KCN和NaN3预处理对烟草愈伤组织抗氰呼吸的影响

用０．５ ｍ ｍ ｏｌ／Ｌ ＫＣＮ 或０．０１ ｍ ｍ ｏｌ／Ｌ ＮａＮ３ 预处理继代培养２５ ｄ 的“甘肃黄花烟草”（Ｎｉｃｏｔｉａｎａ ｒｕｓｔｉｃａ Ｌ． ｃｖ． Ｇａｎｓｕ ｙｅｌｌｏｗ ｆｌｏｗ ｅｒ）愈伤组织对其呼吸作用有明显的影响。ＫＣＮ 和ＮａＮ３ 预处理１２ ｈ 可分别使愈伤组织的总呼吸速率下降１２％ 和１７％ ,细胞色素途径的实际运行量分别下降２２％ 和２８％ ;抗氰交替途径在最大容量（Ｖａｌｔ）基本不变的情况下,实际运行量（ρ·Ｖａｌｔ）不但未随总呼吸速率和细胞色素途径运行量的下降而降低,反而还略有上升。结果导致细胞色素途径对总呼吸的贡献下降而抗氰交替途径对总呼吸的贡献上升。ＫＣＮ 预处理２４ ｈ,细胞色素途径的实际运行量虽略有回升,但仍低于对照;ＮａＮ３ 预处理２４ ｈ 则使细胞色素途径运行继续下降而交替途径运行持续上升。上述结果表明,在继代培养的烟草愈伤组织中,当细胞色素主路途径被部分抑制后交替途径的电子流量明显加强,从而起着一种补偿作用     

Molecular interaction of acrylonitrile and potassium cyanide with rat blood

The interaction of acrylonitrile (VCN) with rat blood has been investigated at the molecular level in an attempt to understand the possible mechanism of its toxicity. The results obtained were compared to those with potassium cyanide (KCN), a compound known to liberate cyanide (CN^?) in biologic conditions. The radioactivity derived from K¹⁴CN was eliminated faster than that from [1-¹⁴C]VCN. Up to a maximum of 94% of ¹⁴C from VCN in erythrocytes was detected covalently bound to cytoplasmic and membrane proteins, whereas 90% of the radioactivity from KCN in erythrocytes was found in the heme fraction of hemoglobin. Determination of specific activity showed that binding occurred more in vivo than in vitro which indicated that the VCN molecule was bioactivated inside erythrocytes. These results indicate that KCN interacts mainly through CN^? liberation and binding to heme, whereas VCN, which binds to cytoplasmic and membrane proteins, may cause damage to red cells by mechanisms other than release of CN^?.     

Alternative Cyanide-sensitive Oxidase Interacting with Photosynthesis in Synechocystis PCC6803. Ancestor of the Terminal Oxidase of Chlororespiration?

Influence of respiration on photosynthesis in Synechocystis PCC6803 was studied by measuring the redox transients of cytochrome f (cyt f) upon excitation of the cells with repetitive single turnover flashes. Upon the addition of KCN the flash-induced oxidation of cyt f was increased and the rereduction of cyt f+ was accelerated. Dependence of these effects on the concentration of KCN clearly demonstrated the existence of two cyanide-sensitive oxidases interacting with photosynthesis: cyt aa3, which was sensitive to low concentrations of cyanide, and an alternative oxidase, which could be suppressed by using 1 mM KCN. The interaction between the photosynthetic and the respiratory electron transport chains was regulated mainly by the activity of the alternative cyanide-sensitive oxidase. The oxidative pathway involving the alternative cyanide-sensitive oxidase was insensitive to salicyl hydroxamic acid and azide. The close resemblance of the inhibition pattern reported here and that described for chlororespiration in algae and higher plants strongly suggest that an oxidase of the same type as the alternative cyanide-sensitive oxidase of cyanobacteria functions as a terminal oxidase in chloroplasts.     

Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

Active Cl− absorption by the Chinese crab (Eriocheir sinensis) gill epithelium measured by transepithelial potential difference

Summary Isolated posterior gills from Chinese crabs (Eriocheir sinensis) acclimated to tap-water were perfused and bathed with full, physiological saline (containing Na⁺ and Cl^–). Under these conditions they developed an outside positive transepithelial potential difference (PD_te). Substitution of Na⁺ by choline on both sides of the epithelium resulted in a substantial hyperpolarization of the PD_te, while substitution of Cl^– by gluconate reverses PD_te to outside negative values.The magnitudes of the potential differences were strongly related to the adaptation media (artificial seawater or tap-water).The KCN-sensitive, outside positive PD_te was shown to be strongly dependent on Cl^–. Application of thiocyanate and 4-acetamido-4-isothiocyanato-stilbene-2,2 disulfonic acid (SITS) to the bath solution resulted in a reduction of the PD_te, while the Cl^–-channel blocker, diphenylamine-2-carboxylic acid (DPC), showed no effect. The PD_te was markedly reduced by acetazolamide, an inhibitor of carbonic anhydrase (CA), and these results are discussed with reference to the presence of a Cl^–/HCO ₃ ^– -exchanger in the apical membrane.Chloride was shown to pass the basolateral membrane via Cl^–-channels: Diphenylamine-2-carboxylic acid (DPC) reduced the PD_te with an IC₅₀ of 3.7×10^–5 mol/l when added to the perfusion saline. A basolateral K⁺-channel and its linkage to Cl^– uptake could be demonstrated by using the K⁺-channel blocker, Ba²⁺, or increased K⁺ concentrations in the perfusion saline (PD_te decrease). Ouabain did not reduce the PD_te under nominally Na⁺-free conditions, indicating that the Cl^– transport is independent of the Na⁺/K⁺-ATPase. In this paper we shall discuss the possible energy sources and linkages between pH regulation and active Cl^– absorption under these experimental conditions.Abbreviations A9C anthracene-9-carboxylic acid - CA carbonic anhydrase - DMSO dimethylsulfoxide - DPC diphenylamino-2-carboxylic acid - PD _te transepithelial potential difference - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TEA tetraethyl-ammoniumchloride     

Alterations in Electron Transport Characteristics during Senescence of Cucumis Cotyledonary Leaves. Analysis of the Effects of Inhibitors

Cotyledonary leaves of Cucumis sativus cv. Poinsette exhibited senescence-induced losses in chlorophyll (Chl) and protein contents within three weeks since germination. Chl and protein concentrations in cotyledonary leaves approached maximum on 6th d after germination and they declined to 50 and 41 %, respectively, by the 20th day of growth. Activities of both photosystem (PS) 2 and PS1 decreased by 33 and 31 %, respectively, on the 20th day, compared to the control 6th day. Changes in sensitivity of PS2 to inhibitors like atrazine and dibromothymoquinone and sensitivity of PS1 to KCN accompanied the changes in PS2 and PS1 activities. Hence both the acceptor side of PS2 and the donor side of PS1 are affected by senescence-induced changes in cucumber cotyledonary leaves.     

Alternative and cytochrome pathway respiration during shoot bud formation in cultured Pinus radiata cotyledons

Respiration rates for excised cotyledons of Pinus radiata cultured in the presence (shoot-forming) and absence (non-shoot-forming) of N⁶-benzyladenine (BA) over a 21-day period were measured using a Clark-type oxygen electrode. The capacities and activities of cytochrome and alternative pathways of respiration were determined from titrations with KCN (1-10 m M ) and salicylhydroxamic acid (2–20 m M ) individually and in combination. Respiration accounted for by alternative (AP) and cytochrome (CP) pathways varied with both culture treatment and age in culture. Rates of total respiration, CP respiration and AP activity rose concurrent with key developmental events of shoot bud formation. The greatest AP capacity was measured at day 3 in shoot-forming tissue. In contrast, for cotyledons cultured under non-shoot-forming conditions, no AP activity was observed after day 3 despite relatively constant AP capacity throughout the culture period. Although initial increases in cotyledon respiration during the culture period may be related to wounding and introduction to a tissue culture environment, later differences in respiratory patterns between shoot-forming and non-shoot-forming cotyledons appear to be associated with the cytokinin-induced developmental changes which give rise to shoot primordia in cultured radiata pine cotyledons.     

Effects of KCN and NaN3 Pretreatment on the Cyanide-Resistant Respiration in Tobacco Callus

The effects of KCN (0.5mmol/L) and NaN3 (0.01 mmol/L) pretreatment on the operation of the alternative pathway in subcultured tobacco (Nicotiana rustica L. cv. Gansu yellow flower) callus were analyzed. After treatment with KCN and NaN3 for 12 h, the total respiration rate (Vt) decreased by 12% and 17%, whereas oxygen consuption by the cytochrome pathway decreased by 22% and 28% respectively. The capacity of the alternative pathway (Valt) remained constant, while the activity of the alternative pathway (ρ· Valt ) inreased slightly. This changing pattern led to a declined contribution of the cytochrome pathway to the total respiration rate and an increased activity of the alternative pathway. Treatment with KCN for 24 h brought about a slight rise of oxygen consumption by the cytochrome pathway as compared with that in callus treated for 12 h, but the oxygen consumption was still lower than that in the untreated callus. Treatment with NaN3 for 24 h resulted in a profound decrease of the cytochrome pathway operation and a continuing increase of the alternative pathway operation. These data indicated that the enhanced operation of the alternative pathway played a compensatory role to the total respiration when the cytochrome pathway was partially inhibited in tobacco callus.     

Cytochrome c is released from coupled mitochondria of yeast en route to acetic acid-induced programmed cell death and can work as an electron donor and a ROS scavenger

To gain insight into the processes by which acetic acid-induced programmed cell death (AA-PCD) takes place in yeast, we investigated both cytochrome c release from yeast mitochondria and mitochondrial coupling over the time course of AA-PCD. We show that the majority of cytochrome c release occurs early in AA-PCD from intact coupled mitochondria which undergo only gradual impairment. The released cytochrome c can be reduced both by ascorbate and by superoxide anion and in turn be oxidized via cytochrome c oxidase, thus working both as a ROS scavenger and a respiratory substrate. Late in AA-PCD, the released cytochrome c is degraded.