活性铝对小麦根生长及酶活性的影响

利用铝形态分布与环境pH的相关性,通过改变染毒液pH条件,研究了不同浓度活性铝对小麦根生长、蛋白质含量及酸性磷酸酶活性的影响,并探讨了不同形态活性铝植物毒性的差异.本实验染毒液中总铝浓度设置为0(CK)、25(T₁)和75μmol·L^-1(T₂)3组,各组pH分别调至4.0、4.5、5.0和5.5.结果表明,微量Al_a与Al_b对小麦根生长均具有抑制作用.但随染毒液中活性铝组分的改变,小麦根蛋白质含量和酸性磷酸酶活性显现相反变化趋势:T₁和T₂组在pH4.0,活性铝主要成分为Al_a时(Al_a浓度高于活性铝浓度的90%),小麦根细胞蛋白质含量显著下降,酸性磷酸酶活性显著上升;T₁和T₂组在pH5.0,Al_a浓度降低至与Al_b浓度接近,且Al_a和Al_b浓度均低于10μmol·L^-1时,根细胞蛋白质含量显著上升,酸性磷酸酶活性显著下降.     

七种抑制剂对两种白蚁谷胱甘肽S-转移酶活性抑制作用的比较

利用分光光度酶动力学方法,确定了白蚁谷胱甘肽S-转移酶（GSTs）的最适反应条件,并进一步研究了7种抑制剂对黑翅土白蚁Odontotermes formosanus (Shiraki)和黑胸散白蚁Reticulitermes chinensis Snyder GSTs活性的体外影响。结果表明：白蚁GSTs测定的最适反应条件为pH 6.5,温度25℃,最适反应时间2 min。黑翅土白蚁GSTs的米氏常数(K_mCDNB和K_mGSH)分别为0.11±0.02 mmol/L和0.81±0.16 mmol/L,最大反应速度(V_maxCDNB和V_maxGSH)分别为425.92±19.67 nmol/(min·mg)和534.86±39.05 nmol/(min·mg)。黑胸散白蚁GSTs的米氏常数(K_mCDNB和K_mGSH)分别为0.12±0.03 mmol/L和1.03±0.31 mmol/L,最大反应速度(V_maxCDNB和V_maxGSH)分别为544.39±37.19 nmol/(min·mg)和715.45±83.68 nmol/(min·mg)。浓度为2×10^-5 mol/L时,槲皮素和辛硫磷对黑胸散白蚁GSTs活性的抑制作用要强于黑翅土白蚁,对黑胸散白蚁GSTs活性的抑制作用分别为62.28％和44.89％,对黑翅土白蚁GSTs活性的抑制作用分别为54.96％和28.36％。高效氯氰菊酯、甲氰菊酯、啶虫脒和单宁酸对黑翅土白蚁GSTs活性的抑制作用要强于黑胸散白蚁,对黑翅土白蚁GSTs活性的抑制作用分别为39.43％,72.07％,52.24％和82.19％;对黑胸散白蚁GSTs活性的抑制作用分别为14.96％,40.23％,39.96％和57.80％。阿维菌素对黑翅土白蚁和黑胸散白蚁GSTs活性的抑制作用没有显著差异,对黑翅土白蚁和黑胸散白蚁GSTs活性的抑制作用分别为76.21％和76.88％。这表明两种白蚁对药剂的敏感性完全不同。实验结果还表明,在3.2×10^-8～2×10^-5 mol/L内,上述植物次生物质和杀虫剂对两种白蚁GSTs活性的抑制率存在明显的剂量-效应关系。     

野生仙人掌多糖对肺鳞癌细胞SK-MES-1 生长的抑制作用

目的:探讨仙人掌多糖对体外肺鳞癌细胞(SK-MES-1)生长抑制作用。方法:体外培养SK-MES-1细胞,四甲基偶氮唑盐(MTT)法观察野生仙人掌多糖对其生长的抑制,计算最低抑制浓度及抑制率;观察不同浓度多糖对细胞形态的影响;SDS-PAGE凝胶电泳简析不同组别蛋白质表达的差异。结果:野生仙人掌多糖24 h和48 h对肿瘤细胞的最低抑制浓度和抑制率分别为0.0625 mg/ml.34.06%和0.0625 mg/ml 35.37%;不同组间蛋白质表达有差异。结论:野生仙人掌多糖对SK-MES-1肺鳞癌细胞有抑制作用。     

纳米TiO_2对油松种子萌发及幼苗生长生理的影响

以油松为材料,研究了不同浓度(50～2 000 mg/L)的纳米TiO2对油松种子萌发及幼苗生长和生理特征的影响,以探讨纳米材料对林木生长发育的调节作用.结果显示:各浓度纳米TiO2处理对油松种子萌发、幼苗生长及生理功能的改善均有一定程度的促进作用,且促进效应随着处理浓度的增加呈先升后降的趋势;1 000 mg/L纳米TiO2处理对种子发芽率、发芽势、发芽指数、活力指数、苗高、鲜重的提高以及霉变率的抑制作用效果最佳,它们依次分别比对照极显著提高28.00%、18.00%、12.36%、126.95%、19.57%、36.89%和下降15.33%(P<0.01);500 mg/L纳米TiO2能最有效增加油松幼苗根长、根系活力及其叶片蛋白质含量、SOD活性、POD活性,降低其细胞质膜相对透性和丙二醛含量,它们分别比相应对照极显著增加111.91%、53.85%、16.32%、170.93%、141.94%和降低45.18%、51.27%.研究表明,适当浓度的纳米TiO2处理不仅能显著促进油松种子的萌发和幼苗生长,而且能改善幼苗的生理功能,增强其抗逆能力.     

壳寡糖对大肠杆菌抑菌活性研究

分析壳寡糖对大肠杆菌抑菌效果的影响因素.采用摇瓶法和ELISA板法对不同浓度的壳寡糖进行抑菌试验;比较不同pH、不同脱乙酰度的壳寡糖对大肠杆菌抑菌效果的差异;比较不同聚合度的单一聚合度壳寡糖抑菌效果的差异.壳寡糖浓度大于5 mg/mL时抑菌效果与同浓度苯甲酸钠相近;pH为4时,0.156 mg/mL的壳寡糖溶液抑菌活性即能超过90%;pH为7时,5 mg/mL的壳寡糖才能达到90%抑菌活性.脱乙酰度为95%时,5 mg/mL的壳寡糖溶液抑菌活性能超过97%;脱乙酰度为45%时,40 mg/mL的壳寡糖溶液抑菌活性仅有56%;聚合度大于4的单一聚合度壳寡糖40 mg/mL时抑菌活性能达到99%.结果表明:提高壳寡糖溶液浓度、降低pH、提高脱乙酰度,能提高壳寡糖的抑菌活性,单一聚合度壳寡糖聚合度越高,对大肠杆菌的抑制作用越强.此外,采用ELISA板的方法进行实验,即节省试药又方便快捷.     

一种新型蚯蚓纤溶酶的部分性质研究

为获得一种高效的溶栓药物。从赤子爱胜蚓(Eiseniafoelida)中分离纯化得到了一种纤溶酶组分。用Lowry法测定蛋白质浓度,SDSPAGE鉴定纯度为98%,表观相对分子质量(Mr)为14850,纤维蛋白平板法测定其总纤溶活性为65.51×103mm2/mg,直接纤溶活性为15.61×103mm2/mg,间接纤溶活性为26.34×103mm2/mg。水解BAEE的米氏常数(Km)为1.82×105mol/L。水解ChromozymPL的米氏常数(Km)3.98×105mol/L,水解ChromozymtPA的米氏常数(Km)5.55×105mol/L活性,N端氨基酸序列测定的结果为VIGGTNAIPGEFPYQ。结果表明该纤溶酶分子量较小,间接活性较高,适宜作为一种新型的溶栓药物。     

薇甘菊挥发油的化感潜力

外来植物薇甘菊(Mikania micrantha Kunth.)已成为华南地区重要的杂草,其挥发油对植物、真菌和细菌均具有生物活性,对植物和水稻稻瘟病菌的抑制活性尤其显著.随着薇甘菊挥发油浓度(200、400、800、1600mg·L^-1)的增加,6种受试植物幼苗的生长随之明显减弱.薇甘菊挥发油(2500g·hm^-2)土壤处理,受试的6种植物鲜重明显减少,出苗时间推迟1～2d.薇甘菊挥发油在中等浓度(400mg·L^-1)时,对水稻稻瘟病菌的抑制作用最强,抑菌率为53.38%;对香蕉枯萎病菌的抑制作用次之,抑菌率为28.66%;对长春花疫病菌的抑制作用最弱,抑菌率为18.69%.     

盐胁迫对豌豆根液泡膜H^＋—ATPase活性及含量的影响

为了阐明液泡膜H^ -ATPase在盐胁迫下的作用和适应性调节机制,对豌豆（Pisum sativum L.）植株进行不同盐浓度和不同盐胁迫时间（1－3d）的处理后,分别测定液泡膜H^ -ATPase的H^ 转运活性、水解性和蛋白含量（A亚基）的变化。结果表明,100mmol/L和200mmol/L NaCl 处理1dH^ -ATPase的水解活性没有变化,而250mmol/L NaCl处理1d引起水解活性降低约25％。100mmol/L NaCl处理2d内水解活性没有变化,而第3天活性下降约20％。但是上述盐胁迫均能提高液泡膜H^ -ATPase的质子转运活性,说明盐胁迫后H^ -ATPase的水解活性和质子转运活性的变化不成比例,盐胁迫可能导致偶联比率的改变。Western blot研究发现,上述盐胁迫对液泡膜H^ -ATPase（A亚基）的含量基本无影响,仅100mmol/L NaCl处理3d后A亚基的量略有下降,这些结果证明,盐胁迫能刺激提高豌豆根液泡膜H^ -ATPase的H^ 泵效率,且泵效率的提高是源于偶联比率的改变,而不是由于ATP水解活性的提高和蛋白含量的增加。     

土壤杆菌固氮生理特性研究

对根癌土壤杆菌C58/pGV3850菌株的固氮生理特性研究结果表明,该菌具有自生固氮活性,其固氮活性的最适pH为8.0,温度为30℃.固氮活性在对数生长后期(14h)最高,延缓期和静止期乙炔还原活性较低.该菌株好氧,通过氧呼吸和吸氢酶的作用达到避氧固氮.当通入氧气超过呼吸耗氧时,对固氮活性产生抑制作用.在培养过程中增加NH_4~+浓度,固氮活性会降低,当达到15mmol/L时完全丧失固氮活性,表现NH_4~+阻遏固氮酶蛋白合成.在乙炔还原测定系统中加入NH_4~+并不影响乙炔还原反应,说明没有NH_4~+关闭现象.培养过程中加入MSX(2.5mg/ml)能解除10mmol/L NH_4~+对固氮酶合成的阻遏作用.固定的游离氮不能以NH_4~+形式分泌于胞外.固氮过程中放出大量氢气,培养16小时产氢量达65μmol H_2/mg蛋白.在限碳条件下(0.2%蔗糖)其吸氢酶活力可达520nmol H_2·ml~(-1)·h~(-1).     

相思豆毒蛋白—a A链在大肠杆菌中的表达及体外生物活性的测定

用RT PCR方法克隆了相思豆毒蛋白 aA链 (ABRaA)的cDNA编码序列 ,并将其重组到原核表达质粒 pET2 8b中。成熟的ABRaA在大肠杆菌中得到高效表达 ,可溶性重组蛋白质的获得率达 4mg/L培养物 ,而且具有较好的纯度。重组蛋白质体外生物活性的测定结果表明 ,其对兔网织红细胞裂解液的体外蛋白质生物合成具有较强的抑制作用 ,IC50 为 0 .0 8nmol/L ,与天然ABRaA的 (0 .0 6nmol/L)相差不大 ;重组ABRaA蛋白表现出较强的N 糖苷酶活性 ,其可切割大鼠肝脏核糖体 2 8SrRNA的A4 32 4位点 ,释放出一条约 4 2 0nt的小片段RNA。这些结果提示重组的ABRaA具有有效的生物活性 ,可以作为一种潜在的肿瘤化疗药物而用于制备免疫毒素。     

Effects of diltiazem or verapamil on calcium uptake and release from chicken skeletal muscle sarcoplasmic reticulum

The purpose of this study was to determine the effects of 2 Ca2+ channel blockers, verapamil and diltiazem, on calcium loading (active Ca2+ uptake) and the following Ca2+ release induced by silver ion (Ag+) and Ca2+ from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle. A fluorescent probe technique was employed to determine the calcium movement through the SR. Pretreatment of the medium with diltiazem and verapamil resulted in a significant decrease in the active Ca2+ uptake, with IC50 of about 290 micromol/L for verapamil and 260 micromol/L for diltiazem. Inhibition of Ca2+ uptake was not due to the development of a substantial drug-dependent leak of Ca2+ from the SR. It might, in part, have been mediated by a direct inhibitory effect of these drugs on the Ca2+ ATPase activity of the SR Ca2+ pump. We confirmed that Ca2+ channel blockers, administered after SR Ca2+ loading and before induction of Ca2+ release, caused a dose-dependent inhibition of both Ca2+- and Ag+-induced Ca2+ release rate. Moreover, if Ca2+ channel blockers were administered prior to SR Ca2+ loading, in spite of Ca2+ uptake inhibition the same reduction in Ca2+- and Ag+-induced Ca2+ release rate was seen. We showed that the inhibition of Ag+-induced Ca2+ release by L-channel blockers is more sensitive than Ca2+-induced Ca2+ release inhibition, so the IC50 for Ag+- and Ca2+-induced Ca2+ release was about 100 and 310 micromol/L for verapamil and 79 and 330 micromol/L for diltiazem, respectively. Our results support the evidence that Ca2+ channel blockers affect muscle microsome of chicken skeletal muscle by 2 independent mechanisms: first, reduction of Ca2+ uptake rate and Ca2+-ATPase activity inhibition, and second, inhibition of both Ag+- and Ca2+-induced Ca2+ release by Ca2+ release channels. These findings confirm the direct effect of Ca2+ channel blockers on calcium release channels. Our results suggest that even if the SR is incompletely preloaded with Ca2+ because of inhibition of Ca2+ uptake by verapamil and diltiazem, no impairment in Ca2+ release occurs.     

Regulation of the apolipoprotein B in heterozygous hypobeta-lipoproteinemic knock-out mice expressing truncated apoB,B81. Low production and enhanced clearance of apoB cause low levels of apoB

Meat quality of pigs is dependent on biochemical and biophysical processes in the time course post mortem (p.m.) and is associated with the intracellular Ca2+ homeostasis. However, there is little known about changes in the Ca2+ transporting proteins controlling the Ca2+ uptake of sarcoplasmic reticulum (SR) in the time course p.m. In this study changes in the Ca2+ transporting proteins were investigated in homogenates of longissimus muscles of 4 malignant hyperthermia susceptible (MHS) and 6 malignant hyperthermia resistant (MHR) Pietrain pigs. Muscle samples were obtained at different time intervals: biopsy 2 h prior slaughtering and from the carcass immediately after exsanguination (0 h), 45 min, 4 h, and 22 h p.m. The SR Ca2+ uptake rate was measured immediately after homogenization with closed calcium release channel (CRC), with opened CRC and without manipulation of CRC. Additionally the SR Ca2+ ATPase activity was determined.The results show: (i) The ability of SR to sequester Ca2+ declined to about 60% in the first 45 min p.m. in MHS samples irrespective of CRC state, whereas in MHR samples this decline was about 5%; (ii) Ca2+ uptake and Ca2+ ATPase activity were not different between the biopsy and 0 h samples, i.e. the stress of slaughter was of no immediate influence; (iii) The Ca2+ ATPase activity of the SR declined at about the same rate as the Ca2+ uptake in both MHS and MHR pig samples in the course of time p.m.; (iv) In samples, taken immediately after exsanguination, the Ca2+ ATPase activity of MHS pigs was higher than that of MHR pigs. However, in samples taken 4 h p.m. Ca2+ ATPase activity of MHS pigs has declined to about 30% of the value at 0 h; (v) The CRC can be closed and opened in all samples up to 22 h p.m. and seems to be fully functional at all sampling times; (vi) The CRC of MHS pigs is almost fully open, whereas the CRC of MHR pigs is only partially open at all sampling times; (vii) The permeability of the SR membrane to Ca2+ (determined as the ratio of SR Ca2+ ATPase with and without ionophore A23187) is the same in both MHS and MHR and did not change with ongoing time; (viii) No uncoupling of uptake from ATP hydrolysis occurred up to 4 h p.m., but the coupling differed between MHS and MHR for all time intervals with lower values for MHS pigs. The results suggest that the decreasing Ca2+ uptake rate of homogenates, sampled at different times p.m., is essentially caused by changes in the Ca2+ pump and not by changes in the CRC or an increased phospholipid membrane permeability to Ca2+.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Preparation and characterization of longitudinal tubules of sarcoplasmic reticulum from fast skeletal muscle

Sarcoplasmic reticulum (SR) serves a central role in calcium uptake and release, thereby regulating muscle relaxation and contraction, respectively. Recently, we have isolated fractions referable to longitudinal tubules (R2) and terminal cisternae (R4), the two major types of sarcoplasmic reticulum (A. Saito et al. (1984) J. Cell Biol. 99, 875-885). The terminal cisternae contain two types of membranes, the calcium pump membrane and the junctional face membrane. The terminal cisternae are filled with electron-opaque contents which serve as a Ca2+ reservoir. The longitudinal tubules consist mainly of the calcium pump membrane. In this study, we describe a new longitudinal tubule fraction (F2) and characterize it together with the R2 and R4 SR fractions. The calcium pump membrane of the longitudinal tubules is a highly specialized membrane consisting of about 90% calcium pump protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive changes in morphology can be observed in the SR fractions referable to osmotic differences during the fixation conditions using either glutaraldehyde-tannic acid or osmium tetroxide fixatives. The changes include swelling or shrinkage and aggregation of the compartmental contents when the fixative contains calcium ions. The two types of SR have different osmotic permeability to the same medium, as indicated by differential swelling or shrinkage. Both longitudinal tubule and terminal cisternae vesicles of SR appear larger and are spherical vesicles when the glutaraldehyde-tannic acid fixative is isotonic as compared with the "standard" fixation method. We have previously reported that the ruthenium red-sensitive calcium release channels are localized to the terminal cisternae. The terminal cisternae as isolated are leaky to Ca2+ since these channels are in the "open state" (S. Fleischer et al. (1985) Proc. Natl. Acad. Sci USA 82, 7256-7259). Thus, the Ca2+, Mg2+-dependent ATPase (Ca2+ ATPase) rate is only slightly enhanced in the presence of a Ca2+ ionophore, which dissipates the Ca2+ gradient across the SR membrane. We now find that preincubation with ruthenium red restores the tight coupling of the Ca2+ ATPase activity to Ca2+ transport. That is to say, ATPase activity is reduced and the addition of ionophore stimulates the Ca2+ ATPase activity 4- to 7-fold. The Ca2+ ATPase activity in longitudinal tubules is already tightly coupled. It is minimal after a Ca2+ gradient has been generated, but can be stimulated 9- to 20-fold when the Ca2+ gradient is dissipated with ionophore. This finding suggests that the Ca2+ ATPase activity in SR is tightly coupled to Ca2+ transport in situ.     

Effects of exercise of varying duration on sarcoplasmic reticulum function

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase

A strong connection with nucleotide activation of Ca2+ATPase and phospholamban inhibition has been found. Phospholamban decreases the number of activatable Ca2+ATPase without affecting substrate affinity or the ability of nucleotide to serve its dual modulatory roles, i.e., catalytic and regulatory. Low concentrations of certain nucleotide mimetics, quercetin, tannin, and ellagic acid, with structural similarity to adenine can unmask phospholamban's inhibitory effect while concurrently acting as competitive inhibitors of nucleotide binding. Micromolar concentrations of tannin (EC50 approximately 0.3 microM) and ellagic acid (EC50 approximately 3 microM) stimulated Ca2+ uptake and calcium-activated ATP hydrolysis at submicromolar Ca2+ in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was followed by pronounced inhibiton at only slightly higher tannin concentrations (IC50 approximately 3 microM), whereas inhibitory effects by ellagic acid were observed at much greater concentrations (IC50 > 300 microM) than the EC50. A complex relationship between compound, SR protein, and MgATP concentration is a major determining factor in the observed effects. Stimulation was only observed under conditions of phospholamban regulation, while the inhibitory effects were observed in cardiac SR at micromolar Ca2+ and in skeletal muscle SR, which lacks phospholamban. Maximal stimulation of Ca2+ATPase was identical to that observed with the anti-phospholamban monoclonal antibody 1D11. Both compounds appear to relieve the Ca2+ATPase from phospholamban inhibition, thereby increasing the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. Tannin, even under stimulatory conditions, is a competitive inhibitor of MgATP with a linear Dixon plot. The subsequent inhibitory action of higher tannin concentrations results from competition of tannin with the nucleotide binding site of the Ca2+ATPase. In contrast, ellagic acid produced a curvilinear Dixon plot suggesting partial inhibition of nucleotide activation. The data suggest that nucleotide activation of Ca2+ATPase is functionally coupled to the phospholamban interaction site. These compounds through their interaction with the adenine binding domain of the nucleotide binding site prevent or dissociate phospholamban regulation. Clearly, this portion of Ca2+ATPase needs further study to elucidate its role in phospholamban inhibition.     

Inhibition of beta-adrenergic stimulated calcium pump of rat cardiac sarcoplasmic reticulum by tricyclohexyltin hydroxide

The effects of tricyclohexyltin hydroxide (Plictran), an organotin acaricide, on 45Ca2+ uptake and Ca2+ ATPase were studied in vitro and in vivo in rat heart ventricular membrane vesicles, primarily sarcoplasmic reticulum. There was a concentration dependent inhibition of both 45Ca2+ uptake and Ca2+ ATPase in vivo as well as in vitro. Isoproterenol, a beta-adrenergic agonist, stimulated 45Ca2+ uptake and Ca2+ ATPase of sarcoplasmic reticulum and this was also inhibited by Plictran. Since cardiac relaxation is mediated by beta-adrenergic stimulation via Ca+ uptake by sarcoplasmic reticulum, the inhibition of calcium pump activity by Plictran may result in alterations in cardiac Ca2+ fluxes leading to cardiac dysfunction.     

Kinetic characterization of the normal and procaine-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump.

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport-coupled Ca(2+)-ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half-maximal inhibition occurred with procaine concentrations close to 15-20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast-kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium-induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca(2+)-dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast-filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.     

Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradually increases because lanthanum phosphoenzyme undergoes hydrolysis much more slowly than does magnesium phosphoenzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cardiac sarcolemmal and sarcoplasmic reticulum membrane vesicles exhibit distinctive (Ca-Mg)-ATPase substrate specificities

The nucleoside 5'-triphosphate (NTP) substrate specificities for Ca-stimulated ATPase and ATP-dependent Ca2+ uptake activities have been examined in cardiac sarcolemma (SL) and sarcoplasmic (SR) membrane vesicles. The results indicate that SL membrane vesicles exhibit a much narrower range of NTP substrate specificities than SR membranes. In SR membrane vesicles, the Ca-stimulated Mg-dependent hydrolysis of ATP and dATP occurred at nearly equivalent rates, whereas the rates of hydrolysis of GTP, ITP, CTP, and UTP ranged from 16-33% of that for ATP. All of the above nucleotides also supported Ca2+ transport into SR vesicles; dATP was somewhat more effective than ATP while GTP, ITP, CTP, and UTP ranged from 28-30% of the activity for ATP. In the presence of oxalate, the initial rate of Ca accumulation with dATP was 4-fold higher than for ATP, whereas the activity for GTP, ITP, CTP, and UTP ranged from 35-45% of that for ATP. For the SL membranes, Ca-activated dATP hydrolysis occurred at 60% of the rate for ATP; GTP, ITP, CTP, and UTP were hydrolyzed by the SL preparations at only 7-9% of the rate for ATP. NTP-dependent Ca2+ uptake in SL membranes was supported only by ATP and dATP, with dATP 60% as effective as ATP. GTP, ITP, CTP, and UTP did not support the transport of Ca2+ by SL vesicles. The results indicate that the SL and SR membranes contain distinctly different ATP-dependent Ca2+ transport systems.