基于线粒体细胞色素b基因序列的版纳鱼螈四个种群遗传多样性与遗传结构研究

版纳鱼螈Ichthyophis bannanicus 是蚓螈类在我国分布的唯一物种,目前已知分布在云南、广西和广东的部分地区.本研究获得4个版纳鱼螈种群的87个个体的线粒体DNA细胞色素6基因1 000 bp序列,对序列变异、遗传多样性、种群结构和种群动态进行了分析.经比对后87个序列发现39个(3.9%)多态性位点,共定义了22个单倍型.序列分析结果显示版纳鱼螈种群单倍型多样性和核苷酸多样性与其它两栖动物接近,说明版纳鱼螈各种群仍保持一定的遗传多样性水平.单倍型网络关系图和系统发生分析均显示来自云南和两广地区的版纳鱼螈分歧明显,已形成了不同的进化谱系.种群遗传结构分析也显示来自云南的勐腊种群与两广各种群之间显著的遗传差异,这提示分布在云南与两广地区的版纳鱼螈应作为不同的管理单元(MU)进行保护.综合两种中性检验(Fu's Fs=9.44,P=0;Tajima's D=1.88,P=0.013)和种群增长指数(g=5415.03±297.55)分析的结果表明,两广地区的版纳鱼螈可能经历近期的种群扩张事件.     

黑麂皖-浙分布中心种群的遗传多样性

本文基于黑麂线粒体控制区序列对皖浙分布中心的4个黑麂种群的遗传多样性和基因流进行了研究。结果显示:4个种群的33个个体中有14个变异位点,占分析序列长度的2·91 %,并由此定义了12个单倍型;遗传多样性检测结果显示4个种群中开化种群具有最高的遗传多样性,应予以优先保护;4个种群间尚存在着一定的基因流,但存在可能由于遗传漂变而产生分化的危险。从Tajima’s D和Fu and Li’s D值的估算结果来看,这4个黑麂种群相对于中性进化的歧异度并没有明显的偏离,具有较为稳定的种群结构(P>0·1),没有明显的证据显示这4个黑麂种群间存在很强的平衡选择。     

黄河上游极边扁咽齿鱼种群遗传结构

极边扁咽齿鱼Platypharodon extremus是黄河上游的特有鱼类,近年来由于过度捕捞、环境变迁等因素,其资源量剧减,种群处于濒危状态。研究中采集了分布于黄河上游的3个种群（n=107）,基于线粒体DNA控制区695bp序列,共检测到101个可变位点,占总分析位点的14.5%,其中58个为简约信息位点。3种群共界定了87个单倍型,种群平均单倍型多样性h=0.995,核苷酸多样性π=0.0129。结果表明,极边扁咽齿鱼的种群遗传多样性水平较高。分子方差分析（AMOVA）表明,3群体间总遗传分化系数Fst=0.0528,群体间尚未有显著的遗传分化。系统发生树显示3种群的单倍型混合分布,各进化枝之间分歧度很低,没有形成明显的单倍型组,也没有显示出单倍型与地理位置的对应关系。3个群体共享1个单倍型（Hap₃1）,推测它们来自于共同的祖先。歧点分布和Fu’Fs中性检测显示极边扁咽齿鱼并未经历种群扩张。     

POPULATION GENETIC STRUCTURE OF ENDANGERED SPECIES PLATΥPHARODON EXTREMUS ( SCHIZOTHORACINAE, CYPRINIDAE )IN YELLOW RIVER, CHINA

极边扁咽齿鱼Platypharodon extremus是黄河上游的特有鱼类,近年来由于过度捕捞、环境变迁等因素,其资源量剧减,种群处于濒危状态.研究中采集了分布于黄河上游的3个种群(n= 107),基于线粒体DNA控制区695bp 序列,共检测到101个可变位点,占总分析位点的14.5％,其中58个为简约信息位点.3种群共界定了87个单倍型,种群平均单倍型多样性h= 0.995,核苷酸多样性π=0.0129.结果表明,极边扁咽齿鱼的种群遗传多样性水平较高.分子方差分析(AMOVA)表明,3群体间总遗传分化系数Fst= 0.0528,群体间尚未有显著的遗传分化.系统发生树显示3种群的单倍型混合分布,各进化枝之间分歧度很低,没有形成明显的单倍型组,也没有显示出单倍型与地理位置的对应关系.3个群体共享1个单倍型(Hap_ 31),推测它们来自于共同的祖先.歧点分布和Fu’Fs中性检测显示极边扁咽齿鱼并未经历种群扩张.     

基于线粒体COI基因的中国西南地区木领针蓟马地理种群的遗传分化分析

【目的】木领针蓟马Helionothrips mube近年来成为芋头Colocasia esculenta上的一种常见害虫。本研究旨在探讨其中国西南地区地理种群间的遗传变异。【方法】通过Sanger法测定了21个地理种群132头木领针蓟马的线粒体COI基因序列,利用MEGA, DnaSP, Arlequin和Network等软件对木领针蓟马种群间的遗传多样性、遗传分化、分子变异等进行分析。【结果】获得的木领针蓟马132条线粒体COI序列(643 bp)中共发现34个变异位点、16种单倍型,其中单倍型H1出现频率最高、分布最广。木领针蓟马总群体的遗传多样性较高(Hd=0.712, Pi=0.00413, K=2.655),遗传分化程度极大(Fst=0.3443),基因交流水平不高(Nm=0.96)。分子方差分析(AMOVA)表明,木领针蓟马的遗传分化主要来自种群内部,Mantel检测出种群地理距离与遗传距离呈正相关。总群体中性检验Tajima’s D值显著负值,Fu’s Fs值不显著,错配分布曲线呈多峰。综合种群间遗传距离、单倍型系统发育树及中介网络图结果,表明四川成都(CHD)、云南昌宁(CHN)和贵州遵义(ZY)3个种群的遗传分化程度均高于其他种群。【结论】中国西南地区木领针蓟马总群体的遗传多样性较高,遗传分化明显,种群间的基因交流受到地理距离的影响,总群体在较近的历史时期内没有出现扩张现象。     

黑龙江省不同山系狍种群遗传多样性分析

狍（Capreolus pygargus）为我国重要的经济动物,并且是国家一级保护动物——东北虎(Panthera tigris altaica)的主要食物之一。因此,深入了解狍各个地理单元内种群的遗传变异,可以为我们制定保护管理策略提供依据,进而恢复珍稀濒危物种的野外种群数量。对32个不同狍个体（来自3个不同山系）的线粒体DNA控制区的部分序列进行了测定和群体分析,发现了50个变异位点,定义了27种单倍型,其单倍型多样性(h)平均值为0.978、核苷酸多样性(π)平均值为0.022 60,种群总体遗传多样性较高;在3个地理单元的狍种群中,大兴安岭地区的狍种群具有较高的遗传多样性,应予以优先保护。从Tajima’s D和Fu & Li’s D值的估算结果表明,这3个狍种群与中性进化的歧异度相比,并没有明显的偏离(P> 0.1),无明显的证据显示这3个狍种群间存在很强的平衡选择。     

基于mtCOI基因序列的中国葱斑潜蝇不同地理种群遗传多样性分析

【目的】葱斑潜蝇Liriomyza chinensis是葱蒜类蔬菜上的重要经济害虫,在我国广泛分布。本研究旨在阐析中国葱斑潜蝇的地理种群遗传分化。【方法】以中国8省12个不同地理种群的253头葱斑潜蝇为样本,测定其mtCOI基因序列;依据获得的mtCOI基因序列,利用MEGA7.0, DnaSP 6.1和Arlequin 3.5等软件对葱斑潜蝇地理种群的遗传多样性、基因流水平和遗传变异等进行分析。【结果】在253头个体的759 bp mtCOI基因片段中,获得13个单倍型,各单倍型间K2P遗传距离均小于0.02;其中单倍型Hap1为12个地理种群所共享,总发生频率高达81.82%。总群体单倍型多样性较低(H_d=0.327),核苷酸多样性(Pi)为0.00159,核苷酸平均差异数(K)为1.21011;葱斑潜蝇总群体遗传分化程度中等(F_ST=0.06971),基因交流较充分(Nm=3.33629)。分子方差分析(AMOVA)结果表明,遗传变异来源为群体内;总群体Tajima’s D检验值为显著负值;Mantel检测结果说明种群间的遗传距离与地理距离没有相关性。【结论】中国葱斑潜蝇地理种群的遗传多样性较低,基因交流较充分,遗传分化程度中等,且地理距离并不影响其遗传分化程度;葱斑潜蝇总群体在较近的历史时期未经历明显的种群扩张和种群增长。     

怒江扎那纹胸鮡的遗传多样性和遗传分化

怒江水电开发将对扎那纹胸鮡产生不利的影响。为了解扎那纹胸鮡遗传多样性和遗传分化情况, 文章测定了采自怒江中下游怒江州地区的贡山、古登和泸水及保山市地区的道街、勐糯和木城6个扎那纹胸鮡群体共102个个体的线粒体Cyt b基因序列。结果显示, 在1 137 bp序列中共检测到87个变异位点, 定义了36个单元型。总样品的单元型多样性(h)和核苷酸多样性(π)分别为0.851±0.028和0.01356±0.0008。扎那纹胸鮡的遗传多样性相对较低, 但怒江州种群遗传多样性显著高于保山市种群。群体间分化指数(FST)(0.475~0.846)明显高于群体内分化指数(0.002~0.108), 且各群体间分化指数和地理距离呈线性正相关。利用AMOVA(Analysis of molecular variance )对遗传分化进行分割, 群体间和群体内分别占53.65%和 46.35%, 群体间遗传分化指数(F_ST)为0.5365 (P<0.01), 扎那纹胸鮡在怒江州和保山种群分化显著。单元型分子系统树和简约网络图显示, 扎那纹胸鮡单元型聚为两个独立的支系: 怒江州支系和保山市支系。这些鱼类至少代表一个管理单位, 但也可能是一个进化显著单位。因此, 建议保护扎那纹胸鮡种群, 在水电工程建设时应充分考虑扎那纹胸鮡种群结构现状, 避免不同区域的种群之间发生基因交流。     

保护珍稀两栖动物-文县疣螈

文县疣螈是中国特有的有尾两栖动物,发现于甘肃省文县,还分布于四川、重庆、贵州、湖南、安徽等地.它是目前世界上最稀少的两栖动物之一,生境的丧失和破碎化、环境质量的急剧恶化、遗传多样性丧失等诸多因素导致其种群数量迅速减少.它还是被关注和研究最少的濒危动物之一,人类的活动是其栖息地丧失和种群数量迅速减少的罪魁祸首.作为一种古老的有尾两栖动物,文县疣螈的进化潜力已经较小,对环境变迁的适应能力较差.以目前的种群数量来看,很可能会在短期内灭绝,对其进行有效的保护已迫在眉睫.     

黑麂（Muntiacus crinifrons）3个种群的遗传多样性

黑麂,为我国特有动物,被公认为目前世界上最珍稀的鹿科动物之一.为了更好地保护黑麂这一珍稀的濒危野生动物,基于黑麂线粒体控制区序列对遂昌分布中心的3个黑麂种群的遗传多样性和基因流进行了研究.结果显示,3个种群的36个个体中有13个变异位点,占分析序列长度的2.71%,且这13个变异位点皆为碱基置换,并未出现碱基插入或缺失的现象,并由此定义了12个单倍型.遗传多样性检测结果显示遂昌种群具有最高的遗传多样性,应予以优先保护.从Tajima′s D和Fu and Li′s D值的估算结果来看,这3个黑麂种群相对于中性进化的歧异度并没有明显的偏离(P＞0.1),没有明显的证据显示这3个黑麂种群间存在很强的平衡选择.3个种群间基因流 Nm均大于1,这3个黑麂种群间存在着较为丰富的基因流.3个黑麂种群单倍型间的序列差异为0.009,这表明这3个黑麂种群的单倍型未出现分化.     

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.     

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.     

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).     

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⁺.     

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.     

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.     

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.     

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.     

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.