竹红菌甲素对红细胞膜和几种磷脂脂质体膜的流动性的光敏损伤

本文以荧光探针为手段,通过测量膜偏振度的变化,探讨了竹红菌甲素光敏作用对红细胞膜和几种磷脂脂质体膜的流动性的损伤。结果表明,甲素光敏作用使不同种类的磷脂(DPPC,DPPC/DPPE,红细胞膜磷脂)脂质体的流动性增加,其对光敏作用的敏感程度为红细胞膜磷脂脂质体显著小于DPPC/DPPE脂质体及DPPC脂质体。对红细胞膜来说,甲素光敏作用使其流动性呈现先降低而后增加的现象。去除膜上的spectrin以及用胰蛋白酶处理可使这种流动性变化的幅度受到抑制。据此,我们认为,膜磷脂,膜蛋白对甲素光敏作用中膜流动性的变化有着不同的影响,膜蛋白,特别是spectrin,是其中极重要的因素。     

竹红菌甲素对红细胞膜蛋白及膜磷脂的光敏损伤

In this paper, using human erythrocyte membrane, the photodamage of Hypocrellin A to membrane protein and phospholipid was studied by measuring the lipid peroxidation, the damage of phospholipid, the change of protein secondary structure, the endogenous fluorescence change and SDS-polyacrylamide gel electrophoresis analysis. These results showed that illumination of erythrocyte membrane in presence of Hypocrellin A can cause lipid peroxidation producing fluorescence adduct and MDA, decomposing in phospholipid composition in which PE and PS were more sensitive than others. Meanwhile, the secondary structure of membrane protein was destroyed and endogenous fluorescence decreased. The photodamage on phospholipid and spectrin occurred more seriously in the case they were embedded in membrane than they were in isolated form. So we suggest that they are interactions existing between proteins and phospholipids to enhance the damage on protein and phospholipid during the HA-sensitized photodamage on membrane.     

竹红菌甲素对红细胞膜蛋白及膜磷脂的光敏损伤

本文以红细胞膜为材料,通过测量磷脂过氧化荧光产物的产生,磷脂组分的损伤以及膜蛋白二级结构的破坏,内源荧光的下降和蛋白SDS-凝胶电泳分析,探讨了甲素光敏作用中蛋白和磷脂的损伤。结果表明:甲素存在时红细胞膜样品照光,使磷脂产生过氧化荧光产物和丙二醛,磷脂组分受到破坏(其中PE与PS较敏感),在膜状态的磷脂比提取的磷脂脂质体中的损伤来得剧烈。同时,膜蛋白二级结构遭到破坏,内源荧光下降。在膜状态中spectrin的损伤比提取出的spectrin的损伤来得严重。据此,我们认为,在甲素光敏作用产生的蛋白和磷脂的损伤过程中,蛋白与磷脂间存在着相互作用和相互影响,使损伤加剧。     

竹红菌甲素对红细胞膜上几种酶光敏失活作用的研究

Hypocrellin A (HA)-sensitized photoinactivation of enzymes in human erythrocyte membrane, including AchE, GPDH, Na(+)-K+ ATPase, Ca2(+)-Mg2+ ATPase were studied in this paper. The sensitivity of these four enzymes inactivated by HA and light are as following order: Ca2(+)-Mg2+ ATPase greater than Na(+)-K+ ATPase greater than GPDH greater than AchE. The relationship among ATPase inactivation, sulfhydryl photoinactivation and lipid peroxidation was also investigated. Results show that SH group photooxidation probably is one of the major reasons of enzyme inactivation whereas lipid peroxidation has little effect. The isolated GPDH was less sensitive than that membrane-bound, GSH, NAD acted protectively on GPDH and ATPase respectively. The evidence of electrophoresis and protein intrinsic fluorescence showed that protein structure did not change significantly even though most activity had lost in case of GPDH.     

竹红菌甲素对红细胞膜上几种酶光敏失活作用的研究

本文比较了竹红菌甲素对人红细胞膜AchE,GPDH,Na~ -K~ ATPase和Ca~(2 )-Mg~(2 )ATPase的光敏失活能力,结果表明甲素对Ca~(2 )-Mg~(2 )ATPase作用最强,Na~ -K~ ATPase次之,GPDH再次之,AchE最不敏感,甲素还引起膜蛋白巯基氧化,膜脂质过氧化。其中,巯基氧化可能是ATPase光敏失活的主要原因,而脂质过氧化对ATPase活力损伤作用不大。游离GPDH不如与膜结合的GPDH敏感。GSH,NAD分别对ATPase,GPDH有保护作用。膜蛋白的电泳及内源荧光证据表明:在GPDH活力受到严重损伤时,酶结构并未发生剧烈改变。     

竹红菌甲素对红细胞膜内脂双层的微扰

In this paper, using human erythrocyte membrane, the effect of Hypocrellin A on the lipid bilayer of the membrane was studied by measuring the change of the fluidity of the membrane, the energy transfer of the fluorescent probes, the shift of the fluorescent emission peaks, and the split of band-a of Hypocrellin A. The results showed that in the presence of HA, the fluidity of erythrocyte membrane was increased, the fluorescence intensity of the probes was decreased, and the fluorescence peaks shifted blue. These phenomena took place more seriously with the increment of HA concentration. Meanwhile, the band-a of HA excitation spectra was splitted. It was suggested from all of the results that HA could significantly perturb the lipid bilayer of erythrocyte membrane, there were interactions existing between the Hypocrellin A and the membrane. The HA was mainly located in the middle range of the membrane lipid bilayer when in high concentration (mainly to the 12-16 positions of the long chain fatty acid).     

竹红菌甲素对红细胞膜内脂双层的微扰

本文以荧光探针为手段,以人红细胞膜为材料,测量了膜偏振度的改变,荧光探针能量转移,荧光峰的蓝移和甲素激发峰的分裂。结果表明在有竹红菌甲素存在时,红细胞膜偏振度增加,探针荧光强度减小,荧光峰蓝移。甲素浓度增加时,上述现象更加明显,即它们之间有正的相关关系。同时,甲素激发光谱的a带发生分裂。据此,我们认为甲素对红细胞膜内脂双层产生明显微扰,甲素与红细胞膜间存在着相互作用。在甲素浓度较大时,它主要是渗入到红细胞膜脂双层的深层部位(膜脂肪酸链的12—16位)。     

竹红菌甲素对体外培养细胞光敏作用的实验研究

本文用免疫荧光细胞化学,流式荧光分析等方法了光敏剂ＨА对培养的ＨｌＬａ细胞的光敏化作用。结果表明,ＨＡ光敏化作用使细胞形态发生变化,细胞表面微绒毛丧失,细胞增殖受到抑制,上述变化随光照剂量增加而加剧,其中对肿瘤细胞的抑制作用较正常二倍体细胞更大,细胞群体中Ｇ１期细胞下降,Ｓ期细胞增多。ＨＡ的光敏化作用还使胞质微管变稀疏,微管中心的亮荧光近乎消失。以上实验说明,ＨＡ的光敏作用对培养细胞的作用引起细胞     

竹红菌甲素在脂质体中的光谱性质和结合能力研究

竹红菌甲素在脂质体中的光谱性质和结合能力研究邹伟,安静仪,蒋丽金（中国科学院北京感光化学研究所,１００１０１）关键词竹红菌甲素;光谱特性;结合;脂质体竹红菌甲素（Ｒ人）是一种新型并配类光疗药物,临床上治疗一些皮肤病效果显著”’,研究表明ＨＡ对癌细胞有...     

Fusion of planar bilayer phospholipid membranes with liposomes]

Lecithine-cholesterol liposomes containing amphotericin B ionoforic marker were used to study the interaction between liposomes and planar phospholipid membranes. The liposomes were shown to increase the permeability of the planar membrane, which may be explained in terms of membrane fusion. Bivalent cations (Mg2+ and particularly Ca2+), dicetylphosphate producing negatively charged groups on the membrane surface and the n-decane suspension in water promote the fusion, whereas the increase of the cholesterol content in the liposomes prevents it.     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

竹红菌素对人红细胞Na^＋—K^＋ATPase和钠通透性光敏损伤的研究

The hypocrellin B (HB)-sensitized photodamage on Na(+)-K+ ATPase and sodium permeability of human erythrocytes by means of NMR and biochemical techniques was studied in this paper. The decrease of the enzyme activity and increase of intracellular sodium concentration were usually observed simultaneously. The evidences suggested that the integrality of membrane phospholipid played an important role in maintaining the physiological sodium content of erythrocytes. The loss of the enzyme activity was a sensitive index compared with the increase of intracellular Na+ concentration during the photosensitization. From the comparison tests among HB, HA, protoporphyrin and bilirubin, we found that HB had more ability to increasing intracellular Na+ concentration than the other photosensitization even though the photodamage on the enzyme activity caused by HB, HA, and protoporphyrin were nearly the same. Besides the photoinactivation of Na(+)-K+ ATPase induced by HB and light, the enzyme was also inactivated in the medium containing HB in absence of light. The active oxygen radicals generated though HB mediated redox-cycling might be involved in the dark inactivation of the enzyme.     

Psoralen sensitized thermoactivated photodamage to leukocyte membranes]

Psoralen sensitized photodamage of rat peritoneal exudate cells was investigated. Irradiation of cells induced latent lesions in membranes which during thermal activation at the post-irradiation stage were transformed into permeability channels for trypan blue. The effect linearly increased with fluence of irradiation which indicates one hit production of thermolabile psoralen photoproducts in the membranes.     

Membrane fluidity and myotonia: Effects of cholesterol and desmosterol on erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia and on phospholipid liposomes

Previous spin-label and electromyographic experiments with rats fed 20,25-diazacholesterol, an inhibitor of the biosynthetic conversion of desmostero[ to cholesterol, demonstrated an increased erythrocyte membrane fluidity and myotonia, a prolonged muscle contraction upon stimulation. The current studies with rats showed normal erythrocyte fluidity in animals fed 20,25-diazacholesterol but maintained on a high-cholesterol diet and no myotonia. Studies of model membrane systems composed of phospholipid vesicles containing desmosterol, cholesterol, or both demonstrated that desmosterol increased membrane lipid fluidity relative to cholesterol, suggesting that in 20,25-diazacholesterol-induced myotonia, in which desmosterot accounts for 85% of the plasma sterol, the increased membrane fluidity previously observed in erythrocytes and sarcolemma m this animal model of human congenital myotonia may be due to desmosterol.     

Incorporation of Na,K-ATPase into human erythrocyte membranes using liposomes

A procedure for incorporation of isolated cattle brain Na,K-ATPase into erythrocyte membranes by proteoliposomes has been elaborated. The Na,K-ATPase activity of proteoliposome-treated human erythrocytes containing incorporated Na,K-ATPase does not exceed that of control erythrocytes. In the erythrocyte membrane the incorporated enzyme exists in a functionally active state and retains the vector properties of the Na+-pump. Exogenous ATP stimulates 22Na influx and 86Rb efflux in and from the erythrocytes.     

Local anesthetics structure-dependently interact with anionic phospholipid membranes to modify the fluidity

While bupivacaine is more cardiotoxic than other local anesthetics, the mechanistic background for different toxic effects remains unclear. Several cardiotoxic compounds act on lipid bilayers to change the physicochemical properties of membranes. We comparatively studied the interaction of local anesthetics with lipid membranous systems which might be related to their structure-selective cardiotoxicity. Amide local anesthetics (10–300 μM) were reacted with unilamellar vesicles which were prepared with different phospholipids and cholesterol of varying lipid compositions. They were compared on the potencies to modify membrane fluidity by measuring fluorescence polarization. Local anesthetics interacted with liposomal membranes to increase the fluidity. Increasing anionic phospholipids in membranes enhanced the membrane-fluidizing effects of local anesthetics with the potency being cardiolipin ? phosphatidic acid > phosphatidylglycerol > phosphatidylserine. Cardiolipin was most effective on bupivacaine, followed by ropivacaine. Local anesthetics interacted differently with biomimetic membranes consisting of 10 mol% cardiolipin, 50 mol% other phospholipids and 40 mol% cholesterol with the potency being bupivacaine ? ropivacaine > lidocaine > prilocaine, which agreed with the rank order of cardiotoxicity. Bupivacaine significantly fluidized 2.5–12.5 mol% cardiolipin-containing membranes at cardiotoxicologically relevant concentrations. Bupivacaine is considered to affect lipid bilayers by interacting electrostatically with negatively charged cardiolipin head groups and hydrophobically with phospholipid acyl chains. The structure-dependent interaction with lipid membranes containing cardiolipin, which is preferentially localized in cardiomyocyte mitochondrial membranes, may be a mechanistic clue to explain the structure-selective cardiotoxicity of local anesthetics.     

The glucose transport activity of human erythrocyte membranes. Reconstitution in phospholipid liposomes and fractionation by molecular sieve and ion exchange chromatography

Human erythrocyte membranes, at a protein concentration of 1–2 g/l, were solubilized with 0.12 M cholate in the presence of 0.06 M phospholipid (egg yolk phospholipids or phosphatidylcholine). More than 40% of the protein was solubilized. Cholate was removed by molecular sieve chromatography, whereby liposomes formed. These liposomes exchanged D-glucose faster than L-glucose. The recovery of glucose transport activity in the reconstituted system was estimated to be higher than 16%.The liposomes were heterogeneous in size, as shown by molecular sieve chromatography on Sepharose 4B, and small liposomes predominated. In liposomes formed with phosphatidylcholine, the distribution of glucose transport activity did not parallel the distribution of protein or phospholipid, and the activity was found mainly in the smallest liposomes. The proteins were incorporated mainly into the liposomes that eluted at the lowest ionic strength upon ion exchange chromatography.The glucose transport activity separated into three main peaks upon ion exchange chromatography of egg yolk phospholipid liposomes. The activity eluted at low ionic strength. The liposomes contained proteins mainly from the 3- and 4.5-regions (nomenclature according to Steck, T.L. (1974) J. Cell Biol. 62, 1–19). The activity peaks were highest in the first part of the chromatogram. The protein distribution did not coincide with the variation in activity over each peak. Therefore, it cannot be excluded that a minor component not seen in the electrophoretic analyses might be responsible for the glucose transport activity.