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    I:Cell Aggregation Induced FGF8 Elevation is Essential for P19 Cell Neural Differentiation II:Visualization of bHLH Transcription Factor Interactions in Living Mammalian Cell Nuclei and Developing Chicken Neural Tube by FRET
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    STUDIES ON THE ENERGY-TRANSDUCING ATPase COMPLEX OF BIOLOGICAL MEMBRANCES:DISSOCIATION AND RECONSTITUTION OF RAT LIVER MITOCHONDRIAL F_1-ATPase
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    STUDIES ON THE ENERGY-TRANSDUCING ATPase COMPLEX OF BIOLOGICAL MEMBRANCES:DISSOCIATION AND RECONSTITUTION OF RAT LIVER MITOCHONDRIAL F_1-ATPase
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  energy
Finite energy band-limited functions are reconstructed iteratively
      
When, in addition, the Tolimieri-Orr condition A is satisfied, the minimum energy dual windowoγ ε L2(?) can be sampled as well, and the two sampled windows continue to be related by duality and minimality.
      
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Chloroplasts, prepared from fresh, prechilled spinach leaves by the method of Jagendorf andAvron, were suspended in Tris buffer (25 μmoles, pH 8.4) containing MgCl_2 (6 μmoles), ADP(3 μmoles), P~(32)-labelled phosphate (3 μmoles), NaCl (25 μmoles) and the different cofactors. Totalvolume of the reaction mixture was 1.25 ml, containing 30-40 μg chlorophyll. Preliminary experi-ments showed that at the low illumination intensity for quantum yield determinations the optimal quan-ties of the co-factors were: PMS...

Chloroplasts, prepared from fresh, prechilled spinach leaves by the method of Jagendorf andAvron, were suspended in Tris buffer (25 μmoles, pH 8.4) containing MgCl_2 (6 μmoles), ADP(3 μmoles), P~(32)-labelled phosphate (3 μmoles), NaCl (25 μmoles) and the different cofactors. Totalvolume of the reaction mixture was 1.25 ml, containing 30-40 μg chlorophyll. Preliminary experi-ments showed that at the low illumination intensity for quantum yield determinations the optimal quan-ties of the co-factors were: PMS or FMN, 0.005 μmole, vitamin K_3, 0.03 μmole, and of the Hilloxidants were: K_3Fe(CN)_6 0.6 μmole, TPN 0.2 μmole. Neon tube with dilute ammoniacal CuSO_4 solution and red-glass filters was used as light source.The wave length range was 620-660 mμ and the intensity was 6-8×10~3 ergs/cm~2/sec. Energydeterminations were made with a blackened constantin-copper thermopile, the absolute energy wascalibrated by the amount of heat produced electrically at the surface of the thermopile. The calcu-lated number of quanta was counter-checked by chlorophyllide actinometer according to Warburg.Light scattering was corrected by the ground glass method of Shibata. Phosphorylation rates weremeasured by ATP~(32) formed according to the method of Nielsen and Lehninger. No incorporation of P~32 was detectable in the dark. To avoid loss of activity preparations were conducted near 0℃ and experiments were completedwithin 10 minutes including isolation of chloroplasts. Representative results are given in Tab. 1. The following conclusions can be drawn. (1) The quantum requirement of cyclic photophosphorylation is between 2.9-6.5 (generally4-5) per molecule of ATP formed, irrespective of the co-factors used,. Although at high lightintensities PMS can be twice as active as vitamin K_3 or FMN, their quantum yields are the same. (2) Same numerical results are obtained with heat deproteinized leaf extract in place of co-factors. (Tab. 3). (3) Non-cyclic photophosphorylation with K_3Fe(CN)_6 or with TPN shows the same quantumrequirement of 4-6. (Tab.1). Apparently one and the same electron transport system is involvedin both types of phosphorylation and there exists probably only one phosphorylation site. However,the possibility of a second easily inactivated site is not excluded. (4) The simultaneously measured Hill reaction requires 9-12 quanta per molecule of O_2evolved with or without photophosphorylation (+ or -ADP, Tab.2). The result corroboratesthose of other workers. It further shows that under the conditions of the present experiment cou-pling is complete (P/2e = 1), and that no extra quantum is needed for the coupled formation ofATP. (5) The quantum requirement of both cyclic and noncyclic phosphorylation increased withdecreasing light intensity within the range used (1.3-6.0×103 ergs/cm~2/sec.) (Tab. 4), whereasthat of O_2 production by the Hill reaction remains constant, thus resulting in a progressive uncou-pling of phosphorylation from the electron transport chain. The relationship between the present results and the quantum requirement of photosyntheticCO_2-reduction is discussed. That at low intensities of illumination used in the present experimentsthe number of quanta required for 2 TPNH and 2 ATP formation equals to that of photosynthesisunder these conditions (~compensation point) indicated that cyclic production of ATP is perhapsnot involved and the extra ATP needed for CO_2-reduction in the Calvin cycle must come fromsome other source, e.g. respiration. That at higher light intensities (several times compensationpoint) the quantum requirement of photosynthesis increases is probably due to the coming into playof the photochemically inefficient cyclic photophosphorylation.

用菠菜叶绿体悬浮液,在红光下(620—660mμ,6—8×10~3尔格/厘米~2-秒)测定同位素P~(32)标记的无机磷酸进入ATP的强度,并根据吸收的光能量换算为形成一个分子ATP所需要的红光量子数。结果指出: (1)循环光合磷酸化作用,不论用何种辅助因素(PMS,维生素K_3,FMN),形成一个分子ATP的量子需要量均在4—5之间(最低一次获得2.9)。叶提取液代替辅助因素,结果亦同。(2)与希尔反应偶联的光合磷酸化作用(希尔氧化剂为K_3Fe(CN)_6或TPN)的量子需要量亦是4—6。同时测定的还原作用指出希尔反应中每放出一个分子O_2,需要8—12个红光量子,表示在试验条件下,二者是完全偶联的(P/2e?1)。没有磷酸化(不加ADP及P_i)时,希尔反应的量子需要量不变,表示偶联的ATP形成不需额外的光量子。(3)光强度减低,则循环与非循环光合磷酸化作用的效率随之降低,量子需要量增加,而希尔反应的效率则不变。从上述结果推论,两种光合磷酸化作用均是通过同一的电子传递系统,在此系统中仅有一个磷酸化部位,除非另有一个部位是极易破坏的。试验结果也对光合作用的量子需要量问题,供给可能的解释。在弱光...

用菠菜叶绿体悬浮液,在红光下(620—660mμ,6—8×10~3尔格/厘米~2-秒)测定同位素P~(32)标记的无机磷酸进入ATP的强度,并根据吸收的光能量换算为形成一个分子ATP所需要的红光量子数。结果指出: (1)循环光合磷酸化作用,不论用何种辅助因素(PMS,维生素K_3,FMN),形成一个分子ATP的量子需要量均在4—5之间(最低一次获得2.9)。叶提取液代替辅助因素,结果亦同。(2)与希尔反应偶联的光合磷酸化作用(希尔氧化剂为K_3Fe(CN)_6或TPN)的量子需要量亦是4—6。同时测定的还原作用指出希尔反应中每放出一个分子O_2,需要8—12个红光量子,表示在试验条件下,二者是完全偶联的(P/2e?1)。没有磷酸化(不加ADP及P_i)时,希尔反应的量子需要量不变,表示偶联的ATP形成不需额外的光量子。(3)光强度减低,则循环与非循环光合磷酸化作用的效率随之降低,量子需要量增加,而希尔反应的效率则不变。从上述结果推论,两种光合磷酸化作用均是通过同一的电子传递系统,在此系统中仅有一个磷酸化部位,除非另有一个部位是极易破坏的。试验结果也对光合作用的量子需要量问题,供给可能的解释。在弱光下光合作用效率高,可能是由于部份ATP来自呼吸;而在强光下效率减低,则是呼吸所供给的ATP不足而必需依靠循环光合磷酸化所致。

The effect of 2,4-dinitrophenol on the oxidation of succinate in rat-liver mitochondria has been studied with the vibrating platinum microelectrode. It has been found that DNP stimulated the rate of respiration only for a short period of about 1 minute, and this was followed by an inhibited phase of the oxygen-uptake. The inhibited phase no longer appeared if inorganic phosphate was added before, together with or shortly after the addition of DNP when the respiration was still in the stimulated phase, whereas...

The effect of 2,4-dinitrophenol on the oxidation of succinate in rat-liver mitochondria has been studied with the vibrating platinum microelectrode. It has been found that DNP stimulated the rate of respiration only for a short period of about 1 minute, and this was followed by an inhibited phase of the oxygen-uptake. The inhibited phase no longer appeared if inorganic phosphate was added before, together with or shortly after the addition of DNP when the respiration was still in the stimulated phase, whereas after the inhibited phase had set in, the addition of inorganic phosphate had no stimulator(?) effect on the rate of respiration. Arsenate could not replace inorganic phosphate in prolonging the stimulating effect of DNP on succinate oxidation. Neither ADP nor ATP could directly restore the respiratory activity of the above system in the inhibited phase. ADP added after DNP and inorganic phosphate was even somewhat inhibitory. Slow increase in respiration of the DNP inhibited system was observed after short incubation with ATP. These facts suggest that the factor which is essential for maintaining a high rate of oxidation in the DNP-treated system is not ATP or inorganic phosphate as such, but is produced from inorganic phosphate during respiration. It has been suggested as a wo(?)king hypothesis that it is a high energy phosphate intermediate of oxidative phosphorylation, which is essential for maintaining a high rate of oxidation of succinate through the phosphorylating respiratory chain.

利用自动記录振动白金微氧电极的方法观察到DNP对綫粒体中琥珀酸氧化的短暫激活和抑制作用诩尤隓NP以后,呼吸明显被激活,但仅持續約1分鉀,随即逐漸受抑制。加入DNP以前,或在加入DNP以后而呼吸仍在激活阶段时加入无机磷酸盐都可以使呼吸激活阶段大大延长,抑制作用延緩出現。但若在DNP加入以后呼吸已被抑制时方行加入无机磷酸盐則不能使呼吸重行激活。砷酸盐不能代替无机磷酸产生相似效应。在呼吸受DNP抑制以后,加入ADP及ATP都不能使呼吸立刻重行激活,但加入ATP經过保温以后可以使呼吸漸漸有所增加。根据本文結果,我們认为在琥珀酸氧化过程中,氧化磷酸化作用中間物x~P可能参与能量反馈作用,当它的衡态浓度因DNP的作用而降低吋,琥珀酸的氧化即受抑制。根据本文結果,我們认为DNP对琥珀酸氧化抑制的各种現象。似乎不能簡单以草酰乙酸堆积完全加以解释。

As reported by Borst and Slater, dinitrophenol (DNP), the uncoupling agent, can under certain conditions cause an inhibition of the oxidation of succinate by intact mitochondria. We have studied this effect of DNP on succinate oxidation by respirationcontrolled rat-liver mitochondria with a vibrating platinum microelectrode and found that, in absence of added inorganic orthophosphate and adenosine diphosphate (ADP), the action of DNP is closely related to its concentrations. When DNP is added in low concentration...

As reported by Borst and Slater, dinitrophenol (DNP), the uncoupling agent, can under certain conditions cause an inhibition of the oxidation of succinate by intact mitochondria. We have studied this effect of DNP on succinate oxidation by respirationcontrolled rat-liver mitochondria with a vibrating platinum microelectrode and found that, in absence of added inorganic orthophosphate and adenosine diphosphate (ADP), the action of DNP is closely related to its concentrations. When DNP is added in low concentration (5-10μM), succinate oxidation is stimulated, the rate of respiration being increased to greater degrees with increasing concentrations of DNP up to 10μM, with no indication of any inhibition. When the DNP concentration is increased to above 30μM, however, stimulation lasts for only a short time, followed by a decrease in the rate of oxidation. The use of still higher concentrations of DNP results in increasingly lower degrees, and shorter periods, of stimulation, followed sooner by inhibition.In agreement with findings reported by other workers, a preincubation with 2mM amytal has been shown to prevent completely the inhibition of succinate oxidation by high concentrations of DNP. In addition, it has been discovered that even after the appearance of the inhibition, amytal is still effective in alleviating the DNP effect, causing a partial restoration of the respiratory rate.The addition of another uncoupling agent, arsenate, in concentrations of 2-10mM also stimulates succinate oxidation and 4mM arsenate is able to elicit a maximal rate of respiration. In contrast with DNP, no inhibition of respiration can.be observed even with the higher concentrations of arsenate used. Nevertheless, after preincubation with 2 mM amytal, the stimulation of respiration caused by arsenate is found to last for only two minutes, thereafter the rate of oxidation begins to decline and respiration gradually becomes inhibited. Adenosine triphosphate (ATP) is able to prevent the appearance of the inhibited phase of succinate oxidation in both cases, i.e. in either DNP or amytal-arsenate treatment, with however, slightly different efficiency. The addition of ATP enables the respiration in presence of high concentrations of DNP to proceed at a good steady rate, not more, however, than 2/3 of the maximal rate obtainable under optimal conditions, while the same amount of ADP is able to abolish completely the inhibiting effect caused by the amytal-arsenate treatment with the ensuing rate of respiration even higher than that elicited by arsenate alone.The characteristics of the inhibiting actions of these two agents on succinate oxidation and the effect of ATP on them have been discussed. The experimental evidence presented is believed to support the postulation that the oxidation of succinate in liver mitochondria requires an intervention of energy. The possibility that under certain experimental conditions, accumulation of oxaloacetate may play a part in affecting the rate of succinate oxidation is considered likely but of only minor importance.

(1)DNP对于琥珀酸氧化的影响随浓度不同而异,低浓度时,呼吸受激活,其程度随浓度升高而增強,不出現抑制現象。高浓度(30μM以上)DNP只引起短时間氧化激活,随即引起抑制,随浓度升高,抑制出現愈早,氧化激活愈小,預先加入Amytal足以防止上述抑制現象,在抑制出現后加入Amytal亦能使琥珀酸氧化部分恢复。(2)砷酸盐激活的琥珀酸氧化仅在有Amytal的条件下,才出現抑制現象。(3)ATP对上述两种情况引起的琥珀酸氧化抑制都具有一定的解除作用。(4)就实驗結果所做分析支持琥珀酸氧化需要能量激活的看法。

 
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