2 型糖尿病患者凝血功能水平与血管病变发生的相关性

目的:探讨2型糖尿病患者凝血功能及血清肿瘤坏死因子α(TNF-α)及白细胞介素18(IL-18)水平与糖尿病血管病变的关系。方法:选择2014年6月-2015年10月在我院接受治疗的2型糖尿病患者83例作为研究对象,根据患者疾病进展情况将其分为血管病变组(45例)和无血管病变组(38例)。测定两组患者凝血酶时间(TT)、活化部分凝血酶原时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(Fg)、血糖(Glu)、糖化血红蛋白(Hb A1c)、肿瘤坏死因子α(TNF-α)及白细胞介素18(IL-18)水平;采用logistics回归分析2型糖尿病患者发生血管病变的危险因素。结果:血管病变组患者Glu,Hb A1c及Fg水平均显著高于无血管病变组,而APTT及PT水平均显著低于无血管病变组,差异均具有统计学意义(P0.05);两组间TT水平比较,差异无统计学意义(P0.05)。血管病变组患者血清中TNF-α及IL-18水平均高于无血管病变组患者,差异具有统计学意义(P0.05)。Hb A1c,Fg,TNF-α以及IL-18水平异常是2型糖尿病患者发生血管病变的独立危险因素(OR=1.23,1.45,2.632,3.884,P0.05)。结论:凝血功能紊乱及炎症反应是2型糖尿病患者发生血管病变的重要因素,临床应给予重视。     

蛇伤凉血合剂对尖吻蝮蛇咬伤患者凝血四项的影响

目的观察蛇伤凉血合剂对尖吻蝮蛇咬伤的临床疗效。方法将60例尖吻蝮蛇咬伤患者随机分为观察组和对照组各30例,两组患者均给予常规治疗,治疗组加服蛇伤凉血合剂,观察比较两组患者就诊时(咬伤2h内)、咬伤后72h凝血酶时间(TT)、凝血酶原时间(PT)、活化部分凝血酶原时间(APTT)、纤维蛋白原(Fg)的变化。结果两组患者就诊时TT、PT、APTT、Fg均在正常范围,两组比较差异无统计学意义。伤后72h,两组患者的TT、PT、APTT均升高,Fg均降低,而且组间比较差异均有统计学意义(P0.05)。结论蛇伤凉血合剂对尖吻蝮蛇咬伤所致凝血功能障碍有防治作用。     

氟比洛芬酯复合小剂量芬太尼对腹腔镜胆囊切除术患者镇痛效果及凝血功能的影响

目的:探讨氟比洛芬酯复合小剂量芬太尼在腹腔镜胆囊切除术后静脉自控镇痛中的应用及对患者凝血功能的影响。方法:选择2015年11月~2016年11月于我院行腹腔镜胆囊切除术的患者102例,随机分为对照组和研究组,每组51例。对照组患者术后采用小剂量芬太尼静脉自控镇痛,研究组患者术后采用氟比洛芬酯复合小剂量芬太尼静脉自控镇痛。观察并比较两组患者手术前后血清纤维蛋白原(Fg),活化部分凝血酶原时间(APTT),凝血酶原时间(PT),血小板计数(PLT),P物质,5-烃色胺(5-HT),白细胞介素-6、8(IL-6、IL-8)水平以及术后并发症的发生情况。结果:术前,比较两组Fg、APTT、PT、PLT、P物质、5-HT、IL-6、IL-8无差异(P0.05);术后,两组Fg、APTT、PT、PLT、P物质、5-HT、IL-6、IL-8均较术前上升,研究组低于对照组,差异均有统计学意义(P0.05)。研究组术后并发症率低于对照组(P0.05)。结论:氟比洛芬酯复合小剂量芬太尼能够提高腹腔镜胆囊切除术患者静脉自控镇痛的效果,改善凝血功能,降低炎症因子水平。     

胰岛素局部应用对糖尿病足患者血清炎性因子、VEGF、氧化应激和创面组织β-catenin表达的影响

目的:研究胰岛素局部应用对糖尿病足(DF)患者血清炎性因子、血管内皮生长因子(VEGF)、氧化应激和创面组织β-catenin表达的影响,旨在为临床DF患者的治疗提供理论依据。方法:将2017年4月至2019年12月于我院接受诊治的90例DF患者纳入研究,按照随机信封法将其分成观察组及对照组各45例。对照组实施负压吸引(NPWT)治疗,观察组则于NPWT治疗的基础上增用胰岛素治疗。比较两组治疗前后创面面积、炎性因子、血管内皮生长因子(VEGF)水平、氧化应激和创面组织β-catenin表达变化情况。结果:治疗后观察组及对照组创面面积均少于治疗前,且观察组少于对照组(均P0.05)。治疗后观察组及对照组血清白细胞介素-1(IL-1)及肿瘤坏死因子-α(TNF-α)均低于治疗前,而VEGF水平高于治疗前(均P0.05);治疗后观察组血清IL-1及TNF-α均低于对照组,而VEGF水平高于对照组(均P0.05)。治疗后观察组及对照组丙二醛(MDA)水平低于治疗前,而超氧化物歧化酶(SOD)水平高于治疗前(均P0.05);治疗后观察组MDA水平低于对照组,而SOD水平高于对照组(均P0.05)。治疗后观察组及对照组创面组织β-catenin表达水平均高于治疗前,且观察组高于对照组(均P0.05)。结论:胰岛素局部应用可促进DF患者的创面愈合,同时有利于下调血清炎性因子水平,减轻氧化应激反应,其主要作用机制可能与上调创面组织VEGF、β-catenin表达有关。     

美沙拉嗪对溃疡性结肠炎患者血清炎症因子与凝血指标的影响

目的:分析美沙拉嗪对溃疡性结肠炎患者血清炎症因子与凝血指标的影响。方法:按抽签法将102例溃疡性结肠炎患者分作对照组和研究组,各51例,对照组治疗采用柳氮磺胺吡啶,研究组治疗采用美沙拉嗪,观察两组治疗前后血清白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子α(TNF-α),血小板(PLT)、纤维蛋白原(Fg)、凝血酶原时间(PT)、D-二聚体(D-D)水平,氧化应激指标,免疫功能,临床疗效及安全性。结果:治疗后,研究组患者血清IL-1β、IL-6、IL-8、TNF-α、Fg、D-D水平及PLT均显著低于对照组,PT高于对照组,差异有统计学意义(P0.05)。研究组氧化应激过氧化脂质(LPO)、丙二醛(MDA)、超氧化物歧化酶(SOD)、免疫功能(CD3~+、CD4~+、CD8~+、CD4~+/CD8~+)改善均优于对照组(P0.05)。研究组总有效率高于对照组,不良反应率低于对照组,均有统计学差异(P0.05)。结论:美沙拉嗪治疗溃疡性结肠炎患者可显著抑制炎症,改善氧化应激状态,纠正凝血功能障碍并改善免疫功能,且临床疗效和安全性均较好。     

壳聚糖创面修复膜凝胶治疗Ⅱ度烧伤的效果及对血清炎症因子水平的影响

为了探讨壳聚糖创面修复膜凝胶治疗浅Ⅱ度烧伤对患者创面愈合及血清肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)、细胞间黏附分子-1(ICAM-1)水平的影响,本研究选取在我院(2014年4月至2016年4月)收治的97例浅Ⅱ度烧伤患者作为研究对象,所有患者均采用抗感染、保暖、止痛、补液等基础治疗,其中49例患者加用壳聚糖创面修复膜凝胶治疗(治疗组)、48例患者采用凡士林油纱布包扎(对照组),对比两组的创面愈合情况、疼痛评分、瘢痕形成情况、血清TNF-α、IL-10、ICAM-1水平。研究发现,治疗组在治疗第7天、第14天的创面愈合率显著高于对照组,具有统计学意义(p0.05);在创面愈合时间上治疗组的显著短于对照组,具有统计学意义(p0.05);在疼痛程度评分上,治疗组在治疗第7天、第14天时均显著低于对照组,差异具有统计学意义(p0.05);治疗前,治疗组和对照组的血清TNF-α、IL-10、ICAM-1水平差异均无统计学意义(p0.05);治疗7 d后,治疗组患者的血清TNF-α、IL-10、ICAM-1水平显著低于对照组(p0.05);治疗后3个月、6个月,治疗组的瘢痕评分均低于同期对照组患者,差异具有统计学意义(p0.05)。壳聚糖创面修复膜凝胶治疗浅Ⅱ度烧伤具有促进创面愈合、减轻烧伤患者炎症反应程度的作用。     

慢性乙型肝炎患者血清白介素17A和胆碱酯酶的表达及临床意义

目的:检测慢性乙型肝炎(CHB)患者血清白介素17A(IL-17A)、胆碱酯酶(CHE)水平,并分析其临床意义。方法:选取2018年1月到2019年3月期间在重庆三峡中心医院接受治疗的CHB患者84例,根据病情严重程度将所有患者分为轻度组30例、中度组28例、重度组26例,另选取同期在重庆三峡中心医院进行体检的健康志愿者50例作为对照组。比较各组的凝血四项指标[纤维蛋白原(FIB)、凝血酶原时间(PT)、凝血活酶时间(APTT)、凝血酶时间(TT)]、肝功能指标[谷丙转氨酶(ALT)、谷草转氨酶(AST)]、IL-17A、CHE水平,采用Pearson相关分析CHB患者血清IL-17A、CHE与凝血四项、ALT、AST的相关性。结果:重度组、中度组、轻度组、对照组的FIB、CHE水平逐渐升高,PT、APTT、TT、ALT、AST、IL-17A水平逐渐降低,两两比较均有统计学差异(P0.05),IL-17A与FIB、CHE呈负相关,与PT、APTT、TT、ALT、AST呈正相关(P0.05);CHE与FIB呈正相关,与PT、APTT、TT、ALT、AST呈负相关(P0.05)。结论:CHB患者血清中IL-17A、CHE水平与患者的肝功能和凝血功能密切相关,联合检测IL-17A和CHE有助于患者的病情评估以及预后判断。     

美沙拉秦对缓解期溃疡性结肠炎患者血清炎症因子的影响

目的:探讨美沙拉秦对缓解期溃疡性结肠炎(UC)患者血清炎性因子水平的影响。方法:收集2013年5月至2016年5月于我院治疗的缓解期UC患者153例,随机分为观察组75例及对照组78例。对照组患者给予常规治疗,观察组在对照组的基础上加用美沙拉秦治疗。观察比较两组治疗前后的血清白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、白细胞介素-17(IL-17)、肿瘤坏死因子-α(TNF-α)水平、疾病活动指数(DAI)评分、临床疗效、病情复发和不良反应的发生情况。结果:治疗后,观察组治疗总有效率为96.0%,明显高于对照组的62.8%(P0.05);观察组的血清IL-6、IL-8、IL-17及TNF-α水平均较治疗前明显降低,且显著低于对照组(P0.05);两组的DAI评分均有明显改善,但观察组的改善幅度明显大于对照组(P0.05);观察组患者的复发率(2.6%)显著低于对照组(21.8%)(P0.05),两组不良反应发生率比较差异无统计学意义(P0.05)。结论:美沙拉秦治疗缓解期溃疡性结肠炎的临床疗效显著,可显著抑制患者的炎症反应,同时可改善患者的临床症状,降低复发率,且安全性高。     

细菌溶解产物联合匹多莫德对反复呼吸道感染儿童炎性因子水平及免疫功能的影响

目的:探讨细菌溶解产物联合匹多莫德(PDT)治疗儿童反复呼吸道感染(RRTI)的临床效果及对血清炎性因子水平及免疫功能的影响。方法:选取我院2015年1月~2017年1月收治的122例RRTI患儿,采用随机数字表法均分为两组。对照组予以PDT治疗,观察组在对照组基础上加用细菌溶解产物治疗。比较两组治疗前后血清白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)、免疫球蛋白(IgA、IgG、IgM)水平的变化及临床疗效。结果:与治疗前对比,两组治疗3个月后血清IL-6、TNF-α水平均显著下降(P0.01),血清IL-10、IgA、IgG、IgM水平均显著升高(P0.01),且观察组血清IL-6、TNF-α水平均显著低于对照组,血清IL-10、IgA、IgG、IgM水平明显高于对照组(P0.01)。治疗3个月后,观察组总有效率为93.4%,较对照组明显升高(80.3%)(P0.05)。结论:反复呼吸道感染儿童应用细菌溶解产物联合匹多莫德治疗更能有效调节机体促炎-抗炎介质平衡、增强免疫功能,临床疗效显著。     

肾康注射液联合羟苯磺酸钙胶囊对PNS并发AKI患者肾功能、凝血功能及炎性因子的影响

摘要 目的：探讨肾康注射液联合羟苯磺酸钙胶囊对原发性肾病综合征（PNS）并发急性肾损伤（AKI）患者肾功能、凝血功能及炎性因子的影响。方法：选取2018年4月~2019年11月期间我院收治的134例PNS合并AKI患者,随机分为对照组（常规治疗基础上予以羟苯磺酸钙胶囊治疗）和联合组（对照组基础上予以肾康注射液治疗）,各67例。治疗14 d后,对比两组患者疗效、肾功能指标[尿素氮（BUN）、血肌酐（Scr）、白蛋白（Alb）]、凝血功能指标[凝血酶时间(TT)、凝血酶原时间（PT）、活化部分凝血酶原时间(APTT)、纤维蛋白原（FIB）]及炎性因子[白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、C反应蛋白（CRP）],记录两组不良反应情况。结果：联合组治疗14d后的临床总有效率高于对照组（P<0.05）。治疗14 d后,两组PT、APTT、TT、FIB、Scr、BUN、IL-6、TNF-α、CRP均较治疗前下降,且联合组低于对照组（P<0.05）。治疗14 d后,两组Alb升高,且联合组高于对照组（P<0.05）。对照组、联合组的不良反应总发生率对比未见统计学差异（P>0.05）。结论：相较于羟苯磺酸钙胶囊单药治疗,PNS合并AKI患者在羟苯磺酸钙胶囊的基础上联合肾康注射液治疗,可有效减轻肾功能损害,改善凝血功能,降低炎性因子水平,疗效明显,且未增加严重不良反应。     

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.