菠菜和水稻细胞色素b-559的分离纯化及其光谱性质研究

采用一种快速简捷的方法从菠菜(Spinacia oleracea L.)和水稻(Oryza sativa L.)中分离纯化了光系统Ⅱ反应中心内的细胞色素b-559,并且研究了其低温可见光区荧光光谱、室温紫外区荧光光谱、吸收光谱以及电泳特性.该方法的主要特点:1.以放氧核心复合物为起始材料以避免其他细胞色素的干扰;2.选用DEAE-Sephacel为层析介质,用等度洗脱除去杂蛋白和叶绿素;3.用同一介质不同条件去除过量的去垢剂.从两种植物中分离纯化的Cyt b-559具有相似的吸收光谱,在非变性电泳中有相同的泳动特征.用修订的适用于分析小蛋白的Tricine-SDS-PAGE证明,从两种植物中分离得到的Cyt b-559都是由两个多肽亚基组成,它们的表观分子量分别为9 kD和4 kD.低温荧光光谱的结果表明,Crt b-559的荧光激发峰位为413nm和439 nm,荧光发射峰位在563 nm和668 nm,首次证明Cyt b-559可以发出荧光并将电子传递给叶绿素.首次通过Cyt b-559的紫外荧光光谱证明Trp残基位于该蛋白的疏水跨膜区内.     

具放氧功能的PSⅡ反应中心复合物的分离及其特性

用Triton X-100处理PSⅡ颗粒,接着通过DEAE-Toyopearl 650S一步柱层析制备PSⅡ反应中心复合物具有良好的DCIP光还原活性和放氧功能,SDS-PAGE分析表明含有47,43,33,32,30kD和9kD6种多肽组分;还具有和前人用其它方法制备的放氧PSⅡ反应中心复合物相同的吸收光谱和荧光发射光谱。圆二色谱检测证明,本法所制备的复合物仍保持色素蛋白固有的α-螺旋结构与反应中心叶绿素的二聚体存在形式。EPR谱检测证明,该复合物具有保持完好的电子供体D存在;Mn~(2 )参与了电子传递。     

从菠菜制备的47kD/D1/D2/Cyt b559 PSⅡ反应中心蛋白复合物

菠菜的PSⅡ颗粒在pH 6.0、有抗坏血酸钠及甘油存在的条件下,用Triton X-100处理后,经过DEAE-Toyopearl 650S离子交换层析柱分离,可得一个由47 kD,D1,D2及Cyt b559组成的PSⅡ反应中心蛋白复合物.纯化的蛋白质复合物在DPC存在下,具明显的光还原DGIP光化学活性,且在暗及光照条件下显示出SignalⅡ_(slow)及Signal Ⅱ_(fast)。低温吸收光谱和荧光光谱表明,复合物中只有叶绿素a存在;用有机溶剂抽提复合物的色素,采用一种灵敏的荧光分析方法并结合分光光度法进行分析,也证实了这点。此复合物有锰的存在,重要的化学成分中Chl a/Pheo a/Cyt b559/Mn原子的摩尔比为:18.4:2:0.8:0.3。这些结果表明;此复合物含有从PSⅡ第二电子供体Z到第一电子受体Q_A的完整光系统Ⅱ电子传递链的所有组分,同时也暗示复合物可能含有锰原子结合部位。为我们(Tang 1985)提出的水裂解系存在于PSⅡ反应中心系之中的观点提供了佐证。     

光系统Ⅱ反应中心复合物中Cytb559的光还原

以分离纯化的光系统Ⅱ反应中心D1/D2/Cyt b559复合物为实验体系,在厌氧条件下,观察到Cytb559的光还原,表明Cyt b559能直接从Pheo~-接受电子,而且Cyt b559的光还原是不可逆的。当外加次级电子受体2,6-二甲基苯醌(DMBQ)与D1/D2/Cyt b559复合物重组之后,Cyt b559的光还原被延迟了,此时电子主要通过DMBQ传递,而且还原的Cyt b559在光照后的暗放置中有部分氧化。作者认为不依赖于醌受体的由Pheo~-到Cyt b559的电子传递是一条新的、次要的电子传递路线,它对光系统Ⅱ反应中心起保护作用。     

菠菜和水稻细胞色素b—559的分离纯化及其光谱性质研究

采用一种快速简捷的方法从菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）和水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）中分离纯化了光系统Ⅱ反应中心内的细胞色素ｂ－５５９,并且研究了其低温可见光区荧光光谱、室温紫外区荧光光谱、吸收光谱以及电泳特性。该方法的主要特点：１．以放氧核心复合物为起始材料以避免其他细胞色素的干扰;２．选用ＤＥＡＥ－Ｓｅｐｈａｃｅｌ为层析介质,用等度洗脱除去杂蛋白和叶绿素;３．用同一     

黄化油菜突变体Cr3529子叶类囊体膜光谱性质研究

以发育10d的黄化油菜突变体为材料,分析了突变体油菜子叶类囊体膜的色素含量、室温吸收光谱、叶绿素荧光发射和激发光谱以及蛋白内源荧光光谱的变化。数据显示：与野生型相比,突变体油菜子叶类囊体膜的光合色素Chl α和Chl b含量均减少．但Chl α／b比值升高;突变体油菜子叶类囊体膜叶绿素捕光能力和受激发能力均下降,且较依赖于Chl α捕光并将光能激发传递给PSⅡ反应中心;突变体油菜子叶类囊体膜的蛋白内源荧光也明显异于野生型。进一步表明突变体油菜子叶类囊体膜蛋白组成发生了改变。     

光系统Ⅱ反应中心CP47/D1/D2/Cyt b-559复合物中色素分子空间取向的线二色光谱研究

线二色光谱(LD)是研究色素分子在光合膜上空间取向和排布的重要手段.采用低温(100K)吸收光谱和线二色光谱技术研究光系统Ⅱ核心复合物CP47/D1/D2/Cyt b-559中色素分子的空间取向.结果表明,在光系统Ⅱ核心复合物CP47/D1/D2/Cyt b-559中680 nm处有吸收的叶绿素分子Qy跃迁与光合膜平面平行.β-胡萝卜素分子有两种不同的空间取向,其中在470和505nm处有吸收的β-胡萝卜素分子(Ⅰ)与光合膜平面近似平行,而在460和490nm处有吸收的β-胡萝卜素分子(Ⅱ)与光合膜垂直.光破坏实验显示垂直取向的β-胡萝卜素分子对强光敏感.680nm处吸收的叶绿素分子成分复杂,可能包含有P680和核心天线CP47蛋白上的色素分子.     

菠菜类囊体LHC的叶绿素-蛋白中的叶绿素排列和方向的研究

用蔗糖梯度离心的方法,从胰蛋白酶处理叶绿体和对照叶绿体膜分离LHC。比较研究了它们的吸收光谱、荧光光谱、圆二色光谱和荧光偏振度的变化。观测到消化叶绿体LHC的吸收光谱除位于652nm的肩消失外,其它特征性吸收均无明显改变,荧光发射峰位和在670nm的C.D.信号峰位与对照相比发生偏移,叶绿素b和吸收≤670 Nm叶绿素a的荧光偏振度降低。结果说明含叶绿素b的蛋白和短波长叶绿素a的蛋白位于类囊体膜的外侧,胰蛋白酶消化引起LHC的构型改变,从而使色素与色素、色素与蛋白的相互关系及叶绿素分子排列和方向发生改变。讨论了LHC上蛋白构型、叶绿素分子机构和叶绿素分子间能量传递的相互关系。提出了蛋白质在色素能量传递过程中的作用。     

温度升高对PSⅡ中CP47和CP47／D1／D2／Cyt b559 Chla能量传递的影响

采用激励光源为4MHz、514．5nm的延时分幅扫描单光子计数荧光装置对从菠菜中分离提纯的核心天线CP47和CP47／D1／D2／Cyt b559复合物的Chla的能量传递进行了研究,得到经20℃、42℃和48℃处理后的最大峰值处的时问常量。分析认为CP47中,20～42℃之间的温度对蛋白质空间结构的改变较小,Chla分子之间的能量传递受到微小的影响,而42～48℃之间的温度引起较大的蛋白质空间结构改变,明显地影响了其中Chla分子问的能量传递。在PSⅡ(2P47／D1／D2／cyt b559复合物中,处理温度的升高使CP47／D1／D2／cyt b559复合物的二级结构、色素分布的空间位置发生变化,从而影响了CP47／D1／D2／Cyt b559复合物中Chla的能量传递以及电荷重组,42℃已对其造成影响,而48℃对其的影响很大。     

光系统Ⅱ反应中心D_1/D_2/cyt b_(559)复合物光破坏的多步反应

光系统Ⅱ(PSⅡ)反应中心D_1D_2’cyt b_(559)复合物在强光照射下色素分子受到破坏,导致在红区(Q_y带)的吸光度值及CD信号的下降,而且在光照后的暗放置过程中这种变化继续进行,吸收差光谱的峰位在680nm处,说明受破坏的很可能是原初电子供体P680.在光照后的暗放置过程中,该反应中心复合物的荧先强度继续升高,而且峰位蓝移.所有这些结果表明,在光照的过程中,PSⅡ反应中心D_1/D_2/cytb_(559)复合物很可能有一个相对稳定的反应中间体形成,从而造成在暗放置过程中该反应中心继续受到破坏,也就是说,PSⅡ反应中心D_1/D_2/cytb_(559)复合物的光破坏不是一步反应,而是一个多步反应.     

大肠癌及癌旁粘膜中CD44、nm23—H1的表达及其临床病理意义

探讨CD     

评介Smith等的热河群^40Ar—^39Ar同位素测年结果

对Smith等1995年热河群40Ar-39Ar同位素测年工作的背景以及各样品的采样地点、层位与测年目的等作了补充介绍.由于Smith等的论文中将3组后期火山岩的测年结果分别误解为义县组下部与上部地层的同位素年龄,因而整体上混淆了义县组不同层位的测年结果.Smith等1995年发表的6组样品的测年数据实际上至少代表了4个与热河群义县组相关层位的40Ar-39Ar同位素年龄122.9±03Ma(凌源大新房子沉积层下伏火山岩)、122.1±0.2～122.5±03Ma(凌源二十里堡沉积层)、121.5±0.9～121.6±0.5Ma(义县金刚山沉积层上覆火山角砾岩)、120.8±0.4～121.4±0.7Ma(后期火山岩).     

FURTHER STUDIES ON THE K+-DEPENDENT SWELLING OF PRIMATE CEREBRAL CORTEX IN VIVO: THE ENZYMATIC BASIS OF THE K+-DEPENDENT TRANSPORT OF CHLORIDE

Abstract— The swelling of intact, exposed primate cerebral cortex perfused in vioo under, isosmotic conditions was a linear function of the concentration of K⁺ in perfusate over the range 25–117 mM. The K⁺-dependent swelling was manifested throughout the depth of the cerebral cortex studied and was associated with an increased content of chloride in the swollen tissue, despite the constancy of the concentration of external chloride. The swelling of the cerebral cortex was a linear function of the temperature of the perfusate over the range 15–38°C, despite the constancy of the concentration of external K⁺. Moreover, the content of chloride in the swollen cerebral cortex was a linear function of the temperature of the overlying perfusate, despite the constancy of the external concentration of chloride. The changes in the contents of Na⁺ and K⁺ in the swollen cerebral cortex perfused with solutions containing constant concentrations of external Na⁺ and K⁺ but differing in temperature suggested that the fluid of swelling in the tissue was rich in both K⁺ and CI^-, as had been shown previously in vitro. Perfusion of the exposed, intact cerebral cortex in uiuo with K⁺-rich fluids usually involved the reciprocal reduction of the concentrations of Na+ in the perfusate to maintain isotonicity. When comparable reductions in the concentration of external Na⁺ were achieved by replacement with choline (instead of K⁺), swelling of the perfused, exposed cortex was significantly less than that attributed to isotonic, K⁺-rich but Na⁺-poor fluids. These observations suggested that it was the elevated levels of K⁺ rather than lowered concentrations of Na+ that promoted the swelling of the perfused cerebral cortex. The apparent rate of influx of ³⁶Cl from the perfusate into the underlying exposed and intact monkey cerebral cortex in vivo was a linear function of the concentration of K⁺ in perfusate over the range 25–117 mM and conformed to Michaelis-Menten kinetics when plotted according to Lineweaver and Burk. Moreover, the apparent influx of chloride from perfusate into swollen cerebral cortex was a linear function of the percentage swelling of cerebral cortex over the range 6–30 per cent. However, the apparent rate of influx of chloride from perfusate into unswollen cortex was not consistent with the linear correlation already described for swollen cerebral cortex. One reason for this discrepancy was the reduction in the size of the true (inulin) extracellular space associated with the K⁺-dependent swelling of cerebral cortex in vivo. The anatomical locus for this K⁺-dependent swelling of cerebral cortex was an expanded glial compartment, as demonstrated by electron-microscopy. The parenteral administration (50 mg/kg) or local perfusion (5 mM) of acetazolamide inhibited the K⁺-dependent swelling of cerebral cortex in vivo. Moreover, administration of acetazolamide inhibited the K⁺-dependent increase in content of C1- and the K⁺-dependent rate of influx of ³⁶Cl into swollen cerebral cortex. We have discussed the possible enzymatic basis of these K⁺-dependent alterations in content of fluid and chloride and transport of chloride in mammalian cerebral cortex in viuo.     

内蒙古锡林河流域主要类型草原土壤中CH4和CO2浓度的变化

 于1999年生长季对内蒙古锡林河流域主要类型草原土壤中CH4和CO2浓度进行测定,结果表明：CH4浓度沿土壤剖面逐渐降低,而且不同土壤深度之间差异显著,而CO2浓度呈现出沿土壤剖面增加的趋势。草甸草原、羊草(Leymus chinesis )草原和大针茅(Stipa grandis)草原土壤中CH4的浓度差异显著,季节变化明显,但是三类草原土壤中CO2浓度变化不大。测定结果还表明：一定时间尺度上,放牧对草原土壤中CH4和CO2的浓度没有显著影响。     

EVIDENCE FOR INVOLVEMENT OF Ca2+ IN THE INCORPORATION OF 32Pi INTO THE PHOSPHATIDYLINOSITOL OF RAT DIAPHRAGM

Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of ³²P_i into phosphatidylinositol (PI) in rat diaphragm incubated in Ca²⁺-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of ³²P_i into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Ca^a+-chelating properties; the inhibition of the incorporation of ³²P_i could be completely reversed by the addition of excess Ca²⁺ but not Mg²⁺. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca²⁺, rather than extracellular Ca²⁺, was involved in the effect. In contrast to its inhibition of the incorporation of ³²P_i EGTA enhanced the incorporation of [³H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [³Hlinositol by the tissue. Our results suggest that the level of intracellular Ca²⁺ plays a role in the regulation of the incorporation of ³²P_i into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of ³²P_i into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of ³²P_i into α-glycerophosphate.     

THE RELATIVE SIGNIFICANCE OF CO2-FIXING ENZYMES IN THE METABOLISM OF RAT BRAIN

To evaluate the relative significance of CO₂-fixing enzymes in the metabolism of rat brain, the subcellular distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase, as well as the fixation of H¹⁴CO₃^? by the cytosol and the mitochondria was investigated. Pyruvate carboxylase and phosphoenol-pyruvate carboxykinase are mainly localized in the mitochondria whereas NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase are present in both the cytosol and the mitochondria. In the presence of pyruvate rat brain mitochondria fixed H¹⁴CO₃^? at a rate of about 170 nmol/g of tissue/min whereas these organelles fixed negligible amounts of H¹⁴CO₃^? in the presence of α-ketoglutarate or phosphoenolpyruvate. Rat brain cortex slices fixed H¹⁴CO₃^? at a rate of about 7 nmol/g of tissue/min and it was increased by two-fold when pyruvate was added to the incubation medium. The carboxylation of α-ketoglutarate and pyruvate by the reversal of the cytosolic NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase respectively was very low as compared to that by pyruvate carboxylase. The rate of carboxylation reaction of both NADP-isocitrate dehydrogenase and NADP-malate dehydrogenase was only about 1/10th of that of decarboxylation reaction of the same enzyme. It is suggested that under physiological conditions these two enzymes do not play a significant role in CO₂-fixation in the brain. In rat brain cytosol, citrate is largely metabolized to α-ketoglutarate by a sequential action of aconitate hydratase and NADP-isocitrate dehydrogenase. The operation of the citrate-cleavage pathway in rat brain cytosol is demonstrated. The data show that among four CO₂-fixing enzymes, pyruvate carboxylase, an anaplerotic enzyme, plays the major role in CO₂-fixation in the brain.     

INCORPORATION OF 3H-LABELLED LEUCINE INTO THE PROTEIN FRACTION IN THE RETINA OF THE HONEYBEE DRONE

Abstract— Protein synthesis in the retina of the honey-bee drone was studied by incubating head slices in labelled leucine and measuring the TCA insoluble radioactivity. It was found that the protein-bound radioactivity in illuminated retinas was half of that in dark-adapted ones. This ratio was not affected by pre-treatment with puromycin. It was therefore concluded that, in the drone, the main influence of illumination is to increase the rate of breakdown of proteins.
Relatively high concentrations of labelled protein were found in dark-adapted retinas when the retinula cells were hyperpolarized by bathing the preparation in a sodiumfree medium; low concentrations were found when retinula cells were depolarized by increasing the extracellular potassium concentration. These findings suggest that protein metabolism of the retina is influenced by the membrane potential of retinula cells.