益生菌治疗溃疡性结肠炎的临床疗效及其机制研究

目的:研究益生菌治疗溃疡性结肠炎的疗效及其机制。方法:选择2011年2月-2015年12月在我院确诊为溃疡性结肠炎的84例患者进行研究,根据治疗方案不同随机分为观察组和对照组,观察组患者接受双歧三联活菌胶囊联合美沙拉嗪治疗,对照组患者接受美沙拉嗪治疗。结果:比较两组患者治疗前后的临床症状评分、外周血CD4~+CD25~+调节性T细胞占CD4~+T细胞的比例以及血清中白细胞介素-10的含量均高于治疗前(P0.05);观察组患者的临床症状评分均低于对照组(P0.05),外周血中Treg细胞占CD4~+T细胞的比例以及血清中白细胞介素-10的含量均高于对照组(P0.05)。结论:双歧三联活菌胶囊联合美沙拉嗪治疗溃疡性结肠炎的疗效优于美沙拉嗪单药治疗,可能与其增加调节性T细胞以及抑炎因子IL-10的含量有关。     

双歧杆菌乳杆菌三联活菌片联合血必净注射液对急性胰腺炎患者炎性介质、细胞免疫指标及肠黏膜屏障功能的影响

目的:探讨血必净注射液联合双歧杆菌乳杆菌三联活菌片治疗急性胰腺炎(SAP)患者的临床疗效。方法:选取2016年9月～2019年4月期间我院收治的SAP患者119例,根据随机数字表法将患者随机分为对照组(n=59)和研究组(n=60),对照组给予血必净注射液治疗,研究组在对照组的基础上联合双歧杆菌乳杆菌三联活菌片治疗,比较两组临床疗效,炎性介质、细胞免疫指标水平、肠黏膜屏障功能及不良反应发生情况。结果:研究组治疗7d后总有效率高于对照组(P0.05)。两组治疗期间未见药品不良反应发生。两组血清内毒素、二胺氧化酶(DAO)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)以及C反应蛋白(CRP)、CD8~+水平均降低,且研究组低于对照组(P0.05),CD4~+/CD8~+、CD4~+、NK细胞水平升高,且研究组高于对照组(P0.05)。结论:SAP患者在血必净的基础上联合双歧杆菌乳杆菌三联活菌片治疗,可提高机体细胞免疫功能,改善炎性因子水平和肠黏膜屏障功能,且用药安全性较好,临床应用价值较高。     

双歧杆菌三联活菌胶囊对溃疡性结肠炎患者血清白介素-6、8和10水平的影响及疗效观察

目的探讨双歧杆菌三联活菌胶囊对溃疡性结肠炎（UC）患者血清白介素（IL）-6、8和10水平的影响及疗效观察。方法选取88例UC患者,随机分为观察组和对照组。两组患者均酌情予以柳氮磺胺吡啶、5-氨基水杨酸和糖皮质激素等常规治疗。观察组患者在此基础上加用口服双歧杆菌三联活菌胶囊420mg/次,3次/d,连用8周。对照组患者除不使用双歧杆菌三联活菌胶囊外余治疗同观察组。观察两组患者治疗前后血清IL-6、8和10水平变化,并比较其临床疗效及不良反应。结果治疗8周后,两组患者血清IL-6、8水平明显下降,IL-10水平明显上升（P〈0.05或P〈0.01）,且观察组下降值或上升值大于对照组（P〈0.05）;观察组患者总有效率明显高于对照组（χ2=4.42,P〈0.05）;治疗中对照组出现不良反应3例,观察组出现5例,症状均较轻,两组患者不良反应发生率比较差异无统计学意义（χ2=0.14,P〉0.05）。结论双歧杆菌三联活菌胶囊治疗UC的疗效确切,安全性较好,作用与其能降低血清IL-6、8水平,提高血清IL-10水平,减轻肠黏膜局部炎症反应密切相关。     

双歧三联活菌胶囊对溃疡性结肠炎患者血清白介素-1β、10和肿瘤坏死因子-α水平的影响及疗效观察

目的探讨双歧三联活菌胶囊对溃疡性结肠炎(UC)患者血清白介素(IL)-1β、10和肿瘤坏死因子(TNF)-α水平的影响及疗效观察。方法选取UC患者76例随机分为观察组和对照组。两组患者均予以常规治疗(根据病情轻重选用抗生素、5-氨基水杨酸、糖皮质激素等治疗)。观察组患者在此基础上加用口服双歧三联活菌胶囊420mg/次,3次/d,连用8周。观察两组患者治疗前后血清IL-1β、10和TNF-α水平变化,并比较其临床疗效。结果治疗8周后,两组患者血清IL-1β和TNF-α水平明显下降,IL-10水平明显上升(P<0.05或P<0.01),且观察组下降值或上升值较对照组更明显(P<0.05);同时观察组患者临床总有效率明显高于对照组(94.74%vs 78.95%)(χ2=4.15,P<0.05)。结论双歧三联活菌胶囊治疗UC具有良好的临床效果,作用机制与其能降低血清IL-1β和TNF-α水平,提高血清IL-10水平,通过纠正促炎症因子与抗炎症因子的比例失衡,减轻肠道黏膜局部炎症反应密切相关。     

枯草杆菌二联活菌肠溶胶囊联合康复新液灌肠治疗溃疡性结肠炎的临床疗效

目的:探讨枯草杆菌二联活菌肠溶胶囊联合康复新液灌肠治疗溃疡性结肠炎的临床疗效。方法:选取2014年5月～2018年6月淮安市淮阴医院诊治的溃疡性结肠炎患者120例,根据患者入院先后顺序分为两组,对照组在常规治疗的基础上给予枯草杆菌二联活菌肠溶胶囊,观察组在对照组的基础上给予康复新液保留灌肠。比较两组患者的治疗总有效率、治疗前后血清白介素-8(IL-8)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平、血小板(PLT)和血酶原时间(PT)、纤维蛋白原(FIB)水平的变化及不良反应的发生情况。结果:治疗后,观察组总有效率为95.00%,对照组为83.33%,观察组显著高于对照组(P0.05)。两组患者治疗后血清IL-8、IL-6、TNF-α水平及PLT、FIB均较治疗前显著下降,且观察组以上指标均显著低于对照组(P0.05),而两组治疗后PT水平均较治疗前显著升高,且观察组显著高于对照组(P0.05)。两组不良反应发生率相比无统计学差异(P0.05)。结论:枯草杆菌二联活菌肠溶胶囊联合康复新液灌肠治疗可显著降低溃疡性结肠炎患者的炎性因子水平,改善凝血功能,提高临床治疗效果,且安全性较高。     

美沙拉嗪联合双歧三联活菌对溃疡性结肠炎患者TNF-α、IL-8及IL-10水平的影响

目的:探讨美沙拉嗪联合双歧三联活菌治疗溃疡性结肠炎(UC)的临床疗效及对患者TNF-α、IL-8及IL-10水平的影响。方法:选择2013年1月~2014年1月我院收治的UC患者60例,随机分为研究组与对照组,每组30例。对照组患者应用美沙拉嗪治疗,研究组在此基础上联合双歧三联活菌治疗。观察两组临床疗效、血清TNF-α、IL-8及IL-10水平变化及药物不良反应。结果:研究组治疗的总有效率为86.67%,显著高于对照组的63.33%(P0.05);治疗后,两组TNF-α、IL-8水平显著降低,IL-10水平显著升高(均P0.01),研究组TNF-α、IL-8显著低于对照组,IL-10水平显著高于对照组(均P0.05);研究组不良反应发生率为6.67%,明显低于对照组的33.33%(P0.05)。结论:美沙拉嗪联合双歧三联活菌对UC患者黏膜的保护作用显著,且有效抑制UC相关的炎性反应,安全可靠,适于临床推广与应用。     

双歧杆菌四联活菌片对乙肝相关慢加急性肝衰竭合并感染患者肠道菌群及免疫功能的影响

目的分析双歧杆菌四联活菌片对乙肝相关慢加急性肝衰竭(HBV-ACLF)合并感染患者肠道菌群及免疫功能的影响,为该病的治疗提供参考。方法选择2017年12月至2018年12月我院收治的106例HBV-ACLF合并感染患者,采用随机数字表法将其分为对照组(n=53)与研究组(n=53)。对照组患者采用常规治疗,研究组患者在对照组基础上口服双歧杆菌四联活菌片,两组患者均治疗1个月。比较两组患者治疗总有效率及肠道菌群(双歧杆菌、肠球菌、乳杆菌、肠杆菌)、免疫功能(CD4~+细胞、CD8~+细胞、CD4~+/CD8~+)、肝功能[丙氨酸氨基转移酶(ALT)、天冬氨酸转氨酶(AST)]情况。结果研究组患者治疗总有效率显著高于对照组(90.57%vs 73.58%,χ~2=5.194,P=0.023)。治疗前,两组患者肠道双歧杆菌、肠球菌、乳杆菌、肠杆菌数量及CD4~+细胞、CD8~+细胞、CD4~+/CD8~+、AST、ALT水平差异无统计学意义(均P0.05)。治疗后,对照组患者肠道双歧杆菌、肠球菌、乳杆菌、肠杆菌数量较治疗前差异无统计学意义(均P0.05),而研究组患者肠道乳杆菌、双歧杆菌数量较治疗前显著升高,肠杆菌、肠球菌数量较治疗前显著下降(均P0.05)。治疗后两组患者CD4~+细胞、CD4~+/CD8~+水平较治疗前显著升高,CD8~+细胞、AST、ALT水平较治疗前显著下降(均P0.05)。治疗后研究组患者CD4~+细胞、CD4~+/CD8~+水平较对照组显著升高,CD8~+细胞、AST、ALT水平较对照组显著下降(均P0.05)。结论双歧杆菌四联活菌片治疗HBV-ACLF合并感染患者的临床疗效较好,能够调节患者肠道菌群,改善患者免疫功能及肝功能,值得临床推广。     

双歧杆菌三联活菌肠溶胶囊对慢性乙型肝炎后肝硬化患者肝功能及血清细胞因子水平的影响

目的探讨双歧杆菌三联活菌肠溶胶囊对慢性乙型肝炎后肝硬化患者肝功能及血清细胞因子水平的影响。方法选取慢性乙型肝炎后肝硬化患者86例,随机分为观察组和对照组。两组患者均予以低盐饮食、保肝和营养支持等常规治疗。观察组患者加用双歧杆菌三联活菌肠溶胶囊0.42 g/次,每天3次,连用8周。对照组患者除不使用双歧杆菌三联活菌肠溶胶囊其他治疗措施同对照组。观察并比较两组患者治疗前和治疗8周后肝功能和血清细胞因子白介素(IL)-1β和肿瘤坏死因子(TNF)-α水平的变化。结果治疗8周后,两组患者丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)和总胆红素(TB)水平较前均有不同程度下降(P<0.05或P<0.01),且观察组下降程度比对照组更明显(P<0.05);同时两组患者血清IL-1β和TNF-α水平较前均有不同程度下降(P<0.05或P<0.01),且观察组下降程度比对照组更明显(P<0.05)。结论双歧杆菌三联活菌肠溶胶囊辅助治疗慢性乙型肝炎后肝硬化患者能明显降低患者肝功能指标,加速肝功能的恢复,并能下调血清细胞因子的水平,抑制肝内炎症反应,加快患者病情的恢复。     

复方谷氨酰胺联合双歧杆菌三联活菌胶囊治疗腹泻型肠易激综合征患者的临床疗效

摘要 目的：研究复方谷氨酰胺以及双歧杆菌三联活菌胶囊联用治疗腹泻型肠易激综合征患者的临床疗效。方法：选择2016年1月～2020年2月安徽省濉溪县医院收治的81例腹泻型肠易激综合征患者,将其随机分为两组。对照组41例患者仅口服420 mg的双歧杆菌三联活菌胶囊,每天3次;观察组40例患者加用500 mg的复方谷氨酰胺,每天3次。检测两组治疗前后的血清内毒素、二胺氧化酶活性及D-乳酸水平;血清白介素-6(Interleukin -6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor alpha,TNF-α)、白介素-8(Interleukin -8,IL-8)、高敏C反应蛋白(High sensitive C reactive protein,hs-CRP)水平;血浆生长抑素和血管活性肠肽水平并比较治疗后临床疗效及治疗过程中不良反应的发生情况。结果：治疗后,观察组的有效率为97.5%,明显高于对照组(75.61%,P<0.05);两组的血清内毒素、二胺氧化酶活性及D-乳酸、IL-6、TNF-α、IL-8、hs-CRP水平、血浆生长抑素和血管活性肠肽水平均较治疗前明显降低(P<0.05),且观察组以上指标均明显低于对照组(P<0.05)。观察组治疗期间的有2例嗜睡、头晕、乏力不适,发生率为5.00 %,对照组有2例头晕,1例头痛、口干,发生率为7.32 %,两组对比无统计学意义（P>0.05）。结论：复方谷氨酰胺联合双歧杆菌三联活菌胶囊治疗腹泻型肠易激综合征患者的临床效果明显优于单用双歧杆菌三联活菌胶囊治疗,其可有效修复肠黏膜屏障损伤,可能与其减轻炎症反应并改善胃肠激素水平有关。     

双歧杆菌三联活菌对胃癌部分切除患者术后 炎症因子、免疫和营养状态的影响

目的 探究胃癌部分切除术后补充双歧杆菌三联活菌对患者营养状态、炎症和免疫因子的影响,为肿瘤术后患者营养治疗支持提供建议。方法 选取2017年1月至2018年12月在我院行部分胃癌切除术的早期胃癌患者114例,采用随机数表法分为对照组(57例)与观察组(57例),两组患者于术后3～5 d开始进食,观察组在此基础上使用双歧杆菌三联活菌片,3粒/次,3次/日,口服,疗程14 d。对比两组治疗前后炎症因子水平(ELISA法测定外周血IL-2、IL-6及TNF-α水平)、免疫细胞水平(流式细胞仪测定外周血CD4~+、CD8~+、CD4~+/CD8~+水平)、营养状态(蛋白质测定仪测定外周血白蛋白和总蛋白水平)及治疗不良反应。结果 治疗前两组IL-2、IL-6及TNF-α水平无明显差异(t=1.861,0.744,1.489;P=0.064,0.457,0.138),治疗后两组上述指标均较治疗前显著下降,其中观察组治疗后上述指标水平显著低于对照组(t=5.784,6.153,8.457;均P0.05);治疗前两组CD4~+、CD8~+、CD4~+/CD8~+水平无明显差异(t=1.301,1.084,0.542;P=0.198,0.283,0.590),治疗后两组CD4~+、CD4~+/CD8~+均较治疗前显著提高,CD8~+显著下降,其中观察组治疗后CD4~+、CD4~+/CD8~+水平显著高于对照组(t=5.205,3.904;均P0.05),CD8~+显著低于对照组(t=6.507;P0.05);治疗前两组白蛋白及总蛋白水平无明显差异(t=1.301,0.651;P=0.198,0.518),治疗后两组上述水平均较治疗前显著提高,其中观察组治疗后白蛋白及总蛋白水平显著高于对照组(t=13.664,8.590;均P0.05);两组不良反应发生率无明显差异(5.26%vs 10.53%;χ~2=1.086,P=0.297)。结论 胃癌部分切除术后补充益生菌可有效调节患者的炎症和免疫状态,显著改善营养状况。     

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.