自身抗体检测对 自身免疫性肝炎的临床诊断价值

目的通过对肝炎患者多种自身抗体检出率的比较,探讨其在自身免疫性肝炎中的临床诊断价值。方法对75例自身免疫性肝炎(AIH)患者,64例非AIH肝炎患者和78例健康体检者进行自身抗体检测。采用免疫印迹法检测抗线粒体抗体-M2(AMA-M2)、抗肝肾微粒体-1抗体(LKM-1)、抗肝细胞胞质抗原-1抗体(LC-1)、抗可溶性肝抗原/肝-胰抗原抗体(SLA/LP);采用间接免疫荧光法检测抗核抗体(ANA),并对各检测指标进行比对分析。结果AIH组患者ANA、AMA-M2、LKM-1、LC-1、SLA/LP检测阳性率分别为100.0%、28.0%、9.3%、1.3%、10.7%均高于非AIH组患者的56.2%、3.1%、0.0%、0.0%、0.0%,且除LC-1外其余差异均具有统计学意义(P0.05)。结论联合检测ANA、AMA-M2、LKM-1、LC-1及SLA/LP对诊断自身免疫性肝炎具有重要的临床意义。     

血清PCT、CRP及IL-6联合检测诊断细菌性血流感染的临床价值分析

目的:探讨血清降钙素原(PCT)、C反应蛋白(CRP)及白介素-6(IL-6)联合检测诊断细菌性血流感染(BSI)的临床价值。方法:选取我院2015年8月到2016年10月收治的疑似细菌性BSI患者216例,入院后均送检血培养,根据培养结果将其分为阳性组(102例)和阴性组(114例)。统计细菌性BSI阳性率、革兰阳性菌感染率和革兰阴性菌感染率;检测血清PCT、CRP、IL-6水平,并比较两组患者的差异,同时绘制ROC曲线并计算出各指标及联合检测的灵敏度、特异度、阳性预测值、阴性预测值及约登指数值。结果:所有疑似BSI患者的细菌阳性检出率为47.22%,革兰阳性菌感染率与革兰阴性菌感染率对比无差异(P0.05);阳性组的血清PCT、CRP、IL-6水平均明显高于阴性组(P0.05);血清IL-6的AUC明显大于PCT和CRP(P0.05);PCT、CRP及IL-6联合检测的灵敏度、特异度、阳性预测值、阴性预测值及约登指数均明显高于单项检测(P0.05)。结论:血清PCT、CRP及IL-6对于BSI均有着一定诊断价值,而各指标联合检测诊断BSI的临床价值更高。     

血清TgAb水平在分化型甲状腺癌术后转移复发中的预测价值

目的:探讨完全切除甲状腺组织后血清甲状腺球蛋白(TG)阴性时,血清抗甲状腺球蛋白抗体(Tg Ab)对分化型甲状腺癌(DTC)术后复发/转移的预测价值。方法:选择2013年4月-2015年4月我院收治的57例完全切除甲状腺组织,TG阴性且Tg Ab阳性的DTC患者临床病历资料,并将其分为复发/转移组(20例)和无复发/转移组(37例)。采用放射免疫分析法测定并比较两组患者的血清TG、Tg Ab水平,分析Tg Ab对DTC复发/转移诊断的灵敏度、特异度、阳性预测值以及阴性预测值,采用Logistic回归分析DTC复发/转移的独立危险因素。结果:复发/转移组的血清Tg Ab水平为72~3850 IU/m L,高于无复发/转移组的18~3638 IU/m L,差异有统计学意义(P0.05)。其中Tg Ab对DTC复发/转移诊断的灵敏度为85.71%,特异度为83.33%,阳性预测值为75.00%,阴性预测值为90.91%。经Logistic回归分析发现,Tg Ab水平为100≤Tg Ab204 IU/m L、204≤Tg Ab≤1000IU/m L、1000 IU/m L是DTC复发/转移的独立危险因素(OR=1.267,2.853,6.791,P0.05)。结论:Tg Ab可作为评估完全切除甲状腺组织、TG阴性且Tg Ab阳性的DTC患者复发/转移的重要指标,其值越高,复发/转移发生的可能性越大。     

唇腺活检、自身抗体在干燥综合征诊断中的意义

目的:探讨干燥综合征(ss)患者的唇腺活检病理表现、自身抗体及其在诊断中的意义。方法:收集85例干燥综合征患者的临床体征、唇腺活检以及其他相关辅助检查,对其唇腺活检病理标本及相关检查进行分析。结果:根据唇腺病理结果,单纯唇腺活检的阳性率为68.2%;单纯血清抗SSA/SSB抗体检出阳性率为78.8%;联合唇腺活检、抗SSA/SSB抗体疾病检出率达94.1%,与单纯唇腺活检或抗SSA/SSB抗体单项阳性的疾病检出率有统计学意义(P0.05);并且唇腺活检阳性患者较阴性患者口干或眼干持续时间长(P0.05);但腮腺ECT异常、关节肿痛、肺脏损害均较唇腺活检阴性组患者累及率高,但差异无统计学意义(P0.05)。结论:唇腺活检、自身抗体是诊断干燥综合征的必要检查,而其联合应用更具有举足轻重的意义。     

超声联合钼靶X线对直径小于1 cm的乳腺癌诊断价值分析

目的:探讨超声联合钼靶X线对直径小于1 cm的乳腺癌诊断价值。方法:选择我院2012年1月至2017年12月收治的66例乳腺疾病患者,所有患者术前均经钼靶X线及彩色多普勒超声检查,分析其病理结果,分析钼靶X线、彩色多普勒超声及二者联合对乳腺肿块的检查结果(边缘毛刺征、血管、淋巴结、微小钙化),比较其对乳腺癌的灵敏度、特异度、准确度、阳性预测值及阴性预测值。结果:66例患者中,经病理检查发现恶性肿瘤34例,良性肿瘤32例。与病理检测相比,彩色多普勒超声联合钼靶X线对乳腺肿块的良恶性检出率无差异性(P0.05),而彩色多普勒超声,钼靶X线的良恶性检出率均显著降低(P0.05)。彩色多普勒超声与钼靶X线良恶性检出率对比无差异(P0.05),但均低于彩色多普勒超声联合钼靶X线的检出率(P0.05)。彩色多普勒超声与钼靶X线对乳腺癌的边缘毛刺征的检出率对比无统计学意义(P0.05);彩色多普勒超声对血管和淋巴结的检出率明显高于钼靶X线,而微小钙化的检出率明显低于钼靶X线,对比差异有统计学意义(P0.05)。彩色多普勒超声联合钼靶X线的灵敏度、特异度、准确度、阳性预测值及阴性预测值均明显高于彩色多普勒超声及钼靶X线(P0.05),钼靶X线及彩色多普勒超声间对比无统计学意义(P0.05)。结论:彩色多普勒超声与钼靶X线对直径小于1 cm乳腺癌的诊断各有优势,二者联合应用的诊断价值优于单一诊断方法。     

外周血中性粒细胞CD4（nCD4）在白血病合并细菌感染患者检测中的应用价值

目的:观察外周血中性粒细胞CD4(neutrophil CD4,nCD4)在白血病合并细菌感染患者检测中的准确度和灵敏度,评价其临床应用价值。方法:筛选我院在2016年1月至2019年1月期间收治的100例白血病患者作为研究对象并纳入研究组,其中合并47例合并细菌感染者纳入研究Ⅰ组,其余53例未合并细菌感染者纳入研究Ⅱ组,另外根据血培养检查结果将研究Ⅰ组分为阳性组(n=12)和阴性组(n=35),同时,筛选同时期进行健康体检者50例纳入对照组;对比降钙素原(Procalcitonin,PCT)、C-反应蛋白(C-reactive protein,CRP)及nCD4水平,并比较阳性组和阴性组的PCT、CRP、nCD4的诊断准确度和灵敏度。结果:研究组的PCT、CRP、nCD4高于对照组(P0.05),研究Ⅰ组的PCT、CRP、nCD4高于研究Ⅱ组(P0.05);研究Ⅰ组中,阳性组的nCD4荧光强度高于阴性组(P0.05);nCD4诊断白细胞细菌感染的准确度、灵敏度、特异度优于CRP及PCT诊断。结论:nCD4在白血病合并细菌感染患者检测中具有较好的临床参考价值,联合PCT、CRP检测能够显著提高检测准确性,可以作为诊断白血病合并细菌感染的诊断指标。     

腔内三维超声与宫腔镜检查对残角子宫畸形的诊断价值

目的:探讨腔内三维超声与宫腔镜检查对残角子宫畸形的诊断价值。方法:收集2009年3月~2013年3月我院收治的疑似残角子宫畸形的患者,分别行腔内三维超声和宫腔镜检查,以病理诊断结果为"金标准",比较两组检查结果的差异。结果:三维超声的灵敏度与宫腔镜比较,差异有统计学意义(P0.05),两组的特异度、准确性、阳性预测值、阴性预测值差异无统计学意义(P0.05);三维超声检查阳性检出率80.65%,阴性检出率19.35%,宫腔镜检查阳性检出率59.68%,阴性检出率40.32%,两组检出率差异无统计学意义(X2=0.498,P0.05)。结论:腔内三维超声诊断残角子宫畸形具有无创、操作简便的特点,但是漏诊率高于宫腔镜结果。     

尿微量蛋白与尿酶联合检测在早期肾脏损害诊断中的临床应用价值

目的:探讨尿微量蛋白与尿酶联合检测在早期肾脏损害诊断中的临床应用价值。方法:尿微量白蛋白(mALB)、视黄醇结合蛋白(RBP)采用免疫透射比浊法测定;尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG)采用终点法测定,尿肌酐(Cr)采用肌氨酸氧化酶法测定。结果:①尿蛋白定性为阴性的高血压、糖尿病、系统性红斑狼疮患者(尿蛋白阴性组)尿mALB/Cr、RBP/Cr、NAG/Cr显著高于正常对照组,两组比较差异具有统计学意义(P均<0.05)。尿蛋白定性为阳性的高血压、糖尿病、系统性红斑狼疮患者(尿蛋白阳性组)尿mALB/Cr、RBP/Cr、NAG/Cr显著高于阴性组,两组比较差异具有统计学意义(P均<0.05)。②应用单个指标或任意两个指标联合诊断早期肾损害的灵敏度较低,应用3个指标联合诊断早期肾损害的灵敏度达到86.30%。结论:尿mALB、RBP、NAG联合检测可以早期、可靠地诊断肾脏损害。     

高分辨率磁共振成像对颅内动脉粥样硬化斑块的诊断价值

目的:探讨高分辨率核磁共振成像(magnetic resonance imaging,MRI)对颅内动脉粥样硬化斑块的诊断价值。方法:选择2016年1月至2018年6月在我院诊治的缺血性脑卒中患者134例作为研究对象,所有患者都给予数字减影血管造影(Digital subtraction angiography,DSA)与高分辨率MRI检查,记录颅内动脉粥样硬化斑块特征与分型,以DSA诊断为金标准,判断MRI的诊断价值(阳性预测值、特异度、灵敏度、阴性预测值)。结果:在134例患者中,高分辨率MRI显示未见斑块62例,Ⅰ型5例、Ⅱ型26例,Ⅲ型26例、Ⅳ型15例,与DSA诊结果一致124例,占比92.5%。高分辨率MRI与DSA诊断颅内动脉粥样硬化斑块的Kappa值为0.89,MRI对各分型的颅内动脉粥样硬化斑块的阳性预测值、特异度、灵敏度、阴性预测值分别为:Ⅰ型87.0%、99.2%、95.8%和99.2%,Ⅱ型81.0%、98.5%、83.4%和98.5%,Ⅲ型82.7%、82.7%、84.0%和95.7%,Ⅳ型100.0%、100.0%、100.0%和100.0%。结论:高分辨率MRI用于诊断颅内动脉粥样硬化斑块的价值与DSA检查有很好的一致性,可反映硬化斑块的分型。     

CCP、AKA、APF联合RF检测对 类风湿性关节炎的诊断意义

目的 探究抗环瓜氨酸肽(CCP)抗体、抗角质蛋白(AKA)抗体、抗核周围因子(APF)抗体联合类风湿因子(RF)检测对类风湿性关节炎(RA)的临床诊断价值。方法 检测94例RA患者和69例非RA自身免疫疾病患者血清中该4项指标,并将该4种指标的敏感度、特异度、阳性预测值以及阴性预测值进行比对分析。结果 RA组患者该4项指标的阳性率均明显高于非RA组(P0.05)。RA组中CCP、AKA、APF和RF的敏感度分别为79.79%、45.74%、48.94%、75.53%,特异性分别为95.65%、94.20%、91.30%、79.71%,其中抗CCP与RF比较差异有统计学意义(P0.05)。RA组中CCP、AKA、APF和RF阳性预测值分别为96.15%、91.49%、88.46%、83.53%。4项指标联合检测中,任一指标阳性即判定RA阳性的敏感度为89.36%,4项指标均为阳性即判定RA阳性的特异度高达98.55%,阳性预测值达96.77%。结论 抗CCP抗体具有较高的灵敏度和特异性。4项指标联合检测可提高RA诊断的准确性,有利于RA患者早期诊断和治疗。     

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.     

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.