electron transport activity was 0.47.
4. About 50% of the membrane preparation was found to consist of inverted vesicles with the hydrogenase and formate dehydrogenase oriented to the inside. The residual part is considered as being incapable of performing energy transduction. The extrapolated P/fumarate ratio valid for the inverted vesicles was 0.94.

     

Asparaginase production by human clinical isolates of Vibrio succinogenes.

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes.

Homogeneols L-asparaginase with anti-lymphoma activity was prepared from Vibrio succinogenes, an anaerobic bacterium from the bovine rumen. An overall yield of pure L-asparaginase of 40 to 45% and a specific activity of 200 +/- 2 IU per mg of protein was obtained. The pure enzyme can be stored at -20 degrees for at least 3 months with no loss of activity. The isoelectric point of the L-asparaginase is 8.74. No carbohydrate, phosphorus, tryptophan, disulfide, or sulfhydryl groups were detected. The enzyme has a molecular weight of 146,000 and a subunit weight of approximately 37,000. The Km of the enzyme for L-asparagine is 4.78 X 10(-5) M and the pH optimum of the L-asparaginase reaction is 7.3. D-Asparagine was hydrolyzed at 6.5% of the rate found with the L isomer. L-Glutamine and a variety of other amides were not hydrolyzed at significant rates; the activity of the enzyme for L-glutamine was 130- to 600-fold less than that of other therapeutically effective L-asparaginases of bacterial origin. The L-asparaginase from V. succinogenes is immunologically distinct from the L-asparaginase (EC-2) of Escherichia coli.     

Effect of medium composition on the growth and asparaginase production of Vibrio succinogenes.

Asparaginase synthesis by Vibrio succinogenes is induced by ammonium ions. Synthesis occurs throughout exponential phase, and in early stationary phase asparaginase accounts for about 5% of the total soluble protein. The organism grows best when fumarate is provided as the terminal electron acceptor of the formate-oxidizing cytochrome system. Yeast extract or enzyme-hydrolyzed proteins are effective nutrient sources. In an ammonium formate-sodium fumarate medium, where maximum growth and asparaginase synthesis occurs, the total enzyme yield (international units per liter of culture) is about one-tenth that obtainable with a good asparaginase-producing strain of Escherichia coli. The energetic inefficiency of V. succinogenes appears to cause a low yield of cells and therefore low total enzyme yield. However, the levels of asparaginase accumulated within cells raise questions about the organism's protein synthesizing system.     

Ultrastructure of Cell Envelopes of Bacteria of the Bovine Rumen

Most of the bacteria found in rumen fluid samples taken from cows fed hay, or a concentrate diet, had cell walls of the gram-negative type. Most were intact, with only a small proportion of lysed cells, and many of the cells contained electron-translucent cytoplasmic deposits similar to the carbohydrate reserve material described in pure cultures of rumen organisms. All of the bacteria observed in these samples had an external "coat" layer outside the outer membrane when fixed in glutaraldehyde and osmium, stained with uranyl acetate and lead citrate, and examined as sectioned material. These coat layers varied from thin (ca. 8 nm) structures to very extensive fibrous systems, sometimes including concentric arrangements and radial fibers extending up to 1,200 nm from the cell. The thin-coat layers sometimes exhibited a rough periodicity. In all, 10 different types of coat layers were distinguishable on a morphological basis. It is proposed that these external coat layers have protective and adherence functions for the rumen bacteria in the environment.     

九节属药用植物化学成分及药理作用研究进展

九节属植物(Psychotria)是我国南方常用中草药。文献调研表明,关于我国境内本属植物相关化学成分及药理作用研究报道很少。为进一步研究开发Psychotria属药用植物资源,对其化学成分及药理作用进行综述,为其深入开发打下基础。