右美托咪定与咪达唑仑对前列腺电切术患者术后认知功能的影响

目的:分析右美托咪定与咪达唑仑对前列腺电切术(TURP)患者认知功能的影响。方法:选择2013年5月-2015年5月在我院接受TURP治疗的良性前列腺增生患者73例作为研究对象。根据麻醉方法不同,将所选患者分为右美托咪定组和咪达唑仑组,分别采用右美托咪定和咪达唑仑麻醉。观察并比较不同时间点两组患者的心率(HR)及平均动脉压(MAP)的变化情况。应用精神状态量表(MMSE)评估两组患者手术前后的认知功能。结果:两组患者术前HR及MAP比较,差异均无统计学意义(P0.05);两组患者手术不同时间点HR、MAP均显著低于术前,且右美托咪定组低于咪达唑仑组,差异具有统计学意义(P0.05);两组患者术前MMSE评分比较,差异无统计学意义(P0.05);两组患者术后MMSE评分均低于术前,且右美托咪定组低于咪达唑仑组,差异均具有统计学意义(P0.05);右美托咪定组术中寒战发生率显著低于咪达唑仑组,差异具有统计学意义(P0.05)。结论:右美托咪定与咪达唑仑对TURP患者均会造成早期认知功能障碍,但右美托咪定的影响较小,患者生命体征较平稳,值得临床推广应用。     

右美托咪定对神经外科手术患者围术期血糖、血清TNF-α和IL-6水平的影响

目的:探讨右美托咪定(DEX)对神经外科手术患者围术期血糖、血清肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平的影响。方法:选取2016年2月-2016年11月我院神经外科收治的患有脑膜瘤拟择期行开颅手术的患者50例,随机分为研究组和对照组各25例。研究组患者于麻醉诱导前10 min以DEX 1.0μg/kg加入0.9%氯化钠注射液中,配成50 mL溶液静脉输注,10 min内输完,而后以0.5μg/kg/h输注DEX。对照组以等速输注等量0.9%氯化钠注射液。记录并对比两组麻醉诱导前10 min(T1)、诱导后(T2)、开皮时(T3)、手术1 h(T4)、手术结束时(T5)心率(HR)、平均动脉压(MAP)及血糖、血清TNF-α和IL-6水平。结果:两组患者HR在T2、T3时间点均高于T1时间点,而研究组患者HR在T2、T3时间点均低于对照组(均P0.05);研究组患者MAP在T3时间点高于T1时间点,对照组患者在T3、T4时间点高于T1时间点,而研究组患者MAP在T3、T4时间点均低于对照组(均P0.05);两组患者在T2、T3、T4、T5时间点血糖均高于T1时间点,而研究组患者在同时间点均低于对照组(P0.05);对照组患者在T2、T3、T4时间点血清TNF-α及IL-6均高于T1时间点(P0.05),研究组患者不同时间点血清TNF-α及IL-6变化无统计学意义(P0.05),且在T2、T3、T4时间点血清TNF-α及IL-6均显著低于对照组(P0.05)。结论:DEX在神经外科手术中有维持患者血流动力学稳定、抗炎、抑制血糖升高的作用,减轻炎症反应和应激反应,从而促进神经外科手术患者术后的恢复。     

右美托咪定对重型颅脑损伤患者术后持续镇静的效果及脑组织的保护作用

目的:探讨右美托咪定对重型颅脑损伤患者术后持续镇静的效果及脑组织的保护作用。方法:将2013年7月至2015年7月医院收治的90例重度颅脑损伤患者按照随机数字表法分为观察组45例和对照组45例,两组患者均常规麻醉后行开颅手术,观察组术后持续输注右美托咪定,对照组术后持续输注芬太尼。比较两组患者手术不同时间点血清S-100β蛋白、MAP、PCO2、Sp O2、HR及脑氧代谢指标变化情况。结果:两组患者术后各时段Sjv O2、Ca-jv DO2、CERO2比较差异无统计学意义,术后各时段两组间Sjv O2、Ca-jv DO2、CERO2比较差异亦无统计学意义(P0.05)。观察组T1~T4时段MAP、HR均显著降低,与T0时段比较比较差异具有统计学意义(P0.05);T1~T4时段观察组MAP、HR显著低于同时段对照组(P0.05)。从T1~T4时段两组患者血清S-100β均呈上升趋势,两组T1~T4时段血清S-100β显著高于T0时段(P0.05);T2~T4时段观察组血清S-100β显著低于同时段对照组(P0.05)。结论:术后持续输注右美托咪定能够对重度颅脑损伤患者能够对患者产生镇静、镇痛作用,且在一定程度上保护了脑组织。     

右美托咪定对腰硬联合麻醉下子宫肌瘤切除术患者术中的镇静效果及综合评价

目的:评价右美托咪定对硬腰联合麻醉下子宫肌瘤切除手术患者术中的镇静效果的影响。方法:选择2013年1月至2016年8月在我院行子宫肌瘤切除术的患者60例,随机分为两组,每组30例。对照组患者静脉注入丙泊酚60μg/kg·h,观察组患者静脉注入右美托咪定0.4μg/kg·h。分别记录患者术前、术中30 min以及术后的Ramsay评分、BIS评分、MAP、HR以及Sp O2等生命指征和术后不良反应发生的情况。结果:实验组患者术中30 min的镇静效果优于对照组,其中Ramsay评分显著高于对照组(P0.05),且BIS评分显著低于对照组(P0.05)。实验组患者术中30 min HR显著低于对照组(P0.05),但是两组之间MAP以及Sp O2却没有明显差异(P0.05)。比较两组患者术后不良反应发生的情况发现两者差异没有明显的统计学意义(P0.05)。结论:右美托咪定在硬腰联合麻醉下子宫肌瘤切除术患者术中可以显著改善镇静作用,值得临床上进一步推广应用。     

喉上神经阻滞复合瑞芬太尼和右美托咪定用于经皮气管切开术的临床效果

目的:探讨喉上神经阻滞复合瑞芬太尼和右美托咪定应用于经皮气管切开术(PT)的临床效果。方法:将60例因呼吸困难行PT的患者随机分为两组。所有患者均喉上神经阻滞,对照组(30例)采用丙泊酚复合瑞芬太尼,实验组(30例)采用右美托咪定复合瑞芬太尼。观察并比较两组患者术前(T1)、麻醉药物注射结束后(T2)、气管切开置入套管时(T3)、手术结束时(T4)收缩压(SBP)、舒张压(DBP)、心率(HR)、血氧饱和度(SpO_2)及平均动脉压(MAP)、警觉/镇静(OAA/S)评分的变化及术中并发症的发生情况。结果:对照组T3、T4时刻SBP、DBP、HR、MAP均较T1时明显升高,且明显高于实验组同时点(P0.05),而实验组T2、T3、T4时SBP、DBP、HR、MAP与T1时刻比较差异无明显统计学意义(P0.05)。实验组术中呛咳、呼吸抑制的发生率显著低于对照组(P0.05)。两组T2～T4时刻OAA/S评分均明显低于T1时刻,且实验组OAA/S评分均明显低于对照组同时刻(P0.05)。结论:喉上神经阻滞复合右美托咪啶和瑞舒芬太尼应用于PT中可维持血流动力学的稳定,减少应激反应,降低术中并发症的发生率。     

右美托咪定联合舒芬太尼用于脊柱手术患者术后镇痛的疗效评价

目的:探讨右美托咪定联合舒芬太尼对脊柱手术患者术后镇痛效果的影响。方法:选择2013年1月至2016年8月在我院行脊柱手术的患者70例,随机分为两组,每组35例。对照组患者术后静脉注入舒芬太尼3μg/kg·h镇痛,观察组患者静脉注入右美托咪定1.5μg/kg·h联合1.5μg/kg·h舒芬太尼。分别记录患者术后6 h,12 h,24 h以及48 h的VAS评分、Ramsay评分、MAP、HR以及SpO_2等生命指征;记录患者术后苏醒期10 min,20 min,30 min以及60 min的RSAS躁动评分;同时记录患者术后48 h不良反应发生的情况。结果:监护仪监护两组患者围手术期的相关生命指征,统计分析结果显示两组患者术后HR,MAP以及SpO_2等指征没有明显的差异(P0.05);比较两组患者结束手术苏醒期的躁动情况,RSAS躁动评分显示实验组患者术后30 min之后较对照组相对安静,没有较明显的躁动行为(P0.05);实验组患者术后的VAS疼痛评分明显低于对照组(P0.05);比较两组患者的Ramsay评分发现实验组患者术后镇静程度显著优于对照组(P0.05);两组患者术后0-24 h使用镇痛泵的有效按压次数存在差异(P0.05);实验组患者术后48 h发生恶心呕吐和寒颤的不良反应发生率明显低于对照组(P0.05)。结论:右美托咪定联合舒芬太尼用于脊柱手术术后镇痛可以显著改善患者镇静状态,降低患者术后发生不良反应的发生率,值得临床上进一步推广应用。     

右美托咪定经鼻滴注对脊柱手术患者围术期血流动力学变化及镇静作用的临床研究

目的:观察右美托咪定经鼻滴注对脊柱手术患者围术期的镇静作用及对血流动力学变化的影响。方法:将90名ASAⅠ~Ⅲ级、18~65岁、拟在全身麻醉下行脊柱手术、且术后须拔除气管导管的患者,随机分为三组:对照组(C组)、右美托咪定1μg/kg组(D1组)和右美托咪定1.5μg/kg组(D2组),n=30。分别记录给药前(T0)、诱导前(T1)、插管前1 min(T2)、插管后1 min(T3)、手术开始即刻(T4)、术毕停全麻药时(T5)、拔管前1 min(T6)、拔管后3 min(T7)和入PACU(T8)时患者的心率(HR)、平均动脉压(MAP)、氧饱和度(SPO_2),术毕停全麻药至拔除气管导管的时间、患者在术后恢复室停留时间、Ramsay镇静评分。结果:T0时3组患者HR、MAP、SPO_2、Ramsay镇静评分无统计学差异(P≥0.05);与C组相比,D1组和D2组各时间点HR、MAP明显降低,Ramsay镇静评分提高(P≤0.05),SPO_2无明显变化(P≥0.05);与D1组相比,D2组各时间点HR、MAP明显降低、Ramsay镇静评分提高(P≤0.05),SPO_2无明显变化(P≥0.05);D1组各时间点HR、MAP、SPO_2、Ramsay镇静评分无明显差异(P≥0.05);D2组HR、MAP、SPO_2、Ramsay镇静评分明显无差异(P≥0.05)。C组T3、T4、T5、T6、T7、T8各时间点HR、MAP均较T1、T2升高,Ramsay镇静评分均明显提高(P≤0.05),SPO_2无明显变化(P≥0.05)。结论:麻醉诱导前40 min右美托咪定经鼻滴注可有效抑制插管和拔管期反应、使血流动力学变化更加稳定,并显著降低降低患者术后躁动的发生率。     

不同浓度的右美托咪定对老年腰椎术患者的镇静效果、氧化应激和血流动力学的影响

目的:探讨不同浓度的右美托咪定对老年腰椎术患者的镇静效果、氧化应激和血流动力学的影响。方法:选取2013年3月-2018年4月期间我院收治的老年腰椎术患者90例为研究对象。根据随机数字表法将患者分为对照组(n=30)、低浓度组(n=30)以及高浓度组(n=30)。低浓度组麻醉诱导前输注0.5μg/kg右美托咪定,高浓度组输注1μg/kg右美托咪定,对照组不输注右美托咪定。比较给药前(T0)、给药后10 min(T1)、给药后30 min(T2)、给药后60 min(T3)的听觉诱发电位指数(AAI)、改良/镇静视觉评分(OAA/S)。比较T0、T3、手术后24h(T4)的三组患者丙二醛(MDA)、超氧化物歧化酶(SOD)水平。比较T0-T3时间点三组患者的心率(HR)、平均动脉压(MAP)情况。观察三组患者术后不良反应发生情况。结果:与T0时间点比较,三组T1-T3时间点AAI、OAA/S均显著降低(P0.05);与对照组相比,低浓度组与高浓度组T1-T3时间点AAI、OAA/S均较低,且高浓度组低于低浓度组(P0.05)。与T0时间点比较,三组T3、T4时间点MDA均显著升高,T3时间点SOD显著降低(P0.05);与对照组相比,低浓度组与高浓度组T3、T4时间点MDA均降低,且高浓度组低于低浓度组(P0.05);高浓度组T3、T4时间点SOD高于低浓度组和对照组(P0.05)。与T0时间点比较,三组T2、T3时间点HR、MAP均降低(P0.05);与对照组相比,低浓度组、高浓度组T2、T3时间点HR均降低,且高浓度组低于低浓度组(P0.05);与对照组相比,低浓度组、高浓度组T2、T3时间点MAP均降低,但高浓度组高于低浓度组(P0.05)。三组患者术后不良反应总发生率比较差异无统计学意义(P0.05)。结论:1μg/kg右美托咪定对老年腰椎术患者的镇静效果较好,可有效维持血流动力学稳定,减轻氧化应激反应。     

右美托咪定对腹腔镜子宫切除术患者血流动力学及应激反应的影响

目的:探讨右美托咪定(Dex)对腹腔镜子宫切除术患者血流动力学及应激反应的影响。方法:选择2012年7月到2014年7月在我院行腹腔镜子宫切除术的36例患者,随机分为实验组和对照组各18例。实验组静脉注射Dex 1μg/kg直到手术结束,对照组同法给予生理盐水。于麻醉前15 min(T0)、插管后5 min(T1)、气腹后20 min(T2)、术后立刻(T3)和术后24 h(T4)四个时间点,分别监测其平均动脉压(MAP)、心率(HR),并测定血浆皮质醇(Cor)、去甲肾上腺素(NE)和白细胞介素6(IL-6)水平。结果:T0时两组患者的MAP和HR比较,无显著性差异(P0.05),对照组T1、T2、T3时明显升高(P0.05),实验组轻微升高,两组相比差异有显著性(P0.05)。T4时两组患者的MAP和HR均恢复至正常水平。T0时两组Cor、NE和IL-6比较,无显著性差异(P0.05),对照组T2、T3、T4时明显升高(P0.05),实验组轻微升高,两组相比差异有显著性(P0.05)。T4时除NE,其他指标均达到峰值。结论:Dex能维持围术期患者血流动力学稳定,降低术中的应激反应。     

不同剂量右美托咪定对妇科腹腔镜手术患者应激反应、血流动力学和术后认知功能的影响

摘要 目的：探讨不同剂量右美托咪定对妇科腹腔镜手术患者血流动力学、术后认知功能以及应激反应的影响。方法：选取2016年3月～2018年5月在我院行妇科腹腔镜手术的150例患者,按照随机数字表法分为甲、乙、丙三组,各50例,甲组在麻醉诱导后以0.8 μg?kg-1?h-1的速率输注右美托咪定,乙组在麻醉诱导后以0.4 μg?kg^-1?h^-1的速率输注右美托咪定,丙组给予患者注射等量生理盐水,对比不同时间点三组患者血流动力学变化情况、应激指标、麻醉恢复时间、气腹时间、拔管时间、麻醉恢复室（PACU）停留时间、改良镇静-躁动评分(RASS)、不良反应发生率,统计患者术后认知功能障碍 (POCD)发生情况。结果：T0（麻醉诱导前10 min）时,三组HR、MAP对比差异无统计学意义（P＞0.05）;T1（气管插管后1 min）、T2（气腹后5 min）时,甲组、乙组HR、MAP均低于丙组,甲组HR、MAP低于乙组（P＜0.05）;T3（术毕）时,甲组、乙组HR均低于丙组（P＜0.05）,甲、乙、丙三组MAP对比,差异无统计学意义（P＞0.05）。甲、乙两组术后1 h、6 h、12 h时RASS评分均低于丙组（P＜0.05）,甲组术后1 h、6 h、12 h时RASS评分低于乙组（P＜0.05）;T1、T2、T3时,甲组、乙组去甲肾上腺素（NA）、促肾上腺皮质激素（ACTH）水平均低于丙组,甲组NA、ACTH水平均低于乙组（P＜0.05）。甲、乙组拔管时间、PACU停留时间均短于丙组,甲组短于乙组（P＜0.05）。甲组、乙组、丙组术后POCD发生率为2.00%（1/50）、10.00%（5/50）、24.00%（12/50）,甲组POCD发生率低于丙组（P＜0.05）。三组麻醉恢复时间、气腹时间、不良反应发生率相比较,差异无统计学意义（P＞0.05）。结论：妇科腹腔镜术患者围术期应用右美托咪定有利于维持血流动力学稳定,减轻应激反应,降低术后认知功能障碍发生率,其中0.8 μg?kg^-1?h^-1右美托咪定作用更明显。     

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.