紫花苜蓿和羊草种子出苗和幼苗生长对土壤含水量的响应

土壤含水量是种子萌发和幼苗生长阶段的关键环境因子。采用室内模拟方法,研究了不同土壤含水量(2.5%、5%、7.5%、10%、20%和30%)对紫花苜蓿和羊草种子出苗和幼苗生长的影响。结果表明:苜蓿和羊草种子的出苗率和幼苗的生长均显著受土壤含水量的影响;随着土壤含水量的增加,苜蓿和羊草的出苗率均呈先上升后下降的趋势;其中,苜蓿种子在土壤含水量为10%~20%时出苗率最高(93%),土壤含水量为30%时最低(2%);在5%土壤含水量时羊草出苗率最大(76%),在30%含水量时最低(9%);随着含水量增加,紫花苜蓿和羊草幼苗根苗长均表现为先上升后下降的趋势,土壤含水量7.5%~10%更适宜于紫花苜蓿种子出苗及幼苗生长,羊草种子的最适土壤含水量为5%~7.5%;综合出苗率和幼苗的生长情况,适宜苜蓿和羊草播种和幼苗生长的土壤水分含量分别为7.5%~10%和5%~7.5%。     

珙桐苗木叶片光合特性对土壤干旱胁迫的响应

通过控制土壤含水量,研究了土壤干旱胁迫对2年生珙桐(Davidia involucrata Baill.)幼苗叶面积、叶绿素含量、光合作用及叶绿素荧光参数的影响,以探讨珙桐光合作用对土壤干旱胁迫的响应机理.结果表明,干旱胁迫后,珙桐叶片失水脱落,各干旱胁迫处理叶面积比对照显著降低23%～98%,但单位面积的叶绿素含量却无显著变化;干旱处理的珙桐幼苗净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、水分利用效率(WUE)显著低于对照(P<0.05);干旱胁迫对珙桐幼苗叶片的初始荧光产量(F0)及最大荧光产量(Fm)没有产生显著影响,却显著降低了PSⅡ最大光化学量子产量(Fv/Fm)、PSⅡ实际光化学量子产量(Yield)、电子传递速率(ETR)及光化学淬灭系数(qP),使非光化学淬灭系数(qN)显著升高(P<0.05).研究发现,土壤水分亏缺对珙桐叶片的光合系统造成了不可逆的伤害,严重抑制了其正常的光合作用和生长发育;珙桐幼苗对土壤干旱胁迫极为敏感.     

呼伦贝尔草甸草原不同土壤水分梯度下羊草的光合特性

为探索气候变化引起的干旱可能对呼伦贝尔草甸草原生产力造成的影响,利用Li-6400便携式光合测定系统对呼伦贝尔草原4个土壤水分梯度下羊草的光合生理指标进行测定.结果表明:羊草叶片净光合速率的日变化在土壤质量含水量为(40±1)%、(20±1)%及(10±1)%的条件下呈双峰曲线,峰值分别出现在8:00和16:00,有明显的光合午休现象,在干旱胁迫(土壤质量含水量为(5±1)%)条件下变化趋势平缓,曲线双峰特征不明显,净光合速率大幅下降;叶片蒸腾速率和气孔导度的日变化趋势均呈双峰曲线;不同土壤水分梯度下羊草叶片胞间CO2浓度的日变化与净光合速率日变化趋势相反.通过光响应的研究表明,土壤水分胁迫使最大净光合速率、光饱和点、表观量子效率以及水分利用效率降低,而光补偿点升高.干旱胁迫降低了呼伦贝尔草甸草原植被的光合生产能力,从而可能导致草地生产力大幅下降.     

野生酸枣光合及叶绿素荧光参数对土壤干旱胁迫的响应

为探究鲁中地区自然条件下野生酸枣光合生理参数对土壤逐渐干旱的响应机制,明确其与土壤水分的定量关系。该研究以2年生野生酸枣盆栽苗为实验材料,采用人工给水和自然耗水相结合法模拟自然条件下土壤干旱过程,分析野生酸枣叶片光合作用和荧光参数对土壤水分含量(RWC)的响应过程及其机制。结果表明:(1)野生酸枣叶片气体交换参数净光合速率(P_n)、蒸腾速率(T_r)、水分利用效率(WUE)均随着土壤含水量的增加表现出先升高后降低的趋势。当RWC38.5%时,P_n下降,而胞间二氧化碳浓度(Ci)上升,气孔限制值(L_s)下降,叶片光合速率下降主要是非气孔限制因素所致;当RWC在38.5%~65.1%范围内,野生酸枣P_n下降,伴随C_i、气孔导度(G_s)均降低,野生酸枣叶片光合速率下降处于气孔限制阶段。(2)通过P_n和WUE对土壤水分的阈值模拟,发现维持野生酸枣叶片具有较高光合生产力的土壤水分范围为46.0%~0.5%,维持其较高水分利用效率的水分范围为56.3%~73.9%。(3)在土壤逐渐干旱过程中(RWC范围为29.9%~86.5%),野生酸枣最大荧光(F_m)、PSⅡ最大光化学效率(F_v/F_m)、PSⅡ实际光化学效率(ΦPSⅡ)逐渐降低,初始荧光(Fo)显著升高,光化学猝灭(qP)先升高再降低,非光化学猝灭(NPQ)在过高(RWC83.7%)或过低(RWC38.5%)的土壤水分下呈现较高值,表现出热耗散增加。研究认为,野生酸枣叶片气体交换参数及叶绿素荧光参数对土壤水分均具有明显的阈值响应,阈值点的土壤相对含水量大约为38.5%,低于阈值时叶片光合速率由气孔限制转向非气孔限制,PSⅡ受到损伤,电子传递受阻,光合机构受到破坏。     

不同土壤水分条件下硅对紫花苜蓿水分利用效率及产量构成要素的影响

硅是地壳中含量仅次于氧的元素,植物不可能在无硅的环境中生长.通过盆栽试验研究了不同土壤水分条件下硅对紫花苜蓿(Medicago sativa)水分利用效率及产量构成要素的影响.结果表明,在土壤含水量为田间最大持水量的35%和80%的条件下,硅对紫花苜蓿水分利用效率和生物量没有显著影响,而在土壤含水量为田间最大持水量的50%和65%的条件下,硅显著提高了紫花苜蓿水分利用效率和生物量(p<0.05),紫花苜蓿水分利用效率的增幅分别为35%和20%,主要途径为降低叶片蒸腾速率;紫花苜蓿生物量增幅分别为41%和14%,主要通过促进分枝和株高生长,而不受单枝生物量的影响.因此硅对紫花苜蓿水分利用效率和生物量的有益作用与其生长环境中的土壤水分条件密切相关.     

三峡库区消落带芦苇(Phragmites communis (reed))的光合生理响应和叶绿素荧光特性

模拟三峡库区消落带土壤含水量变化特征,设置了T1(淹水超过土壤表面2cm)、T2(土壤含水量为田间持水量的70%～100%)、T3(土壤含水量为田间持水量的40%～60%)3个不同处理组,用嘉陵江江水灌溉芦苇(Phragmites communis (reed)),研究了三峡库区不同消落带带位土壤不同含水量条件下芦苇穗期的光合生理生态响应机理和适应对策.结果表明,土壤不同含水量对芦苇植株光合色素、叶片气体交换参数、资源利用效率以及叶绿素荧光参数有不同影响.其中,T1的光合色素含量最低,T3的类胡萝卜素含量最高.在T1条件下,芦苇表现出较低的光能利用率(LUE)、CO2利用率(CUE)、净光合速率(Pn)、电子传递速率(ETR)和光化学荧光猝灭系数(qP),但与其它耐水淹植物相比,T1条件下的穗期芦苇仍具有较高的光合速率(17.067μmol·m-2·s-1),说明芦苇具有较强的耐水淹能力.在T2和T3条件下,芦苇具有较高的光能利用率(LUE)、CO2利用率(CUE)、净光合速率(Pn)、电子传递速率(ETR)和光化学荧光猝灭系数(qP).水分利用效率(WUE)大小顺序为T3>T1>T2.虽然T1的PSII有效光化学量子产量(F′v/F′m)最高,T2的光化学荧光猝灭系数(qP)最大,而T3的电子传递效率(ETR)和非光化学淬灭系数(NPQ)最高,表明在高光强和高温条件下,T3具有较强的热耗散能力,能有效保护光合机构,因而光合速率最高(20.47μmol·m-2·s-1).研究证实芦苇不仅具有耐水湿的特点,还具有耐旱性,芦苇适合作为三峡库区消落带植被恢复建设禾本科先锋物种.     

外源亚精胺对盐胁迫下黄瓜幼苗光合作用的影响

采用营养液栽培,研究了外源亚精胺对50mmol·L-1NaCl胁迫下黄瓜幼苗植株生长、叶片叶绿素含量、光合气体交换参数和叶绿素荧光参数(PSⅡ光化学效率)的影响。结果表明:NaCl胁迫显著降低了黄瓜植株生长量、净光合速率(Pn)、气孔导度(Gs)、胞间CO2浓度(Ci)(P<0.05),但对PSⅡ实际光化学效率(ФPSⅡ)、光化学淬灭(qP)、有效光化学效率(Fv′/Fm′)、非光化学淬灭(qN)和PSⅡ最大光化学效率(Fv/Fm)无显著影响(P>0.05);外源亚精胺显著提高了盐胁迫下黄瓜植株生长量、叶绿素含量、净光合速率、气孔导度、胞间CO2浓度,增加了ФPSⅡ、qP、Fv′/Fm′,降低了qN(P<0.05);外源亚精胺对Fv/Fm影响不显著(P>0.05)。外源施加亚精胺可增强盐胁迫下黄瓜植株的光合能力,主要是由于减弱了盐胁迫对植株的气孔限制,但对PSⅡ实际光化学效率影响较小,且叶面喷施比根施处理对改善盐胁迫下植株的生长和光合作用更有效。     

干旱胁迫对黑果枸杞幼苗光合特性的影响

以当年生黑果枸杞幼苗为试验材料,通过称重控水的方法设置对照(土壤含水量为32.96%~35.35%)、轻度干旱胁迫(土壤含水量为21.18%~22.32%)、中度干旱胁迫(土壤含水量为12.20%~13.82%)和重度干旱胁迫(土壤含水量为7.89%~8.73%)4个水分梯度,研究了干旱胁迫对黑果枸杞叶片光合色素、光合特性、叶绿素荧光特性的影响,以揭示黑果枸杞对干旱胁迫的适应能力和适应机制。结果显示:(1)随着干旱胁迫强度的增加,黑果枸杞幼苗叶片叶绿素含量、类胡萝卜素含量均呈显著下降趋势。(2)黑果枸杞幼苗叶片净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(G_s)在中度和重度干旱胁迫下显著下降;其胞间CO_2浓度(C_i)、水分利用效率(WUE)随干旱胁迫强度的增加而逐渐增加,而气孔限制值(L_s)随干旱胁迫强度的增加而逐渐降低。(3)随着土壤含水量的降低,黑果枸杞幼苗叶片初始荧光(F_0)和非光化学猝灭系数(q_N)逐渐增加,而其最大荧光(F_m)、PSⅡ最大光化学效率(F_v/F_m)、实际光化学效率(Ф_(PSⅡ))和光化学猝灭系数(q_P)均逐渐降低。研究表明,在干旱胁迫条件下,黑果枸杞叶片过多的能量以热的形式被耗散,反应中心开放程度降低,从而避免PSⅡ反应中心受到损伤,表现出一定的耐旱性;黑果枸杞生长所允许的最大土壤水分亏缺为7.89%,维持黑果枸杞具有较高的WUE和P_n的土壤水分阈值为12.20%~13.82%。     

水分胁迫对羊草光合产物分配及其气体交换特征的影响

 通过对典型草原优势植物种羊草(Leymus chinensis)的盆栽实验,模拟5个土壤水分梯度（分别为土壤持水量的75%～80%(对照)、60%～65%、50%～55%、35%～40%和25%～30% ）对羊草叶片相对含水量、光合速率、光合产物分配和种群CO2交换速率的影响。结果表明：随着土壤水分胁迫的增加,羊草叶片相对含水量呈先增加而后下降的单峰型变化,且在50%～55%处理下达到最大;叶片光合速率随着水分胁迫的增加而减小,且75%～80%、60%～65%、50%～55%的水分处理与35%～40%、25%～30%的水分处理的叶片光合速度日动态规律不同。羊草总生物量及根、鞘、叶生物量均随着水分胁迫的增加呈下降趋势。干旱促进早期羊草根的分配和根冠比增加, 但到后期却使它们降低, 表明羊草在受到较长期的持续干旱后通过增加根部的比重来提高抗旱性的能力逐渐降低。羊草根茎的生物量和分配随着土壤水分含量降低均呈现出先增加而后下降的趋势,羊草根茎的生物量在50%～55%处理下达最大（1.28 g·株-1）,而羊草根茎的分配在35%～40%处理下达最大（48.5%）。羊草种群CO2的净交换速率随着水分胁迫的增加而减小,其日交换量随着水分胁迫的增加而增加,且在60%～65%处理下达到最高,而后呈下降趋势,并在25%～30% 处理下为负值。研究结果表明,土壤持水量的40%可能是羊草对于水分变化响应的阈值。     

紫花苜蓿(Medicago sativa)对干旱胁迫的光合生理响应

紫花苜蓿是重要的豆科牧草,具有较强的抗旱性,然而干旱仍是制约紫花苜蓿生产的主要逆境因子。通过盆栽试验,以抗旱性强弱不同的两种紫花苜蓿为试验材料,对干旱胁迫下紫花苜蓿的光合生理进行较为系统的研究,结果表明：（1）干旱胁迫下两种紫花苜蓿叶片净光合速率（Pn）、蒸腾速率(E)、气孔导度(Gs)、叶绿素含量(Chl)都有不同幅度的下降;叶绿体超微结构遭到破坏。相对于抗旱性弱的苜蓿,抗旱性强的苜蓿随干旱胁迫程度的加深,净光合速率下降较慢,叶绿体的外形及基粒结构受到的影响较小。（2）轻度干旱胁迫下气孔限制是两种紫花苜蓿Pn降低的主要因素,中度和重度干旱胁迫下非气孔限制是Pn降低的主要因素。（3）对叶绿素荧光参数的研究表明：干旱胁迫下两种紫花苜蓿PSⅡ反应中心光化学效率(Fv/Fm)、PSⅡ潜在活性(Fv/Fo)降低。总体上抗旱性强的紫花苜蓿Fv/Fm和Fv/Fo下降幅度小,PSⅡ利用光能的能力及PSⅡ的潜在活性均较强。PSⅡ光化学淬灭系数（qP）、非光化学淬灭系数（qN）的变化表现为干旱胁迫下两种紫花苜蓿qP值降低、qN值升高,总体上抗旱性强的紫花苜蓿qP降低的幅度低且qN升高幅度大,表明抗旱性强的紫花苜蓿PSⅡ反应中心电子传递活性受到的影响小,光合机构的损伤程度低。     

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.     

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.