海洋弧菌QY105中褐藻胶寡糖酶OalA的基因克隆、重组表达与性质研究?

目的 褐藻胶寡糖酶可将褐藻胶多糖和寡糖降解为单糖,但现有的褐藻胶寡糖酶数量极少且活力较差。本文旨在从海洋弧菌QY105中克隆寡糖酶OalA的编码基因,重组表达并研究其酶学性质。方法 利用简并PCR和SiteFinding-PCR从海洋弧菌QY105中克隆得到褐藻胶寡糖酶OalA的编码基因,在大肠杆菌中进行重组表达,对重组酶进行分离纯化及酶学性质研究。结果 褐藻胶寡糖酶OalA基因全长2082 bp,编码一条含有693个氨基酸残基的多肽链,理论分子量为79.17 kDa,理论等电点为4.98,属于多糖裂解酶第15家族（PL-15）。重组OalA比活力为9.2 U?mg?1,最适温度30 ℃,在10 ℃的酶活力达到最高酶活的45%,最适pH7.6,在低于30 ℃和pH6~10范围内稳定,偏好降解polyM。结论 OalA具有低温酶的特点,且对polyM具有偏好性,可用于褐藻胶单糖制备、PL-15家族结构与功能相互关系研究等领域。     

链球菌噬菌体透明质酸裂解酶的异源表达及酶学性质表征

透明质酸酶是一类能降解透明质酸的酶的总称，在生物医药及药物递送等多方面具有重要的应用价值。为了挖掘高活性、高底物特异性及高稳定性的透明质酸酶，对来自链球菌噬菌体的透明质酸裂解酶(HylP)进行了异源表达及酶学性质表征。结果表明HylP由337个氨基酸残基构成，是多糖裂解酶家族16(PL16)新成员，重组HylP可在大肠埃希菌BL21(DE3)中可溶性表达，纯化产率可达到30 mg/L，最适酶活反应条件为40℃、pH 6.0，且在35℃及pH 6.0～7.0的中性条件下较为稳定。HylP专一性作用于透明质酸，比活力为24 u/mg；该酶的米氏常数(K_m)为0.13 mg/mL，催化常数(k_cat)为13.80 s^-1，具有较好的催化活性。链球菌噬菌体来源的HylP具有中温、高特异性、高酶活的特性，在绿色制备功能性低分子透明质酸上有潜在应用价值。     

噬琼胶卵链菌β-琼胶酶基因YM01-5的克隆表达及其酶学性质的研究

目的 从青岛近海海水中分离鉴定了1株能够降解琼胶的海洋新菌—嗜琼胶卵链菌（Catenovulumagarivoransgen. nov. sp. nov.）YM01T,并对其进行了全基因组测序。本文对该菌的1个β-琼胶酶基因YM01-5进行了克隆表达,并对重组琼胶酶的酶学性质进行了研究。方法 利用镍柱亲和层析对重组琼胶酶进行了分离纯化,采用DNS法测定重组酶的酶学性质,薄层层析(TLC)和质谱(MS)法对AgaYM01-5的酶解产物进行分析。结果 β-琼胶酶基因YM01-5全长2 412 bp,编码803个氨基酸,预测分子量为91.6 kDa,其催化模块属于糖苷水解酶GH50家族。重组琼胶酶YM01-5酶学性质的研究结果表明,该酶的最适温度为40 ℃,最适pH为9.0。在35 ℃以下具有良好的热稳定性,pH 6～10之间保持较高的稳定性,在该范围的pH缓冲液中放置12 h后,YM01-5仍能保持80%以上的酶活力。此外,该重组琼胶酶降解琼脂糖的Km、Vmax值分别为15.6 mg/mL和188 U/mg, 对降解产物的薄层层析及质谱分析结果表明,β-琼胶酶基因YM01-5以外切酶的形式作用于琼脂糖产生新琼二糖作为终产物。结论 该酶降解产物单一,有利于琼胶寡糖的制备,具有较高的工业应用潜力,它的发现也为研究菌株YM01中琼脂糖的代谢通路提供了参考。     

催化根皮素生成三叶苷的来源于穿心莲的糖基转移酶UGT74L2及其酶学研究

根皮素-4’-O糖基转移酶(P4’-OGT)是三叶苷生物合成途径中最后一步关键酶,可在体外催化根皮素生成三叶苷,但目前仅有少数P4’-OGT被鉴定。本研究利用已报道的苹果中P4’-OGT (MdPh-4’-OGT)序列对穿心莲转录组进行筛选,获得两条同源基因UGT74L2和UGT74L3。系统发育树分析表明,UGT74L2和UGT74L3与其他物种中已鉴定功能的UGT74家族聚为一类。体外酶促反应显示,UGT74L2可特异性催化根皮素生成三叶苷,而UGT74L3无产物产生。通过Ni-NTA亲和色谱纯化获得可溶性UGT74L2重组蛋白,以根皮素为底物进行酶促动力学研究。研究结果表明,UGT74L2酶促反应最适温度为40℃,最适pH值为8.0 (Tris-HCl体系)。Ca²⁺、Mn²⁺、Co²⁺对UGT74L2的活性有一定的抑制作用,而Mg²⁺可提高UGT74L2的活性,其他金属离子对UGT74L2活性无明显影响。酶促动力学参数测定结果显示,K_m值为29.84μmol·L     

纳豆菌糖苷酶的分离纯化及酶学性质研究

目的：通过对纳豆糖苷酶的酶学性质研究,获得其适宜条件,为其各方面研究应用提供理论基础。方法：根据不同蛋白质分子理化和生物学性质差异,应用SDS-PAGE凝胶电泳技术和DEAE-cellulose DE-52柱分离方法建立纳豆糖苷酶的分离纯化方法,确定相对分子量;以酶活力为指标研究其酶学性质。结果：纳豆糖苷酶的相对分子质量约为65.6kDa。最适温度为40℃,最适pH为5.0,在20℃~40℃、pH5~pH7、低金属离子浓度条件下稳定性较好;,金属离子浓度达到5mmol/L时Na 、K 、Ca2 对酶有激活作用,Mn2 、Cu2 对酶有抑制作用。结论：纳豆糖苷酶的热稳定性较好;对较强酸碱性环境敏感,耐受性较差;较高浓度金属离子对该酶有明显激活或抑制作用。     

耐热脂肪酶产生菌FS1403的分离筛选和16SrDNA基因序列的分析

从菲律宾火山口土样中分离、筛选获得一株产脂肪酶的耐热菌FS1403,对该菌脂肪酶的酶学性质初步研究表明:酶的最适作用温度为55℃,60℃处理1h保持70%酶活性,为中度耐热脂肪酶.酶的最适pH值为8.0,在pH 7～pH 10的条件下酶活都比较稳定.采用细菌16SrDNA通用引物,PCR扩增、克隆并分析了该菌的16SrDNA基因序列,同源性和系统发育学分析表明菌株FS1403与Bacillus subtilis具有最紧密亲缘关系.     

重组尿酸酶部分理化性质及其初步药效学研究

产朊假丝酵母尿酸酶由基因工程菌表达，文章对重组蛋白的亚基相对分子质量、最适作用温度、最适作用pH及其热稳定性和pH稳定性等理化性质进行了考察。成功构建了小鼠高尿酸血症动物模型，并用于重组尿酸酶的初步体内药效学研究。结果：经SDS-PAGE检测重组尿酸酶亚基的表观相对分子质量为32．4k。酶的最适作用温度和pH分别为40℃和8．0，且在pH6-12，温度20℃～60℃条件下比较稳定。初步体内药效学研究结果表明，重组尿酸酶可以显著降低模型小鼠体内的血尿酸水平。     

生鲜葱白自身酶解条件的研究

目的:通过对生鲜葱白自身酶解条件进行初步研究,为进一步研究葱白中的酶活性提供依据。方法:利用丙酮酸与2,4-二硝基苯肼-氢氧化钠系统发生显色反应原理,采用分光光度法测定丙酮酸的生成量来推算酶活力,同时根据丙酮酸的生成量间接反映含硫化合物的生成量,考察不同温度、pH、金属离子、时间以及不同灭活方式对酶解的影响。结果:温度在25～55℃之间酶活性较高,超过65℃后急剧下降;pH在6～8之间时,酶活性受到的影响比较小;Fe³⁺、Cu²⁺这两种金属离子对鲜葱白中酶活性有一定抑制作用,而Ca²⁺、Zn²⁺对葱白中酶的活性影响不是很大;酶促反应进行迅速,30min内完成;对比三种灭活方式的影响是微波>有机溶剂>高温。结论:葱白中酶有着其最适宜的反应温度、pH以及离子活性中心。     

青霉素酶分离纯化及酶学性质研究

本文对蜡状芽孢杆菌CMCC(B)63301产青霉素酶的分离纯化工艺及酶学性质进行了研究。粗酶液通过硫酸铵分级盐析、超滤除盐浓缩、SephadexG-75凝胶层析纯化后得到的青霉素酶液的比活力为68.2×106u/mg,纯化倍数为11.32,酶活回收率为35.04%。经SDS-PAGE检测为单一区带。该酶作用的最适温度为37℃,最适作用pH范围为6.5～7.0,在0～20℃下存放比较稳定。酶液中添加5%NaCl或5%甘油可提高酶的保存稳定性。     

可溶性固定化木瓜蛋白酶的制备及性质研究

目的 制备一种可溶性固定化木瓜蛋白酶(PP)并研究其部分酶学性质.方法 将PP偶联在羧甲基纤维素醋酸琥珀酸酯(AS-L)上,制备得到水溶性固定化PP,并测定游离和水溶性固定化PP最适作用pH、最适作用温度、Km值、热稳定性、酸碱稳定性、低温稳定性和回收稳定性.结果 最适作用pH分别为6.0、5.0,最适作用温度分别为60℃、70℃,Km值分别为2.53、3.07 mg·ml-1;可溶性固定化PP在60℃条件下,保温12 h,仍有62%的活性;在4℃条件下,30 d后活性保留达90%以上;在pH6时稳定性最好,pH4～7范围内也较稳定(相对活性＞80%).结论 可溶性固定化PP能够在大于pH5.5的溶液中完全溶解,最适作用pH范围变窄,最适作用温度和Km值升高;热和酸碱的稳定性均明显增强.     

Chemical modification of the histidine residue in phospholipase A2 from the Hemachatus haemachatus snake venom

The major phospholipase A₂ (DE-I) was isolated from Hemachatus haemachatus (Ringhals) venom and further purified on SP-Sephadex C-25. The homogeneity was verified by disc electrophoresis and the isoelectric point determined to be 7·35. The specific activity was 2950 U mg protein at 30°C and pH8·0.

The purified DE-I was subjected to chemical modification with p-bromophenacyl bromide at pH 8·0. Both the enzymatic activity and lethal toxicity were lost; however, the antigenic specificity remained unchanged. Although both ANS and Ca²⁺ showed pronounced protection on the inactivation process, the mechanism of ANS protection is different from that of Ca²⁺. Amino acid analysis showed only one out of four His-residues was modified and the modified residue was identified to be His-47 in the sequence.

Ultra-violet difference spectra revealed that both native and His-modified DE-I were perturbed by the presence of Ca²⁺ and the data indicated that a charge effect was responsible for the typical tryptophan blue shift of the Ca²⁺ bound native DE-I. However, the modified enzyme lost the characteristic tryptophan blue shift suggesting that a titratable group in the vicinity of a tryptophan residue was unable to exert a charge effect.

DE-I not only enhanced the emission intensity of ANS dramatically but also shifted the maximum from 480 to 450 nm. On the other hand, the His-modified DE-I did not increase the emission intensity at all. It is suggested tentatively that the hydrophobic pocket in which ANS bound may be the site of the enzyme that interacts with phospholipid.     

Effect of polyhistidine-tagging site on the stability of recombinant alginate lyase from Streptomyces sp. ALG-5

The purpose of this study was to investigate the effect of polyhistidine (His)-tagging site on the stability of alginate lyase from a marine bacterium Streptomyces species ALG-5 by the combined use of microchip electrophoresis and enzymatic depolymerizing activity assay. In microchip electrophoresis, C-terminally His-tagged alginate lyase (C-His-AL) was more stable than N-terminally His-tagged alginate lyase (N-His-AL) after the incubation in 50?mM potassium phosphate buffer (pH?7.0) at 37°C for 14?days. When the enzymatic depolymerizing activity of the same samples was measured, the activity of C-His-AL was not significantly changed for 14?days, whereas N-His-AL showed substantially declined activity after incubation. Consequently, this study demonstrated that the C-terminally His-tagging is more efficient than N-terminally His-tagging for preparing stable ALG-5 alginate lyase.     

Activity of the enzymes of the antioxidative system in cadmium-treated Oxya chinensis (Orthoptera Acridoidae)

One purpose in this research was to determine the toxic effects of Cd on antioxidant enzymes of Oxya chinensis (Orthoptera: Acridoidae). Changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPx) were measured in O. chinensis insects injected with Cd²⁺. Fifth-nymphs of O. chinensis insects were injected with Cd²⁺ at different concentrations (0, 0.55 × 10⁻⁴, 1.10 × 10⁻⁴, 1.65 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴ g g⁻¹). An increase in SOD activity in O. chinensis was observed at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ Cd²⁺. The SOD activity was lower at 2.20 × 10⁻⁴and 2.75 × 10⁻⁴ g g⁻¹ than that at 1.10 × 10⁻⁴ and 1.65 × 10⁻⁴ g g⁻¹. It appears that SOD had a positive protective effect at low Cd²⁺ concentrations, and that this effect disappeared at high Cd²⁺ concentrations. CAT activity was accelerated to varying degrees at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ for males and at 1.10 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴g g⁻¹ for females. CAT showed a strong detoxification effect with all treatments. GPx activity decreased with increasing Cd²⁺ concentration with all treatments for males and at 2.20 × 10⁻⁴ and 2.65 × 10⁻⁴g g⁻¹ for females. We showed that GPx activity had a weak detoxification function with all treatments for males and at high Cd²⁺ for females. Thus, CAT had a strong detoxification effect, whereas SOD had a medium and GPx had a weak detoxification effect. Among the three enzymes, CAT played an important role in the damaging mechanisms of reactive oxygen species in O. chinensis insects. Alterations of the antioxidant enzyme level under environmental stresses are suggested as indicators of biotic and abiotic stress.     

Flow-injection amperometric determination of dopamine in pharmaceuticals using a polyphenol oxidase biosensor obtained from soursop pulp

The amperometric determination of dopamine (Do) in pharmaceuticals formulations by flow injection analysis (FIA) is proposed. An enzymatically modified carbon paste electrode constituted by 25% (w/w) of polyphenol oxidase obtained from Annona muricata L. tissue, 30% (w/w) of graphite, 30% (w/w) of silicone and 15% (w/w) of 7,7,8,8 tetracyanoquinodimethane (TCNQ), was used as flow-through detector. The flow amperometric detection was carried out at a potential of 0.10 V (vs. Ag/AgCl) when an injected sample volume of 250 μl was inserted on a 0.3 M phosphate buffer carrier solution (pH 7.8) flowing at 2.5 ml/min. The developed biosensor showed good stability and reproducibility, enabling up to 500 determinations in 60 days, without considerable loss of enzymatic activity. The FIA system presented a linear response to Do concentrations in the interval from 2×10⁻² to 2×10⁻⁴ M, with relative standard deviations lower than 1.5%. The kinetic parameter K_M for the soluble and immobilized enzyme was 1.45×10⁻² and 1.91×10⁻² M, respectively. In the analyses of different commercially pharmaceutical formulations a relative deviation lower than about 3.4% was obtained.     

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents

, , , and . Effect of a phospholipase A₂ with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon 27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A₂ with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca²⁺-activated K⁺current; the sensitivity for voltage activation of the K⁺ current was enhanced by increasing free Ca²⁺ in the recording pipette from 10⁻⁸ M to 2 × 10⁻⁶ M. In contrast, peripheral blood lymphocytes possessed voltage-activated K⁺ currents that were inhibited by increasing intracellular Ca²⁺.

Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10⁻⁶ M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K⁺ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K⁺ currents in human lymphocytes, suggesting a specific increase in intracellular Ca²⁺ levels in both cell types.

The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca²⁺ levels. Exposure of either lymphocytes or epithelial cells to toxin (10⁻⁶ M) resulted in a transient increase in Ca²⁺. However, while the Ca²⁺ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca²⁺-regulated K⁺ channels may reflect an irreversible action of the B.fasciatus phospholipase A₂ on a Ca²⁺-dependent regulatory process.     

A protein kinase C inhibitor attenuates cyanide toxicity in vivo

We have examined the effect of pretreatment with a potent protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), against metabolic alterations induced by sodium cyanide (NaCN), 4.2 mg/kg, in brain of anesthetized male micropigs^© (6–10 kg). Brain high energy phosphates were analyzed using a ³¹P nuclear magnetic resonance (NMR) spectroscopic surface coil in a 4.7 Tesla horizontal bore magnet. H-7, 1 mg/kg, was given intravenously (i.v.) 30 min before NaCN challenge (H-7 + CN⁻). Prior to NaCN, H-7, or H-7 + CN⁻ administration, baseline ³¹P resonance spectra of 1-min duration were acquired for 5–10 min, and continued for an additional 60 min following i.v. NaCN injection, each animal serving as its own control. Peaks were identified as phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr) and adenosine triphosphate (ATP), based on their respective chemical shifts. Without H-7 pretreatment, NaCN effects were marked by a rising Pi and a declining PCr peak 2 min after injection, with only 2/5 of the animals surviving the 60 min experiment. Through a pretreatment period of 30 min, H-7 did not affect baseline cell energy profile as reflected by the ³¹P-NMR spectra, but in its presence, those changes (i.e. diminishing PCr and rising Pi peaks) elicited by NaCN were markedly blunted; 4/5 of the animals in this group survived the NaCN challenge. It is proposed that H-7, a pharmacologie inhibitor of PKC, may be useful in CN⁻ antagonism, underscoring the role of PKC in cyanide intoxication.     

Solid-phase electrochemical enzyme immunoassay with attomole detection limit by flow injection analysis

A sandwich electrochemical enzyme immunoassay with flow injection analysis for the model antigen mouse IgG has been developed with alkaline phosphatase as the enzyme label. The enzyme substrate, 4-aminophenyl phosphate and its enzymatic reaction product, 4-aminophenol have been studied by cyclic and hydrodynamic voltammetry. The determination of 4-aminophenol by flow injection analysis with electrochemical detection (FIAEC) has a linear range of 5.0 × 10⁻⁸ to 1.0 × 10⁻⁵ M, a detection limit of 2.4 × 10⁻⁸ M, and a sample throughput of 72 samples/h. The detection limit is set by a background capacitance response, which depends on the ionic strength difference between the sample and the mobile phase. The sandwich immunoassay has been characterized with respect to substrate concentration for the enzymatic reaction, detection limit, dynamic range and sources of error. Mouse IgG can be determined with a detection limit of 0.81 pg ml⁻¹ by a 30-min substrate incubation time and a six orders of magnitude linear dynamic range.     

超高效液相色谱-质谱联用法快速测定生脉饮中7种木脂素的含量

目的：建立生脉饮（红参、五味子、麦冬）中五味子甲素、五味子乙素、五味子丙素、五味子醇甲、五味子醇乙、五味子酯甲、五味子酚的含量测定方法。方法：采用超高效液相色谱-质谱联用（UPLC-MS）法进行定量分析,色谱柱ACQUITY UPLC BEH C₁₈（2.1 mm×50 mm,1.7 μm）,以乙腈（A）-0.1%甲酸水溶液（B）为流动相梯度洗脱：1~5 min,A-B（40：60）;5~7 min A-B（40：60~70：30）;7~10 min A-B（70：30）;10~11 min 100% A;11~12 min A-B（40：60）,流速为0.3 mL·min^-1,进样量2 μL,柱温30℃,采用ESI离子源在正离子模式下选择离子扫描模式进行定量分析。结果： 7种木脂素在一定范围内线性良好,相关系数均大于0.999,加样回收率在95.15%~100.84%之间。结论：该方法快速、灵敏度高、选择性好,适合测定生脉饮中木脂素的含量。     

The effects of five phospholipases A2 from the venom of king brown snake, Pseudechis australis, on nerve and muscle

The effects on vertebrate neuromuscular function of five homologous phospholipases A₂ (PLA₂) (Pa-3, Pa-8, Pa-9C, Pa-10F and Pa-12B) from the venom of the Australian king brown snake, Pseudechis australis, were determined. These isoenzymes (0.2–1.6 μM) reduced, with different potencies, responses of chick biventer cervicis preparations to nerve stimulation and to exogenously applied acetylcholine, carbachol and KCl in a time- and concentration-dependent way but with different potencies. They also blocked twitches of mouse hemidiaphragm preparations evoked by nerve and by direct muscle stimulation. Pa-8 was the most active and Pa-9C was the least potent. There was a strong correlation between the enzymatic activity and the effect of toxins on the responses of mouse hemidiaphragm to direct muscle stimulation, but weak correlation between the effects on indirect responses and enzymatic activity. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations showed that Pa-10F and Pa-12B at 0.2 μM significantly reduced quantal content after 10 min. Pa-8 (0.2 μM) reduced the amplitude of endplate potentials by about 25% and abolished miniature endplate potentials within 15 min. Pa-3 (0.2 μM) and Pa-9C (0.8 μM) also significantly reduced quantal content by about 30% of control after 30 min. Among these toxins, Pa-3 and Pa-8 at 0.2 μM depolarised mouse muscle fibres after 30 min. Extracellular recording of action potentials at motor nerve terminals of mouse triangularis sterni preparations indicated that these isoenzymes reduced the waveforms associated with both Na⁺ and K⁺ conductances. Since no facilitatory effect on the release process has been observed, the apparent blockade of K⁺ conductance by some of these toxins may not be a selective action on K⁺ channels, but may be secondary to membrane depolarisation. An in vivo study with Pa-8 and Pa-10F demonstrated myotoxic effects. Light microscopic examination showed a degeneration of mouse and rat skeletal muscle fibres caused by Pa-8 and Pa-10F. For the in vivo study, rats received 80 μg/kg of the toxins s.c. and mice were injected i.m. with the toxins (40 μg/kg). Myotoxicity appears to be the predominant effect of these five toxins.     

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro.

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca²⁺ (Fura2) and Na⁺ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o⁻ after exposure to H₂O₂ or ·OH (H₂O₂+Fe²⁺). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H₂O₂ or ·OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl⁻. Ca_i also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Na_i on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, ·OH inhibited the Na⁺ current (a measure of Na⁺-K⁺-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na⁺/K⁺-pump may contribute to the sustained elevation of Na_i. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.