云南滇龙胆居群表型多样性及其与环境关系研究

为揭示滇龙胆天然居群表型变异程度和变异规律,以云南省5个区域天然分布的20个野生居群400个单株20个表型性状指标为调查研究对象,并用变异系数、Shannon-Weiner多样性指数和巢式方差分析对滇龙胆居群间和居群内的表型变异进行分析,用相关性分析对滇龙胆表型性状与地理气象因子间的变异格局进行分析,用类平均聚类法对20个滇龙胆居群进行分类.结果表明:5个区域的滇龙胆以楚雄地区变异最大,变异系数为39.8%,变异最小的是昆明地区,为31.4%;不同居群的20个表型性状变异程度差异明显,变异系数在14.4%～91.8%之间,平均为40.4%,20个居群的平均变异为26.8%～37.0%;滇龙胆地理居群的表型分化较高,20个性状居群间的分化系数平均为73.14%,变化范围为36.03%～91.94%,居群间变异高于居群内;滇龙胆20个表型性状的总的多样性指数平均为2.547,5个不同分布区域多样性指数存在差异,最大为楚雄(1.271 4),最小为玉溪(1.266 7);表型性状变异受经度和降雨量影响较大,与纬度、海拔和温度相关性不显著;通过UPGMA聚类,滇龙胆被分成3个组,性状的表型特征并没有依地理距离而聚类....     

云南岩陀及其近缘种质资源群体表型多样性

为有效保护和持续利用药用植物云南岩陀及其近缘种质资源提供基础数据,采用巢式方差分析和聚类分析等方法对岩陀及其近缘种质资源共4个种(包括变种)的15个居群150个单株16种表型性状进行表型多样性分析.结果表明:不同种间表型性状变异均超过20％,变异由大到小依次为光腹鬼灯檠、岩陀、羽叶鬼灯檠、七叶鬼灯檠;居群间表型性状变异较高,其地上部分干重、单株根状茎数变异较大,变异系数均超过50％;小叶表面毛被状态变异系数为100％、小叶背沿脉柔毛色变异系数为0,因此这些性状为种和变种分类的重要依据;4个种的居群内变异系数均大于居群间,变异主要来源于居群内.种的表型多样性指数相对较高,其中根粗最高,叶表面毛被状态和叶背面沿脉柔毛色最低,总体平均多样性指数为1.39;不同种间表型多样性指数变化在1.23-1.44,岩陀最高,七叶鬼灯檠最低;通过聚类分析可将15个居群分为4类.结果暗示:岩陀及其近缘种质资源的遗传改良应适当地减少抽样居群数,增加居群内的家系数,重视居群内优良单株的选择;种质资源的保护应尽量保护一个居群的完整性.     

太白杜鹃天然居群的表型多样性

为揭示太白杜鹃(Rhododendron purdomii)天然居群的表型变异程度、变异规律,以秦岭山区的7个天然居群为研究对象,对其花序和叶片的10个数量性状和8个质量性状进行方差分析、相关分析与聚类分析,探讨居群间与居群内表型多样性的变异特点。结果显示:(1)在太白杜鹃的质量性状中,花色与花瓣内斑在居群间与居群内均存在丰富变异,花朵香味、花序形状、花冠形状、花梗颜色、叶片形状和株型等仅在居群间存在明显变异,在居群内则趋于一致性;其数量性状在居群间与居群内也存在广泛变异,10个数量性状在居群间和居群内平均值差异全部达显著水平;在7个居群内数量性状的变异系数为0～55.3%,其中花梗长、花丝长、花瓣宽与叶柄长变异较大,花朵数变异较小。(2)相关性分析表明,太白杜鹃花瓣长和花瓣宽分别与经度存在极显著与显著负相关关系,叶片长、叶片宽和叶片长宽比与海拔均存在显著负相关关系,而其它性状与地理因子的关系密切。(3)聚类分析显示,太白杜鹃的7个天然居群可以初步分为两大类,但性状的表型特征并没有依地理距离而聚类。研究表明,太白杜鹃天然居群的表型性状在居群内与居群间均存在丰富的变异,而且其与居群特点、分布生境等关系密切。     

滇牡丹天然居群的遗传多样性分析

以云南中部及西北部的6个滇牡丹(Paeonia delavayi)天然居群为研究对象,进行株高、新枝长等9个表型性状的表型多样性分析和ISSR分析。结果表明:9个表型性状变异幅度为0.9%~39.8%,平均值达到了18.9%;居群间生殖器官的变异较大,居群内营养器官更容易产生变异。利用居群间欧式距离进行聚类分析,6个居群聚为4个类群,没有与实际地理位置相吻合,说明表型特征的性状与地理距离的相关性不大。遗传多样性分析结果表明:利用筛选得到的10条引物,在取自6个自然居群、180个个体中,检测到56个多态位点。在居群水平上,多态位点百分率(PPB)为60.2%,Nei's基因多样性指数(H)和Shannon's信息指数(I)分别为0.281和0.414。在物种水平上,Nei's基因多样性指数(H)和Shannon's信息指数(I)分别为0.409和0.596。居群间的遗传分化系数(Gst)达0.319。结果显示,表型性状在居群间和居群内均存在广泛变异。滇牡丹遗传多样性水平较高,居群间遗传分化较大,滇牡丹并不濒危。     

湖南白檀居群形态多样性及与环境的相关性

为了解白檀在湖南地区不同环境的适应性及遗传变化程度,对湖南地区6个白檀天然居群的189个个体的11个表型性状进行形态遗传多样性分析。结果表明,11个表型性状均具有丰富的遗传多样性(H'=1.389),居群间的平均方差分量为52.60%,居群内的平均方差分量为37.03%,说明在白檀形态性状多样性分布上是居群间多样性程度大于居群内,即居群间的形态变异是白檀形态变异的主要来源。聚类分析结果表明,6个居群的表型性状并没有严格依地理距离聚类,主成分与相关分析结果显示在11个表型中树型因子是主要形态变异因子,年降雨量与树型因子呈显著正相关,其他表型变异受遗传的影响可能大于受环境的影响。     

披碱草属野生居群表型多样性及其与环境关系研究

以来自8省区38个披碱草属野生居群的22个表型性状为调查对象,利用变异系数及多样性指数法对性状内和性状间及种间的表型变异进行分析,并对表型性状与地理气象因子间的关系进行相关性分析,用平均聚类法对38个居群进行分类,以揭示不同来源披碱草属种质资源表型多样性。结果显示:(1)营养器官性状中旗叶至穗基部长变异系数最高(44.695%),穗部性状中的每穗小穗数变异系数最高(77.005%)。(2)披碱草、麦宾草、圆柱披碱草与老芒麦这4个种间总体平均多样性指数为0.920～1.560,披碱草最高,麦宾草次之,圆柱披碱草最低。(3)表型性状变异受海拔、经度及年均温的影响较大。(4)UPGMA聚类结果将所有材料分成4组,披碱草属植物性状的表型特征没有严格按地理距离而聚类。研究表明,披碱草属植物野生种质材料具有丰富的表型多样性,表型性状与环境之间关系密切,采集过程中应尽量保持居群的完整性,最大程度地保护中国披碱草属牧草的遗传多样性。     

都市园林植物云南樱花的表型多样性研究

云南樱花(Prunus yunnannensis)为蔷薇科(Rosaceae)多年生落叶乔木,是中国西南地区特有的重要园林观赏植物,其在昆明园林绿化中具有较高的应用价值。以昆明地区的云南樱花为研究对象,研究了6个栽培居群120个单株的16个表型性状的多样性,分析了16个表型性状的相关性和广义遗传力,并对6个栽培居群进行了聚类分析,旨在探明其在资源应用过程中的表型变异程度及变异规律,为今后优良性状选育与种质推广提供科学依据。结果表明:云南樱花在居群间及居群内均存在丰富的表型多样性,除雌蕊长以外,其它15个表型性状在各居群之间均存在显著性差异,其中各性状的平均变异幅度为7.73%~94.04%,在居群内的变异幅度为20.74%~28.22%,各性状居群内的变异明显小于居群间,16个表型性状的广义遗传力介于0.54~0.97之间,其中叶长的广义遗传力最大,而雌蕊长的广义遗传力最小。对云南樱花16个形态指标的相关性分析表明,大部分形态指标之间均存在显著的相关性。利用居群间欧氏距离进行的UPGMA聚类分析结果表明,云南樱花6个栽培居群可以聚为2类。     

新疆东天山地区宽刺蔷薇居群表型多样性分析

对新疆东天山地区6个宽刺蔷薇（Rosa platyacantha）居群进行叶片、花序、果实、种子等11个表型性状的遗传多样性分析,结果表明：宽刺蔷薇各表型性状无论在居群间还是在居群内均表现出显著或极显著差异,存在丰富的表型多样性。6个居群11个性状的平均表型分化系数为27.50%,表明居群内多样性较居群间多样性更为丰富。各表型性状平均变异系数为16.51%,离散程度相对较低。对表型性状进行的变异系数多重比较和主成分分析均显示,果实相关性状的变异是造成宽刺蔷薇表型变异的主要来源。性状相关性分析进一步发现,生殖生长与前期的营养生长并无明显相关关系。聚类分析结果表明,6个宽刺蔷薇居群的表型性状并没有严格依地理距离聚类,而是受到遗传因素与环境条件特别是海拔因子的共同影响。     

北沙柳种质资源居群表型多样性

该研究采用单因素方差分析、巢式方差分析、群落多样性指数分析等方法,以国家北沙柳种质资源库内13个居群的494个无性系为实验材料,通过表型性状(叶面积、叶周长、叶柄长、叶长、叶宽、长宽比、开枝角度、株高和地径)比较分析,探讨居群间和居群内表型分化程度、表型多样性和地理变异,为北沙柳种质资源遗传改良和生产提供理论依据。结果显示:(1)北沙柳表型性状变异丰富,变异系数范围为17.64%~28.79%,平均为22.53%。(2)在13个居群中,居群P2的Simpson、Shannon和Brilliouin平均多样性指数最高,居群P13最低;表型性状中分枝角度多样性指数最大,地径多样性指数最小。(3)表型性状分化系数为0.265 4,即北沙柳种质资源居群间表型变异为26.54%,居群内表型变异为73.46%。(4)主成分分析表明,叶面积、叶周长、叶长、叶柄长和叶宽对分组的贡献率较大;聚类分析将13个北沙柳居群可划分为四组;Mantel检验表明,地理距离与表型距离(欧氏距离)相关性不显著(r=0.192 3,P=0.082)。研究认为,居群内不同无性系的选育是北沙柳定向育种的主要研究方向;边缘居群的表型性状具有形成地理变异的趋势;遗传多样性高是北沙柳适应性强的物质基础。     

新疆野扁桃天然居群形态变异的研究

为了从数量上分析新疆野扁桃(Amygdalus ledebouriana)天然居群表型性状在居群间和居群内的变异,我们于2007年对分布存新疆的5个野扁桃天然居群的8个表型性状进行了测量和比较分析.结果表明:新疆野扁桃表型性状在居群间和居群内均存在着较丰富的差异,居群内的变异大于居群间的变异,居群间的分化相对较小;利用居群问欧氏距离进行UPGMA聚类分析表明,5个天然居群可以划分为3类,表型性状的欧式遗传距离与地理距离相关不显著.主成分综合分析结果显示:新梢长宽比、叶片长宽比、果核千粒重、果核长宽比及花冠直径等5个表型性状指标是反映新疆野扁桃表型差异的主要因素.     

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.     

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.     

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.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.