Effects of acute gamma-irradiation on extracellular adenine nucleotide hydrolysis in developing rat brain

Cell membrane is highly sensitive to irradiation which, acting directly or indirectly, may disturb functions of constitutive proteins including membrane enzymes. Plasma membrane surface-located enzyme chain of ecto-nucleotide triphospho diphosphohydrolases (NTPDases) and 5′-nucleotidase are involved in termination of cell purinergic signalization by hydrolyzing extracellular, excitatory adenosine triphosphate (ATP), as well as nucleotide di-, and mono-phosphate (ADP and AMP) to neuroprotective adenosine. Extracellular ATP, ADP, and AMP hydrolyzes were examined in purified synaptic plasma membranes after whole-body acute irradiation. All measurements were done 24 h after irradiation of developing (15-, 30-day-old) and adult (90-day-old) rats with low (50 cGy) and high (2 Gy) dose of gamma-rays. Both, high and low doses inhibited nucleotide hydrolyses in 15-day-old rats; in 30-day-old rats low dose of radiation inhibited ADP and AMP hydrolyses while high dose inhibited only ATP hydrolyse. In adult rats high dose induced no effects, while low dose stimulated nucleotides hydrolyses. According to obtained results it was concluded that ecto-nucleotidases of young rats are more sensitive to irradiation, since even low dose induces inhibition of ecto-nucleotidases activities. Ionizing radiation, by decreasing brain nucleotide hydrolyses in developing rats, induces accumulation of ATP and decreases production of adenosine in synaptic cleft which could be neurocytotoxic. On the contrary, in adult rats low dose of radiation stimulates NTPDase and 5′-nucleotidase activity and protective adenosine production which indicates protective and adaptive mechanisms developed in adult brain neuronal cells. The article is published in the original.     

UVA irradiation induces energy-independent phospholipid-flip in mammalian plasma membrane

Translocation from the outer to the inner membrane leaflet (flip) of phospholipids after ultraviolet A (UVA) irradiation was investigated in Chinese hamster ovary cells. Fluorescent 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzox- adiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (NBD-labeled phosphatidylserine [NBD-PS]) was used to assay transbilayer lipid movement. A marked increase in flip of NBD-PS was observed immediately after low-dose UVA irradiation which was not lethal and returned to the basal level after 6 h. UVA-induced flip was not attributed to the increase of permeability by UVA irradiation because cells that were negative for staining with propidium iodide also showed increased flip of NBD-PS. Furthermore, the enhancement was independent of adenosine 5'-triphosphate, demonstrating the lack of involvement of phospholipid translocase. Marked increases were also observed in flip of both NBD-phosphatidylethanolamine and NBD-phosphatidylcholine immediately after UVA irradiation, showing that the increase was independent on the head groups of phospholipids. These findings indicated that UVA changes the flip-flop of phospholipids and that the cell membrane is a molecular and cellular target of UVA.     

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.     

Haematoporphyrin derivative (Photofrin II) photosensitization of isolated mitochondria: inhibition of ADP/ATP translocator

To gain further insight into the mechanism by which irradiation of mitochondria in the presence of haematoporphyrin derivative (Photofrin II) (PF II) causes impairment of mitochondrial oxidative phosphorylation, the rate of ADP/ATP exchange via the ADP/ATP translocator was measured fluorometrically is isolated rat liver mitochondria. In accord with noncompetitive inhibition, PF II photosensitization decreases the maximum rate of exchange Vmax (20.8 and 9.6 nmol ATP effluxed min-1 x mg protein in the control and after 2 min irradiation, respectively) without changing the ADP affinity for the carrier (Km = 5 microM in both cases). Comparison of the rate of oxygen uptake by mitochondria stimulated by either ADP or by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) confirms that the adenine nucleotide carrier is a major target of photodynamic action which causes oxidative phosphorylation impairment.     

高效液相色谱法测定细胞内三磷酸腺苷及其代谢物的含量（英文）

研究了三磷酸腺苷(ATP)及其代谢物在细胞内的含量以及2-叔丁基-1,4-苯醌(TBBQ)对ATP及其代谢产物在细胞内含量的影响。建立了一种高效液相色谱法(HPLC)用于快速分离、检测细胞内ATP及其代谢产物(二磷酸腺苷(ADP)和一磷酸腺苷(AMP))的含量。使用岛津高效液相系统及艾杰尔Venusil MP C18柱,采用等度洗脱的方式。流动相A相为50 mmol/L磷酸氢二钠和15 mmol/L三甲胺(TEA),用醋酸(HAc)调节pH至7.88;流动相B相为甲醇。采用建立的高效液相色谱法得到了3种代谢物的工作曲线,相关系数高(R~2≥0.999 6),MRC-5细胞中3种代谢物的含量均在线性范围(0.1~100μmol/L)内。该方法检出限低。采用预冷的80%(体积分数)甲醇水溶液提取细胞内的代谢物。该研究建立的方法成功地应用于检测MRC-5细胞中的ATP、ADP和AMP的含量,结果表明,TBBQ会对ATP、ADP、AMP在细胞内的含量产生影响,但TBBQ浓度和ATP、ADP以及AMP在MRC-5细胞内浓度的关系比较复杂。     

Convenient syntheses of adenosine 5′-diphosphate,adenosine 5′-methylenediphosphonate,and adenosine 5′-triphosphate

Adenosine 5′-tosylate is converted to adenosine 5′-diphosphate (ADP), adenosine 5′-methylenediphosphonate, and adenosine 5′-triphosphate (ATP) in good yields by direct displacement with the appropriate inorganic salt.     

PHOTOLYSIS AND HYDROLYSIS OF ADENOSINE 5'-PHOSPHATES

Abstract— Mild acid hydrolysis of adenosine 5'-di-, tri- and tetra-phosphate (ADP, ATP and ADTP) occurs predominantly in the side chain, yielding orthophosphate and adenosine 5'-mono-phosphate (AMP). Photolysis results in breaking of the 5'-C—O—P bond, yielding orthophosphate for AMP and mainly condensed phosphates for ADP, ATP and ADTP, together with decomposition products of adenosine. The photolytic breaking of the 5'-C—O—P bond is not enhanced by excitation of the strongly absorbing adenine ring followed by energy transfer but results from direct excitation of the very weakly absorbing ribosephosphate- moiety of the molecule. The arguments for this are: (i) cyclic adenosine 3'5'-mono-phosphate, which is sterically hindered for direct energy transfer from the adenine ring to the phosphate-ester bond, is photolyzed at approximately the same rate as AMP, and (ii) the rate of disappearance of the strong absorption band due to the adenine ring is higher than the rate of liberation of orthophosphate.     

Polymer evolution of a sulfonated polysulfone membrane as a function of ion beam irradiation fluence

Ion beam irradiation was used to modify the surface of a sulfonated polysulfone water treatment membrane. A beam of 25 keV H⁺ ions with three irradiation fluences (1 × 10¹³ ions/cm², 5 × 10¹³ ions/cm², and 1 × 10¹⁴ ions/cm²) was used for membrane irradiation. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analyses were performed on the virgin and irradiated membranes in order to determine the changes to chemical structure incurred by ion beam irradiation. The results show that some of the sulphonic and CH bonds were broken and new CS bonds were formed after irradiation. Atomic force microscope (AFM) analyses show that membrane roughness decreased after irradiation. A significant increase in flux after ion beam irradiation was also observed, while the amount of cake accumulation on the membrane was decreased after ion beam irradiation. Hydrophobicity, pore size distribution and selectivity of the membrane were not affected by ion beam irradiation.     

Evolution of a polysulfone nanofiltration membrane following ion beam irradiation

Ion beam irradiation was used to modify the surface of a sulfonated polysulfone water treatment membrane. A beam of 25 keV H (+) ions with four irradiation fluences (1 x 10 (13), 5 x 10 (13), 1 x 10 (14), and 5 x 10 (14) ions/cm (2)) was used to study the effects of ion beam irradiation on chemical structure, surface morphology, microstructure, and performance. XPS and ATR-FTIR analyses were performed on the virgin and irradiated membranes in order to determine the changes to chemical structure incurred by ion beam irradiation. The results showed that some sulfonic and C-H bonds were broken and new C-S bonds were formed after irradiation. AFM analysis showed that the roughness of the membranes decreased after irradiation, and the decrease in surface roughness was proportional to the increase in irradiation fluence. An increase in flux after ion beam irradiation was also observed along with a smaller flux decline during operation. Flux was not a function of irradiation fluence. Hydrophobicity, pore size distribution, and membrane rejection efficiencies were not affected by ion beam irradiation. Overall, irradiation led to an improvement in membrane performance.     

On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography

Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.     

IRRADIATION IN THE FROZEN STATE: A TECHNIQUE FOR DIRECT PHOTOAFFINITY LABELING

UV irradiation of rabbit muscle phosphofructokinase (PFK) in the presence of adenosine 3',5'-cyclic phosphate (cAMP) resulted in the covalent attachment of this ligand molecule to the enzyme protein. Irradiation in the frozen ice state enhanced the rate of this incorporation more than 10-fold above that achieved in aqueous solution, without significantly affecting the rate of photodestruction of the protein. [³H]-cAMP and [³²P]-cAMP were each incorporated into PFK at identical rates in the frozen state. Rates of photoincorporation in the frozen and liquid states were both half-maximal at a free ligand concentration approximately equal to the dissociation constant of cAMP and PFK. Adenosine diphosphate (ADP) and adenosine monophosphate (AMP), both of which are known to compete for cAMP binding to PFK, inhibited photoincorporation of cAMP. Guanosine monophosphate (GMP), inosine monophosphate (IMP), and guanosine 3',5'-cyclic phosphate (cGMP), which do not compete for cAMP binding, had no effect on photoincorporation of cAMP. Irradiation of [³H]-AMP or [³H]-ADP resulted in photoincorporation into PFK at 0°C, with enhancement at — 77°C similar to that noted with cAMP.     

Determination of the big head carp myofibrillar (Aristichthys nobilis) adenosine triphosphatase activity by ion chromatography

A method for the determination of adenosine triphosphatase (ATPase) activity of myofibrils of big head carp by using ion chromatography was introduced. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and orthophosphate (Pi) were separated completely. Recoveries for ATP, ADP and Pi were 98+/-5%, 97+/-4% and 98+/-5%, respectively. Pi liberated from ATP during reaction was monitored by ion chromatography using the suggested method. This method was applicable to the determination of myofibrils ATPase activity for quick quality evaluation of surimi.     

蜂王浆中磷酸腺苷的提取及超高效液相色谱分析

比较了高氯酸提取、热水提取和热硫酸镁溶液提取3种提取方式对蜂王浆中磷酸腺苷三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和单磷酸腺苷(AMP)的提取效果,发现在低温(低于4 ℃)下以5%高氯酸的提取效果最佳。采用超高效液相色谱-紫外检测法分析蜂王浆中的ATP, ADP和AMP的含量。以BEH Shield RP18柱(100 mm×2.1 mm,1.7 μm)为分析柱,以50 mmol/L的磷酸二氢铵(pH 6.5)和乙腈为流动相进行梯度洗脱,3种磷酸腺苷在4 min内实现了较好的分离。以加标王浆样品作添加回收率测定,ATP, ADP和AMP的回收率分别为84.1%～94.3%,86.2%～93.7%和91.0%～104.3%,相对标准偏差均小于10%。方法已被用于一些实际样品的分析,以了解ATP, ADP和AMP在蜂王浆样品中的分布情况。     

Treadmilling of actin filaments via Brownian dynamics simulations

Actin polymerization is coupled to the hydrolysis of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) and inorganic phosphate (P(i)). Therefore, each protomer within an actin filament can attain three different nucleotide states corresponding to bound ATP, ADP/P(i), and ADP. These protomer states form spatial patterns on the growing (or shrinking) filaments. Using Brownian dynamics simulations, the growth behavior of long filaments is studied, together with the associated protomer patterns, as a function of ATP-actin monomer concentration, C(T), within the surrounding solution. For concentrations close to the critical concentration C(T)=C(T,cr), the filaments undergo treadmilling, i.e., they grow at the barbed and shrink at the pointed end, which leads to directed translational motion of the whole filament. The corresponding nonequilibrium states are characterized by several global fluxes and by spatial density and flux profiles along the filaments. We focus on a certain set of transition rates as deduced from in vitro experiments and find that the associated treadmilling (or turnover) rate is about 0.08 monomers per second.     

Mitochondrial Responses of Normal and Injured Human Skin Fibroblasts Following Low Level Laser Irradiation—An In Vitro Study

Laser irradiation has proved to be very efficient in speeding and improving the quality of healing in pathological conditions of diverse etiologies. However, the mechanisms by which the beneficial effects are attained are not clear. Mitochondria are the primary phototargets during irradiation. The study aimed to establish if laser irradiation had an effect on hypoxic and acidotic cells. The study also aimed to use existing information regarding the possible mechanism of action (established in wounded cells) and apply these principles to acidic and hypoxic irradiated cells to determine whether laser has a stimulatory or inhibitory effect. Cell cultures were modified to simulate conditions of hypoxia (hypoxic gas mixture 95% N₂ and 5% O₂) and acidosis (pH 6.7) whereas the central scratch model was used to simulate a wound. Cells were irradiated with a helium–neon (632.8 nm, 3 mW cm^?2) laser using 5 or 16 J cm^?2 on days 1 and 4. Mitochondrial responses were measured 1 or 24 h after laser irradiation by assessing changes in mitochondrial membrane potential (MMP), cyclic AMP, intracellular Ca²⁺ and adenosine triphosphate (ATP) cell viability. Hypoxia and acidosis significantly reduced MMP when compared with normal nonirradiated control cells. Wounded, hypoxic and acidotic cells irradiated with 5 J cm^?2 showed an increase in mitochondrial responses when compared with nonirradiated cells while 16 J cm^?2 showed a significant decrease. The study confirmed that laser irradiation with 5 J cm^?2 stimulated an increase in intracellular Ca²⁺ which resulted in an increase in MMP, ATP and cAMP, which ultimately results in photobiomodulation to restore homeostasis of injured cells.     

布朗动力学理论模拟动态肌动蛋白纤维的增长

肌动蛋白的聚合耦合三磷酸腺酐(ATP)分子水解成二磷酸腺苷(ADP)分子和磷酸(Pi)的释放两个过程. 因此, 肌动蛋白纤维上的原聚体存在三种不同状态, 即分别结合ATP, ADP/Pi和ADP分子. 原聚体的不同状态导致纤维具有不同的空间图谱, 这些状态的空间分布将影响纤维的各种行为. 为此,建立了相应的分子模型,在布朗动力学模拟中实现了遵循时间演化的连续马尔可夫随机过程的解聚和水解反应; 重点阐述了如何实现纤维两端的聚合和解聚达到化学平衡的方法, 并系统研究了纤维在结合ATP分子的肌动蛋白单体溶液中的增长行为.     

ENERGY-REGULATED FUNCTIONAL TRANSITIONS OF CHLOROPLAST ATPASE

The functional transitions of the membrane-bound chloroplast ATPase (CF₁) as influenced by low ADP and uncoupler concentrations are investigated by measurements of initial and steady-state ATP hyrolysis and concomitant membrane energization. Following activation of latent ATP hydrolysis by light in the presence of dithioerythritol, the resulting steady-state ATP hydrolysis depends on the dark-period ( t _d) bteween light activation and ATP addition. ADP, added during t _d, inhibits this activity ( K _i about 2 μ M ) and induces a lag in the onset of ATP hydrolysis. The extent of membrane energization as monitored by an aminoacridine fluorescent probe is proportional to the ATPase activity.
An uncoupler amplifies the inhibitory effect of ADP if added during f _d, whereas it induces the normal stimulation of ATP hydrolysis in the absence of ADP. The ADP effect, which is different from product inhibition, is interpreted as a conformational interaction with CF₁ causing an increase of the energy threshold required for the inactive → active transition of the CF₁ molecules. These results are in harmony with currently proposed models of CF₁ regulation by adenine nucleotides based on binding studies.
The inactive → active transition of CF₁ conformation is investigated by analysis of the lag in the onset of ATP hydrolysis at different ADP concentrations and by means of varied light pulses and single-turnover flashes, using the electric potential indicating absorption change at 515 nm as a probe for the onset of ATP hydrolysis. The half-time of the process leading to fully (re)activated ATP hydrolysis is about 0.25 s. The ATP-dependent flash-induced inactive → active transition occurs within a few turnovers of electron flow.     

Membrane Potential Photoresponse of Crowned Malachite Green Derivatives Affording Perfect Photoswitching of Metal Ion Complexation

Photoirradiation effect on potential response to metal ion concentrations and photoinduced potential change were investigated with poly(vinyl chloride) membranes based on a Malachite Green derivative carrying a bis(monoaza-15-crown-5) moiety, by comparing other Malachite Green derivatives. The Malachite Green carrying a bis(crown ether) moiety caused a potential response to potassium ion concentration changes under dark condition. In the membrane potential response, a clear-cut photoinduced switching of potential response was realized by the membrane of Malachite Green carrying a bis(crown ether) moiety, which exhibited no potential response to potassium ion concentrations (0 mV/decade) on UV irradiation. On the other hand, a Malachite Green carrying a monocyclic benzocrown ether moiety showed a considerable dependence of the membrane potential on the metal ion concentrations under both dark and UV irradiation conditions.     

Near-infrared induced membrane surface electrostatic potential, fluorescent measurements

The change of the electrostatic surface potential induced by near-infrared radiation was monitored by the fluorescence probe technique. Fluorescence intensity of 1-anilinonaphtalene-8-sulfate (ANS) was studied in the pH range 4.8–9.5 before and after exposition to NIR (700–2000 nm). The intensity of fluorescence changed (decreased after exposition on radiation) only at pH 7.4. The effect is due to decreasing concentration of ANS in liposome membrane after irradiation. The modified distribution of ANS in liposome membrane upon irradiation is attributed to the dehydration of membrane surface. Dehydratation diminishes the electrostatic surface potential about 36±15 mV.     

ADENYLATE KINASE BOUND TO THE CHROMATOPHORE MEMBRANES OF Rhodobactor spheroides G1C

Transformation of adenylates (AMP, ADP and ATP) by washed chromatophore membranes of Rhodobactor spheroides G1C in the dark and in the light indicated the functions of ATPase (ADP + Pi in equilibrium ATP) and of an adenylate kinase (2ADP in equilibrium AMP + ATP). The activity of adenylate kinase of the chromatophores was not inhibited by AP5A, and persisted even after sonication in the presence of EDTA or CaCl2; the results suggested the presence of an adenylate kinase bound to the chromatophore membrane. In search of the enzyme, the supernatant after sonication of the chromatophores in the presence of EDTA was subjected to a molecular sieve and then to ion-exchange HPLC; a fraction with high specific adenylate kinase activity, containing a very sharp peak at 55 kDa, was isolated. Preliminary characterization indicated that it is different from the well-documented water-soluble 33 kDa adenylate kinase.