应用稳定同位素技术构建胶州湾食物网的连续营养谱

根据2011年春季和秋季在胶州湾进行的渔业资源综合调查,应用稳定同位素示踪技术,分析了胶州湾主要渔业生物的碳、氮稳定同位素比值(δ13C,δ15N),并计算其营养级,进而构建胶州湾食物网的连续营养谱。分析的生物种类包括浮游植物、浮游动物、大型无脊椎动物和鱼类,其生物量之和占总渔获量的95%。结果表明,胶州湾食物网的δ13C值范围是-25.63‰—-17.16‰,跨度为8.47‰,平均值为(-19.42±1.80)‰;δ15N值范围是4.15‰—14.11‰,跨度为9.96‰,平均值为(11.98±1.77)‰。胶州湾食物网中的主要生物种类可以划分为4个营养组群,即初级生产者、初级消费者、次级消费者以及顶级捕食者。δ15N值分析显示,胶州湾主要生物种类的营养级范围是1.10—4.03。与文献中基于胃含物分析计算的营养级相比较,37个种类中有29种的营养级分析结果基本一致(在0.5个营养级的误差范围之内)。因此,氮稳定同位素法是一种研究水生生态系统食物网营养位置的有效方法。其中,有8种鱼类的营养级与历史文献相比有所下降,分析方法的不同可能是原因之一,此外,这些鱼种摄食饵料生物营养级的下降也是导致其营养级降低的另一个主要原因。根据营养级计算的结果,构建了胶州湾食物网的连续营养谱,胶州湾食物网中,绝大多数生物种类都属于初级和中级肉食性种类。     

大连海域食物网连续营养谱

根据2008年1月—2017年6月在大连海域收集的因搁浅、误捕及救助无效而死亡的斑海豹、江豚、小须鲸等海洋哺乳动物及2016年秋季和2017年春季在该海域进行的渔业资源调查,应用稳定同位素技术,分析了大连海域海洋哺乳动物及主要生物样品的碳、氮稳定同位素比值(δ¹³C、δ¹⁵N),并计算其营养级,进而构建大连海域食物网的连续营养谱.结果表明: 大连海域食物网的δ¹⁵N值范围为8.0‰～14.7‰,δ¹³C值范围为-21.1‰～-16.7‰.主要生物种类可划分为初级消费者、次级消费者及顶级捕食者3个营养组群.δ¹⁵N值分析显示,主要生物种类的营养级范围为2.63～4.59,其中,小须鲸、江豚、斑海豹的营养级依次为3.16、4.11、4.25,棘皮动物为3.24～3.84,头足类为3.81～3.93,腹足类为3.65～4.13,双壳类为2.63～3.15,甲壳类为3.58～4.12,鱼类为3.20～4.59.营养结构特征显示,初级消费者主要为双壳类,次级消费者主要为小须鲸、头足类、棘皮类、腹足类、甲壳类,顶级捕食者主要为江豚、斑海豹、鱼类.随着江豚体长的增加,δ¹⁵N值有增大趋势,说明随着江豚生长和摄食能力的增强,其摄食的食物趋向于更高营养层次的生物.研究建立了大连海域食物网的连续营养谱,可以为海洋哺乳动物和渔业资源的保护提供依据.     

枸杞岛近岸海域食物网的稳定同位素分析

为了探明岛礁近岸海域食物网主要生物类群之间的营养关系,对2012年11月和2013年2月枸杞岛近岸的消费者及其潜在食物源的稳定碳、氮同位素组成进行了分析,并利用氮稳定同位素数据计算了消费者的营养级。结果表明:枸杞岛近岸海域消费者潜在食源浮游植物、POM、SOM和大型海藻的δ13C值范围为-21.7‰~-14.7‰,浮游动物、大型无脊椎动物和鱼类等消费者的δ13C值范围为-21.1‰~-13.7‰。初级生产者的C/N平均值为8.5,消费者的C/N平均值3.7,差异极显著(P0.001)。根据消费者的δ15N值,可将枸杞岛近岸海域的消费者分为滤食性浮游动物、食藻或碎屑的小型底栖动物、杂食性的大型无脊椎动物和鱼类以及凶猛的肉食性鱼类4大类,消费者共有4个营养等级,黑鲷的营养级最高为4.33。     

利用碳氮稳定同位素技术分析东海银鲳食性

利用稳定同位素技术分析了东海银鲳(Pampus argenteus)及其可能摄食饵料的碳、氮稳定同位素比值,以期探讨东海银鲳可能的食物来源.结果表明:东海区银鲳平均碳(813C)和氮(δ15N)稳定同位素比值分别为-18.22‰、8.16‰,其可能摄食饵料的δ13C值变化范围为-17.33‰～-21.58‰,差值为4.25‰,δ15N值变化范围为3.89‰～7.96‰,差值为4.07‰;东海银鲳可能的食物来源主要为箭虫、虾类、水母类、头足类、仔稚鱼和浮游动物等,其中箭虫为主要可能的食物来源,其贡献率为24%～78%,平均贡献率为57%;银鲳可能摄食的其他饵料中,贡献率从大到小依次为虾类、水母类、头足类、仔稚鱼、＞1000 μm浮游动物、500～1000 μm浮游动物和100～500 μm浮游动物,其平均贡献率分别为11.8%、8.4%、7.1%、5.0%、4.9%、3.2%和2.6%.由以上结果可知,银鲳是一种广食性鱼类,其饵料种类较多,碳和氮的来源均较为复杂.     

饶河枯水期主要鱼类营养级位置及其影响因素

稳定同位素技术已经越来越多地被用来研究淡水生态系统的结构与功能。利用氮稳定同位素技术测定了枯水季节饶河鱼类等消费者的营养级位置,比较上、中、下游及入湖口鱼类营养级的空间差异,并分析了影响饶河鱼类营养级位置的主要因素。研究结果表明,饶河鱼类的δ15N值范围为4.7‰—15.6‰,大部分鱼类的δ15N值集中在10‰—14‰之间,其中鄱阳湖间下鱵的δ15N值最大,为(15.6±1.6)‰;乔木湾鲫的δ15N值最小值,为(4.7±0.9)‰。根据δ15N值计算可知,饶河鱼类占有3—4个营养等级。75%的鱼类种类所占的营养级大于3,而营养级小于2的鱼类种类不到10%,可能与枯水期鱼类活动范围受限,种间捕食作用增强,肉食性或饥饿现象增加有关。另外,饶河鱼类的营养级也存在着空间差异,表现为鄱阳湖湖区和入湖口处的鱼类营养级比上、中、下游的鱼类营养级要大。该结果与颗粒有机物POM的δ15N值呈现一致的变化,反映了饶河鱼类的营养级位置主要受到食物来源的影响,与鱼类的个体大小无明显相关。     

草海湿地食物链稳定碳氮同位素特征与食物链结构

以国家级自然保护区贵州威宁草海为研究对象,利用稳定同位素技术,分析草海湿地食物链碳(δ~(13)C)、氮(δ15N)同位素特征,计算各生物类群营养级别,建立草海食物链结构。结果表明:草海湿地生态系统中δ13C比值范围为-27.56‰～-13.25‰(均值±标准差,-21.52‰±3.61‰);δ15N值范围为0.32‰～15.14‰(8.69‰±3.92‰),δ13C与δ15N呈显著负相关(r=-0.423,P0.01)。草海湿地食物链中消费者营养级处于0.8～3.7,其中:鱼类营养级为0.8～2.5,相对其他地区偏低;底栖动物营养级为2.0～2.8,鸟类营养级为1.0～3.7。鱼类和底栖动物的营养级别均表现为肉食性杂食性植食性。草海食物链结构复杂,主要的两条碳流动途径分别为:底泥和浮游植物→浮游动物→鱼类→鸟类以及水生植物→鱼类和鸟类。     

应用碳、氮稳定同位素研究稻田多个物种共存的食物网结构和营养级关系

稳定碳、氮同位素比值分析技术是研究生态系统中物质循环与能量流动的有效技术。δ13C值常用来分析消费者食物来源,δ15N值常用来确定生物在食物网中的营养位置。应用稳定同位素技术分析了稻-鱼(R-F)和稻-鱼-鸭(R-F-D)两种稻作方式下,稻田多个物种共存的食物网结构和营养级关系。结果表明,R-F中SOM的δ13C值为(-27.7±0.3)‰,与R-F-D(-27.4±0.4)‰,相差不大;R-F中POM的1δ3C值为(-27.4±0.8)‰,低于稻鱼鸭共生田(-26.7±0.5)‰;δ15N值计算发现R-F内浮游动物的营养级位置在2.24±0.16,鱼的营养级位置在3.07±0.26,均高于其在R-F-D内的营养级。在R-F-D内,由于鸭的引入,和R-F相比,鱼的营养级降低为2.63±0.13。     

川东南小型水库营养结构特征的稳定C、N同位素分析

为了解我国水库生态系统营养结构特征, 研究应用稳定同位素技术分析了四川省东南部的典型小型水库松林水库中不同水生生物碳、氮稳定性同位素比值。基于Bayesian混合模型(SIAR)分析了不同消费者基础碳来源, 并计算了δ13C–δ15N同位素生态位中6个营养结构量化指标。结果表明: 调查期间松林水库处于富营养化状态; 初级生产者POM(主要成分为浮游藻类)、固着藻类、喜旱莲子草Alternanthera philoxeroides和水蓼Polygonum hydropiper的δ13C值范围为–29.20‰—18.81‰, δ15N值范围为4.01‰—12.73‰; 其中POM和固着藻类是多数消费者的主要碳源; 松林水库食物网营养级长度为3级, 以杂食性鱼类为优势类群并存在营养冗余现象, 暗示了该生态系统鱼类群落结构的相对稳定性; 入侵物种福寿螺Pomacea lineata、罗非鱼Oreochromis spp.与土著物种铜锈环棱螺Bellamya aeruginosa、鲫Carassius auratus等之间存在明显的同位素生态位重叠现象。建议加强水生生物资源管理, 减少外来物种入侵对当地土著物种的保护具有重要意义。     

应用碳氮稳定同位素研究斑海豹(Phoca largha)的食性

通过检测以毛鳞鱼和大西洋鲱饲养6个月以上的幼年和成年豢养斑海豹(Phoca largha)肌肉、肝脏、肾脏、肺、毛发、触须和趾甲等组织的δ13C和δ15N值,获得各组织的分馏系数,结合辽东湾海域斑海豹肌肉、肝脏和肾脏等组织及辽东湾海域潜在食物的δ13C和δ15N值,推测斑海豹在辽东湾海域的食物来源。结果发现:豢养斑海豹对食物中的δ13C值各组织分馏系数依次为触须(3.5‰)毛发(3.2‰)趾甲(3.0‰)肌肉(1.3‰)肺(1.0‰)肝脏(0.5‰)肾脏(0.3‰);对食物中的δ15N值各组织分馏系数依次为肾脏(2.8‰)肝脏(2.7‰)肌肉、趾甲和触须(均为2.6‰)肺(2.4‰)毛发(1.8‰);豢养斑海豹幼崽肌肉、肾脏和肝脏组织的δ13C值分别为-22.4‰、-23.0‰和-22.1‰,母兽乳汁的δ13C值为-24.8‰;辽东湾海域成年斑海豹肌肉、肾脏和肝脏组织的δ13C值分别为-18.6‰、-19.1‰和-18.7‰。通过豢养斑海豹肌肉组织δ13C的分馏系数(1.3‰)推测辽东湾海域斑海豹的食物以鱼类为主,喜食中上层和中下层鱼类,同时也摄食一些头足类和虾类。     

冶金区节肢动物碳、氮稳定同位素组成及营养级关系

通过测定辽宁省葫芦岛锌厂不同生境植物和主要消费者的碳、氮稳定同位素比值,研究冶金区不同消费者碳、氮稳定同位素组成及不同消费者之间的关系.结果表明:节肢动物δ13C、δ15N值的变化范围分别为-12.61‰～-29.63‰、1.73‰～9.94‰,变化幅度较大.研究区植物以C3植物居多,导致不同群落生境动物δ13C值较低;螳螂、蜘蛛δ15N比值高于其他动物;通过营养级模型计算可知不同消费者之间的关系,蝗虫、蚱蜢等草食性动物形成第二营养级,即初级消费者;第三营养级包括蜘蛛、螳螂,为次级消费者.     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

AutoFACT: AnAutomaticFunctionalAnnotation andClassificationTool

Background  

Assignment of function to new molecular sequence data is an essential step in genomics projects. The usual process involves similarity searches of a given sequence against one or more databases, an arduous process for large datasets.     

Characterization of cytochrome P-450SCC-containing liposomes

Purified cytochrome $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}\text{-}\text{containing liposomes}$ showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was less stable than $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. Liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was subjected to $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ treatment. This reagent destroyed the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. These results suggest that the heme is located in the proximity of the $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.     

A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens

The partial purification of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in $\text{p-}\text{nitrophenylphosphatase}$ activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ which can be phosphorylated by reaction with [γ-³²P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the $\text{I}{}_{50}$ for $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and $\text{p-}\text{nitrophenylphosphatase}$ inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from other tissues such as oligomycin, Ca²⁺, vanadate, $\text{N-}\text{ethylmaleimide}$, $\text{p-}\text{chloromercuribenzenesulfonic acid}$ (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K⁺ are partially effective in activating the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-ATPase and $\text{p-}\text{nitrophenylphosphatase}$ activities. The K⁺ congeners were relatively more effective in supporting $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ compared to $\text{p-}\text{nitrophenylphosphatase}$ activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, $\text{p-}\text{nitrophenylphosphatase}$ activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Quenching of the fluorescence of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Comparison of parameters estimated from A/Ci and A/Cc curve analysis

The parameters estimated from traditional A/C _i curve analysis are dependent upon some underlying assumptions that substomatal CO₂ concentration (C _i) equals the chloroplast CO₂ concentration (C _c) and the C _i value at which the A/C _i curve switches between Rubisco- and electron transport-limited portions of the curve (C _i-t) is set to a constant. However, the assumptions reduced the accuracy of parameter estimation significantly without taking the influence of C _i-t value and mesophyll conductance (g _m) on parameters into account. Based on the analysis of Larix gmelinii’s A/C _i curves, it showed the C _i-t value varied significantly, ranging from 24 Pa to 72 Pa and averaging 38 Pa. t-test demonstrated there were significant differences in parameters respectively estimated from A/C _i and A/C _c curve analysis (p<0.01). Compared with the maximum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (V_cmax), the maximum electron transport rate (J_max) and J_max/V_cmax estimated from A/C _c curve analysis which considers the effects of g _m limit and simultaneously fits parameters with the whole A/C _c curve, mean V_cmax estimated from A/C _i curve analysis (V_cmax-C _i) was underestimated by 37.49%; mean Jmax estimated from A/C _i curve analysis (J_max-C _i) was overestimated by 17.8% and (J_max-C _i)/(V_cmax-C _i) was overestimated by 24.2%. However, there was a significant linear relationship between V_cmax estimated from A/C _i curve analysis and V_cmax estimated from A/C _c curve analysis, so was it J_max (p<0.05).     

Regulation of rat brain (Na+ + K+)-ATPase activity by cyclic AMP

The interaction between the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5′-AMP, cyclic GMP or 5′-GMP, could inhibit the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854–3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, resulted in a decrease in overall $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has no effect on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme, there appeared to be a decrease in the Na⁺-dependent phosphorylation of the Na⁺-ATPase enzyme, while the K⁺-dependent dephosphorylation of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was unaffected.