亚高山暗针叶林林缘华西箭竹对岷江冷杉幼苗更新的影响

大量研究表明在箭竹-冷云杉林下,竹类对冷杉的影响大于其它树种。在卧龙自然保护区岷江冷杉(Abies faxoniana)林下,华西箭竹(Fargesia nitida)为灌木层优势种。为了进一步了解箭竹对岷江冷杉幼苗更新的影响,该文采用样带法在四川省卧龙自然保护区亚高山岷江冷杉林林缘,针对华西箭竹和岷江冷杉幼苗展开调查研究。以竹丛覆盖区域为边界,分别向竹丛外2 m (带1)、1 m (带2),向竹丛内1 m (带3)、2 m (带4)、3 m (带5)做5条长30 m的相邻样带,每条样带划分为30个连续的1 m×1 m小样方。对岷江冷杉幼苗数量动态、种群结构、生长与生物量的研究表明: 1)离竹丛越远,大年龄岷江冷杉幼苗数量越多;岷江冷杉幼苗转化率(下一径级苗木数量与前一径级苗木数量之比)随年龄增加而减小,随离竹丛距离增大而增大。2)苗木高度与径级结构相似,随深入竹丛,幼苗数量峰值的径级和高度级分布从较大径级(Ⅲ)向小径级(Ⅰ)变化。3)华西箭竹的存在抑制了岷江冷杉小径级幼苗(Ⅰ)的高生长,基径与高度的回归也表明离竹丛越远,高生长相对更大;但一定密度华西箭竹(带4)增大了大径级(Ⅲ)岷江冷杉幼苗冠幅的扩展。4)岷江冷杉幼苗总生物量随华西箭竹密度的增高而减小,华西箭竹的大量存在抑制岷江冷杉幼苗对地下生物量的分配。总的看来,一定密度华西箭竹环境下岷江冷杉幼苗的生长和更新较好,华西箭竹大量分布的区域,岷江冷杉幼苗的更新和生长受到抑制,可能会影响岷江冷杉种群的扩散以及群落的发展。     

甘南高山林线岷江冷杉—杜鹃种群结构与动态

高山林线是一种典型的生态交错带,是对气候反映最敏感的地区之一。甘肃南部高山林线区域主要以原始岷江冷杉种群和杜鹃种群为优势种,通过对岷江冷杉和杜鹃种群建立静态生命表,绘制存活曲线描述其结构特征,利用种群数量动态预测时间序列分析定量研究未来的发展趋势。结果显示:(1)岷江冷杉种群幼苗比较丰富,能很好的维持种群个体的自疏死亡,存活曲线呈Deevey-Ⅲ型;杜鹃种群幼苗缺乏,存活曲线趋向于Deevey-Ⅰ型;死亡曲线和危险率曲线都随着龄级的增加而增加,杜鹃种群的死亡率在各个龄级一直大于岷江冷杉种群,危险率在Ⅱ龄级以后杜鹃种群也始终大于岷江冷杉种群。(2)岷江冷杉种群结构动态变化指数Vpi大于修正后的种群结构动态变化指数V′pi且大于0,而杜鹃种群结构动态变化指数Vpi小于修正后的种群结构动态变化指数V′pi且小于0,则岷江冷杉种群属于增长型,杜鹃种群属于衰退型,岷江冷杉、杜鹃随机干扰极大值分别为0.027、0.011,说明二者对外界干扰均比较敏感。(3)杜鹃种群时间序列预测为前期幼苗比较缺乏,中期稳定,后期衰退的动态特征,而岷江冷杉种群表现出各龄级时间变化较小,幼苗个体数较多,种群为稳定增长型,岷江冷杉更能适应甘肃南部高山林线区域当前环境。     

林窗对岷江冷杉幼苗生存过程的影响

岷江冷杉林是我国西南亚高山暗针叶林的主体之一,其健康的种群动态和更新策略在维持长江上游生态安全方面具有重要意义。为了解岷江冷杉更新过程中林窗对幼苗种群动态的影响,通过调查林窗内和林下岷江冷杉更新幼苗的存活情况,采用数量分析方法研究了幼苗的年龄结构和动态特征。结果表明:林窗内和林下岷江冷杉幼苗的年龄结构金字塔均为两头小中间大的纺锤型,存活曲线均为DeeveyⅡ,生命期望以1龄级的最高、林下种群的高于林窗种群。幼苗的死亡率、消失率、累计死亡率和危险率均随龄级的增加而增大,生存率和死亡密度随龄级的增加而减小;岷江冷杉幼苗种群的数量化动态指数Vpi=0.1059,为增长型种群,在外界随机事件干扰时也可持续增长,尤以林窗种群抵御随机干扰、持续增长的特点更为突出;岷江冷杉幼苗种群的周期波动主要受基波控制,林窗种群在9龄级和11龄级处的波动还受谐波的影响。林窗扩大了岷江冷杉更新幼苗的数量规模、提高了种群抵御外界随机干扰的能力。因此,在岷江冷杉林更新管理中,应高度重视林窗的作用。     

川西亚高山天然林岷江冷杉的更新结构与数量动态

岷江冷杉(Abies fargesii var.faxoniana)的天然更新是川西亚高山地区天然次生林生态恢复的关键。为揭示川西亚高山4种典型天然林林型(杜鹃-岷江冷杉林、箭竹-岷江冷杉林、红桦-岷江冷杉林和红桦林)中岷江冷杉的更新结构和其幼龄植株的生存过程,基于4块1 hm²样地的调查,通过单因素方差分析和多重比较对比不同林型中岷江冷杉的更新结构和更新质量;通过对数回归建立了岷江冷杉幼龄植株高度与年龄间的关系;通过建立幼龄植株的静态生命表、绘制存活曲线和计算数量动态指数分析其生存特征和存活瓶颈期。结果表明：4种林型中岷江冷杉的更新质量和幼龄植株存活状态依次为杜鹃-岷江冷杉林>红桦林>红桦-岷江冷杉林>箭竹-岷江冷杉林。各林型中岷江冷杉幼龄植株的死亡率与相邻龄级间的个体数量动态(V_n指数)随龄级增加的波动大。综合死亡率与V_n指数的龄级差异,杜鹃-岷江冷杉林和箭竹-岷江冷杉林中幼龄植株存活的年龄瓶颈期分别为第21—25年(树高0.66—1.04 m)和第11—15年(树高0.46—0.73 m);红桦-...     

岷江上游林线附近岷江冷杉种群的生存分析

岷江冷杉 (Abiesfaxoniana) 林是青藏高原东南缘亚高山暗针叶林的主要类型之一, 在岷江上游地区, 岷江冷杉林一般在 2 80 0~ 380 0m之间呈带状分布并且在阴坡形成林线。为了对岷江上游林线地段的岷江冷杉种群进行生存分析, 在岷江上游林线附近弓杠岭 (33°0 2′39″N, 10 3°4 3′11″E) 设置了 10个样地, 使用侧生轮枝计数或WinDENDRO系统对个体年龄进行确定。调查数据经匀滑技术处理, 编制了岷江冷杉种群特定时间生命表, 绘制了岷江冷杉种群的存活曲线、死亡率曲线和消失率曲线。结果表明:1) 岷江上游林线地段岷江冷杉种群存活曲线趋于Deevey_Ⅲ型。 2) 林线地段岷江冷杉种群生存率呈单调下降趋势, 生存率下降趋势前期高于后期, 说明岷江冷杉种群幼苗死亡率高, 种群后期比较稳定。3) 林线地段岷江冷杉林整个生长期中出现了两个死亡高峰期, 一个出现在幼苗向幼树过渡时期 (0~ 4 0年), 另一个出现在中龄时期 (180年) 。岷江上游林线地段岷江冷杉种群幼苗死亡率较高的原因可能和林线地段的温度较低、积雪厚度、风向、水分等环境条件有关。     

塔里木河上、中游胡杨种群结构与统计分析

在新疆塔里木河的上游-阿瓦提县和中游-轮台县胡杨林自然保护区内设置1．2hm^2的样地,应用相邻格子法进行每木调查获取野外资料,编绘了不同生境胡杨种群的特定时间生命表、存活曲线和大小、年龄结构图。结果表明：不同生境胡杨的种群结构差异明显。阿瓦提县胡杨种群大小、年龄结构呈金字塔型,幼苗储备丰富,缺乏老树,林分较年轻,为增长种群。种群从Ⅰ～Ⅱ级发育过程中死亡率较高,中龄个体生命期望寿命较高,存活曲线符合DeeveyⅢ（凹）型。轮台县胡杨种群大小、年龄结构呈中部大、两端小的正态分布,幼苗相对较少,林相成熟,为稳定种群。种群从Ⅵ～Ⅶ级发育过程中死亡率较高,生命期望寿命随年龄的增大而降低,存活曲线经模型检验符合DeeveyⅡ（直线）型。不同生境种群的消失率与死亡率曲线变化一致,均出现两个高峰,峰值大小明显不同。种群数量动态是胡杨生物学特性和环境条件共同作用的结果。     

濒危植物秦岭冷杉种群数量动态

为了对濒危植物秦岭冷杉种群数量动态评价和预测,通过样地调查和数据统计,研究了秦岭冷杉种群的年龄结构、静态生命表及其与环境因子关系,运用时间序列模型预测了种群数量动态.结果表明,多数秦岭冷杉种群幼龄级个体数较少,中老龄个体数量较大,呈衰退趋势.仅处于低海拔地区的秦岭冷杉-木蓝-苔草群丛中的种群(D种群)由于立地条件较好,幼龄级个体数量相对丰富,种群稳定.不同秦岭冷杉种群生命表和存活曲线的分析表明,尽管生境条件差异,但存活曲线基本接近DeeveyⅢ型;不同种群偏离典型存活曲线的程度与幼苗缺乏程度有关,一般Ⅲ～Ⅴ龄级死亡率较高.时间序列分析表明,在未来20、40和80年中,不同秦岭冷杉种群均会呈现老龄级株数先增后减的趋势,种群稳定性长期维持困难.对影响秦岭冷杉种群增长的10个环境因子通过主成分分析(PCA)发现,乔木层盖度、土壤有机质含量和空气湿度对种群发挥有利影响,而人为干扰和光照强度对秦岭冷杉种群增长发挥不利影响.应充分利用秦岭冷杉性喜荫、耐寒、种子活力较强的特点,加强现有林分就地保护,重点是具有结实能力的中老龄个体;在阴坡地带,对林下灌木比较密集的群丛,通过砍灌、清理林下活地被物等抚育措施,为幼苗发育创造良好的环境条件;就地采种育苗,扩大人工种群.     

湖北神农架地区巴山冷杉（Abies fargesii）种群结构特征与空间分布格局

巴山冷杉群落是湖北神农架地区主要的群落类型之一,从生命表特征、年龄结构、存活曲线以及种群和不同龄级个体空间分布格局等方面研究了湖北神农架地区巴山冷杉的种群结构特征与空间分布规律.研究结果表明:巴山冷杉的种群年龄结构图呈现的是幼苗比例最大,中龄树、幼树和成龄树比例次之的规律;巴山冷杉幼苗储备丰富,巴山冷杉自身的生物学特性及环境因素是形成这种现象的主要原因;湖北神农架地区巴山冷杉种群的存活曲线接近于Deevey C型,I径级巴山冷杉死亡率最高,II径级开始巴山冷杉幼树死亡率大幅度下降,直至老龄期死亡率逐渐升高;巴山冷杉种群不同年龄级的个体呈现出集群分布或随机分布,处于不同发育阶段的个体群,其空间分布格局是随时间变化的.     

塔里木河上游胡杨种群结构与动态

采用样带调查法对塔里木河上游3个断面8.5 hm2天然胡杨林进行野外调查,绘制了胡杨种群年龄结构图和存活曲线,结合种群动态量化方法与时间序列预测,分析种群结构与动态特征。结果表明:塔里木河上游胡杨种群密度随径级增大而减小,自疏指数接近-3/2,个体胸径达24 cm后种群密度保持相对稳定。中、下段胡杨种群年龄结构呈金字塔型,幼龄级所占比例较大、林分年轻;存活曲线为Deevey C型,且动态指数0,为增长种群。受所处生境条件影响,上段不同生境胡杨种群年龄结构差异明显。阿瓦提县胡杨种群结构呈金字塔型,幼龄级比例高达77.2%,存活曲线属Deevey C型,为增长种群;南口镇与托海乡胡杨种群结构呈壶型,幼龄级比例极低、中大龄级比例高达84.7%,存活曲线属Deevey A型,为衰退种群;沙黑里克镇胡杨种群结构呈钟型,存活曲线属Deevey A型,为暂时稳定种群。种群数量动态与时间序列分析表明,中、下段胡杨更新资源丰富,未来种群能够稳定增长;上段南口镇、托海乡与沙黑里克镇幼龄级数量随时间推移减少,种群向大老龄级发展,未来种群将走向衰退。总之,上段胡杨种群因更新资源不足而趋于衰退,未来保持适当的人工辅助恢复措施和加强保护现有植株及其生境是保持胡杨种群自然更新和进行种群恢复的关键。     

濒危植物连香树种群结构与动态研究

以空间代替时间的方法调查了喇叭河自然保护区和宝兴县两个地区连香树种群的结构.绘制大小结构图,编制静态生命表,绘制存活曲线、消失率曲线和死亡率曲线,并进行种群动态谱分析.结果表明:两个地区连香树种群幼苗个体比例都比较大,但死亡率很高;存活曲线介于Deevey Ⅰ型和DeeveyⅡ型之间;消失率和死亡率曲线变化趋势一致;种群动态过程存在周期性,种群数量动态不仅受基波的影响,而且还显示出了小周期的波动.     

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.     

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⁺.     

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).     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Occurrence of N-malonyl-D-alanine in pea seedlings

One of the ninhydrin-negative alanine conjugates isolated from pea seedlings was identified as $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$.The identification of this conjugate was carried out by a comparison of its gas-liquid chromatographic and mass spectrometric properties, and its nuclear magnetic resonance and infrared spectra with those of synthetic $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$. The alanine in the conjugate was shown to be present as the D-isomer by enzymatic and chromatographic analyses.     

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.     

The effects of multiple features of alternatively spliced exons on the KA/KSratio test

Background  

The evolution of alternatively spliced exons (ASEs) is of primary interest because these exons are suggested to be a major source of functional diversity of proteins. Many exon features have been suggested to affect the evolution of ASEs. However, previous studies have relied on the K _A /K _S ratio test without taking into consideration information sufficiency (i.e., exon length > 75 bp, cross-species divergence > 5%) of the studied exons, leading to potentially biased interpretations. Furthermore, which exon feature dominates the results of the K _A /K _S ratio test and whether multiple exon features have additive effects have remained unexplored.     

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.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(1) ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C₁₂E₈) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K⁺-stimulated Na⁺-influx is found to be about 35 μmol Na⁺ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na⁺ transported per ATP molecule hydrolyzed.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca²⁺ and decreased approx. 1000-times when the Ca²⁺ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca_iZ) decreases in the order of $\text{Ca}{}_3\text{Z}\text{>}\text{Ca}{}_4\text{Z}\text{>}\text{Ca}{}_2\text{Z}\text{?}\text{CaZ}$. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca²⁺-ATPase in the range of 10^?7–10^?6 M Ca²⁺, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca²⁺-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca²⁺-transport ATPase in erythrocytes at the prevailing Ca²⁺ concentration (probably 10^?7 – 10^?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca²⁺-ATPase requires that the Ca²⁺ concentration rises to 10^?6 – 10^?5 M.