[Analysis of free-radical products of near-UV irradiation at 77 K of frozen adenine and adenosine diphosphate solutions containing inorganic phosphate].

Irradiation by near-UV light at 77 K of aqueous solutions of inorganic phosphate in weakly acidic conditions in the presence of the photosensitizers adenine and adenosine diphosphate results in the formation of free radicals of these compounds, photosensitized free radicals of phosphate itself, and H* and OH* radicals. The relative concentrations of free radical products were estimated by the analysis of total ESR signals registered in the region of g = 2.00 in the photosystems Ade + Pi and Adphi + Pi using the original computer program of ESR spectra simulation.

Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation.

We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y(1) and P2Y(12) nucleotide receptors, 2-MeSADP, by blocking the beta-phosphate group with a 1-(3,4-dimethyloxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itself inactive at human P2Y(1) and P2Y(12) receptors expressed heterologously in 1321N1 astrocytoma cells or in washed human platelets, this derivative readily regenerates the parent agonist upon mild irradiation with long-wave UV light (360 nm). The functional effect of the regenerated agonist was demonstrated by a rise in intracellular calcium mediated by either P2Y(1) or P2Y(12) receptors in transfected cells. Washed human platelets exposed to a solution of MRS2703 were induced to aggregate upon UV irradiation. At 1.0 microM MRS2703, full aggregation was achieved within 1 min of irradiation. Thus, this caged nucleotide promises to be a useful probe for potent P2Y receptor activation with light-directed spatial and temporal control.

Modification of the intrinsic fluorescence and the biochemical behavior of ATP after irradiation with visible and near-infrared laser light.

In this work, the effects of visible (655 nm) and near-infrared (830 nm) light on ATP in solution were examined. The addition of irradiated ATP to the hexokinase reaction caused significant differences in the reaction rates and in the Michaelis-Menten kinetic parameters, k(m) and v(max). Irradiated ATP cleavage by hexokinase occurred in less time. Changes were wavelength and dose dependent. Excitation of ATP with a 260 nm wavelength ultraviolet light induced a fluorescence emission that was decreased when Mg2+ was added due to ion binding of the phosphates, which are the structures that modify the fluorescence produced by the adenine dipoles. The irradiation of this ATP.Mg2+ solution using 655 and 830 nm light increased the fluorescence by a possible displacement of Mg2+ from the phosphates. In conclusion, visible and near-infrared light modifies the biochemical behavior of ATP in the hexokinase reaction and the fluorescence intensity of the molecule thus altering the Mg2+ binding strength to the oxygen atoms in the phosphate group.

An x-ray diffraction study on the ADP-induced conformational change in skeletal muscle myosin.

Effects of ADP on the conformation of myosin cross-bridges were studied in x-ray diffraction experiments on single skinned fibers of frog skeletal muscle by photorelease of ADP from caged-ADP. The experiments were performed at the third-generation synchrotron radiation facility SPring-8 with a time resolution of 5 ms. The intensity of the third-order meridional reflection from myosin filaments (at 1/14.4 nm(-1)) increased promptly after the ADP release with a time constant smaller than 5 ms, which was similar to that of tension decline. The results show that ADP binding induces a conformational change of myosin in skeletal muscle fibers.

Adenilate pool and thylakoid ATP-synthase content in pea leaves under clinorotation.

It is known that plant resistance to stress factors is connected with energy metabolism. The energy stored in the process of photophosphorylation in the form of ATP is used then to support respiration, transpiration, organic compound synthesis, growth and development as well as to restore cell structure after its damage under extremal environmental factors. Transformation of light energy into chemical energy of ATP is catalyzed by thylakoid membrane enzymatic complex of ATPsynthase-CF1CF0. Its activity and amount in the thylakoid membrane depends on plant growth conditions. The aim of this work was investigation of clinorotation effect on light-induced dynamics of adenyl nucleotides (AMP, ADP and ATP) and estimation of CF1CF0 content in thylakoids of pea leaves grown under slow clinorotation and vertical control.

[The acute action of gamma radiation at a dose of 1 Gy on the enzymatic activity of serum ATPase and ADPase]. / Ostroe vozdeistvie gamma-izlucheniia v doze 1 Gr na fermentativnuiu aktivnost' syvorotochnoi ATFazy i ADFazy.

Studies were carried out to investigate the activity of ATPase and ADPase in rat blood serum after strong (137Cs) on the rate of 1 Gy. Rats were examined for various postirradiation periods up to 3 months. Two-fold decreasing of serum ATPase activity was recorded within the two weeks after gamma-irradiation. The rate of 3H-ADP enzymatic hydrolysis in the serum of irradiated animals remained unchanged as compared to controls.

Conformational changes of arginine kinase induced by photochemical release of nucleotides from caged nucleotides--an infrared difference-spectroscopy investigation.

The conformations of arginine kinase (AK) in AK x Mg x ADP, AK x Mg x ATP, AK x Mg x ADP x NO3-, AK x Mg x ADP x Arg and AK x Mg x ADP x NO3- x Arg complexes were investigated by measuring their reaction-induced infrared difference spectra (RIDS). The photochemical release of ATP from ATP[Et(PhNO2)] and of ADP from ADP[Et(PhNO2)] produced distinct RIDS of AK complexes, suggesting that binding of ADP and ATP promoted different structural alterations of the enzyme active-site. Small infrared changes in the amide-I region were observed, indicating that about 5-10 amino acid residues were involved in the nucleotide-binding site. These infrared changes were due to the structural alteration of the peptide backbone caused by the nucleotide-binding and to the coupling effects between the nucleotide-binding site and the other substrate (Arg or NO3-)-binding site. ATP binding to AK (as well as ADP-binding to AK in the presence of NO3-) induced protonation of a carboxylate group of Asp or Glu, as evidenced by the appearance of the 1733-cm(-1) band, which was not observed with the AK x Mg x ADP, AK x Mg x ADP x Arg and AK x Mg x ADP x NO3- x Arg complexes. The RIDS of the AK x Mg x ADP x NO3- x Arg complex showed new infrared bands at 1622 cm(-1) (negative) and at 1613 cm(-1) (positive), which were not seen in the RIDS of other complexes (without NO3- or/and Arg). In the transition-state-analog complex of AK, no protonation of the carboxylate residue (Asp or Glu) was observed, and the binding site of NO3- or the gamma-phosphate group of nucleotide was altered.

Orientation changes in myosin regulatory light chains following photorelease of ATP in skinned muscle fibers.

The orientation of the light-chain region of myosin heads in muscle fibers was followed by polarized fluorescence from an extrinsic probe during tension transients elicited by photolysis of caged ATP. Regulatory light chain from chicken gizzard myosin was covalently modified with iodoacetamidotetramethylrhodamine and exchanged into skinned fibers from rabbit psoas muscle without significant effect of the tension transients. Fluorescence polarization ratios Q parallel = (parallel I parallel-perpendicular I parallel)/ (parallel I parallel+perpendicular I parallel) and Q perpendicular = perpendicular I perpendicular - parallel I perpendicular)/ (perpendicular I perpendicular + parallel I perpendicular), where mIn denote fluorescence intensities for excitation (pre-subscript) and emission (post-subscript) parallel or perpendicular to the fiber axis, were simultaneously measured at 0.5 ms time resolution. Q perpendicular decreased and Q parallel increased promptly after ATP release in the presence or absence of CA2+, indicating changes in orientation of the light-chain region associated with ATP binding or cross-bridge detachment. Little further change in the Q signals accompanied either active tension development (+Ca2+) or the final relaxation (-Ca2+). The Q and tension transients slowed when liberated ATP concentration was reduced. Assuming that ATP is released at 118 s-1 (20 degrees C), the apparent second-order rate constants were 3-10 x 10(5) M-1 s-1 for Q parallel, 1-5 x 10(5) M-1 s-1 for Q perpendicular, and 0.5-2 x 10(5) M-1 s-1 for the convergence of tension traces starting from different rigor values. Fitting of model orientation distributions to the Q signals indicated that the angular disorder increases after ATP binding. This orientation change is specific to ATP because photo release of ADP caused much smaller changes in the Q signals.

Alterations in adenylate ratios in plant cells after accelerated ion irradiation.

Levels of adenylate metabolism have been studied in cells of Nicotiana tabacum growing in vitro, and in root apex extracts of Pisum sativum irradiated at the 95-in. isochronous cyclotron U-240, Institute for Nuclear Research, Ukrainian National Academy of Sciences, Kyiv. Particle beams of accelerated helium ions with energy 9.34 keV/micrometer were used. Replacement and rapid freezing of the irradiated plants samples in liquid nitrogen were carried out with a manipulator and a remote control system. After doses of 5, 20, 50, and 100 Gy of gamma-irradiation, as well as 50 and 100 Gy 4He irradiation, the cellular ATP/ADP ratio increased during early stages of the response. This effect was absent at higher doses and after exposure to sparesly-ionizing radiation, when a rapid decline in the cellular ATP concentration and the ATP/ADP ratio occurred.

[Effect of gamma radiation on levels of adenine nucleotides in erythrocytes of healthy individuals after submaximum physical exertion]. / Wplyw promieniowania gamma na stezenie neukeotydów adeninowych w erytrocytach ludzi zdrowych poddanych submaksymalnemu wysilkowi fizycznemu.

The authors studied the effect of gamma radiation and submaximum physical exercise on adenosine-5'-triphosphate (ATP), adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP) contents in erythrocytes of healthy males. Twenty one men aged 20-22 years were examined. They underwent physical exercise at doses of 2 w/kg body weight for 15 min. Erythrocytes were exposed to gamma radiation (500 gy doses) from 60Co source. The concentration of adenine nucleotides in erythrocytes was measured by the Boehringer Mannheim tests. The submaximum physical exercise was found to decrease ATP content and to to increase ADP and AMP in erythrocytes. Gamma radiation at 500 Gy dose was found to decrease ATP concentration in erythrocytes both at rest and after submaximum exercise and to increase AD content. It was revealed that AMP content increased at rest and decreased after submaximum exercise in irradiated erythrocytes.

Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate.

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with [3H]ADP. Addition of vanadate (Vi) and Co2+ enabled [3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.[3H]ADP.Vi complex allowed it to be purified free of excess [3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped [3H]ADP became covalently attached at the active site. All of the [3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped [alpha-32P]ATP [Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using [3H]UDP as a photolabel [Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.

Electrotransfer of [32P]NAD allows labeling of ADP-ribosylated proteins in intact Chinese hamster ovary cells.

CHO-K1D cells electroporated in buffers containing [32P]NAD incorporated the label in a voltage-dependent manner. Electroporation with 650 V/cm at 1460 microF in Ham's F12 medium supplemented with 10 mM Hepes, pH 7.1, resulted in a greater than 20-fold increase in [32P]NAD uptake, while decreasing relative cellular survival by only 6%. Exposure of cells to gamma irradiation (20 Gy) prior to electroporation increased the steady-state level of poly(ADP-ribosylated) nuclear proteins two- to four-fold over that of unirradiated control cells. These data indicate that electrotransfer of [32P]NAD is a simple and rapid means of labeling the cellular NAD pool and should prove useful in the analysis of the relationship between poly(ADP-ribosylation) of nuclear proteins and DNA repair.

Influence of light on long-term ADP phosphorylation.

ADP phosphorylation coupled to cyclic electron transport was studied over a long period using immobilized chromatophores from Rhodopseudomonas capsulata. Photophosphorylation of ADP as a function of time was continuously measured under different conditions of continuous illumination and concentration of oxygen. Using a red cutoff filter, it was possible to sustain ADP phosphorylation at a maximal rate for more than 10 days, whereas inactivation had always been observed after 5 days when white light was used. The influence of light nature on inactivation was demonstrated using photopigment absorption spectra.

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes.

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.

[Adenosine triphosphate synthesis induced by ultraviolet irradiation]. / Sintez adenozintrifosfata, stimulirovannyi ul'trafioletovym oblucheniem.

Effect of UV-irradiation on non-aqueous solutions of adenine nucleotide mixture of the presence of inorganic phosphate was studied. Solutions of ADP sodium salts containing AMP and ATP admixtures and of inorganic phosphate in DMSO and DMPA and ethylene glycol were illuminated with the lamp dPIII-500. It has been shown that along with nucleotide disintegration and complex formation ATP synthesis takes place, which is evidenced by an increase of relative ATP content in experimental specimens.

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP.

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F1) as the triphosphate, either in the absence or presence of Mg2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the alpha and beta subunits, mainly (63%) to the alpha subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the alpha and 2 mol to the beta subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the alpha-subunits hinders binding to the beta subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1.

Influence of light on activity of adenylate kinase and pterin-protein complexes from pea chloroplasts.

The activity of adenylate kinase (AK) and of pterin-protein complexes (PPC), whose proteins have adenylate kinase activity comparable to that of the enzyme was studied. It was established that light inhibits adenylate kinase activity and that this effect is partially eliminated by phosphate ions. The forward and reverse reactions catalyzed by AK and PPC were studied and it was found that the activity of native protein complexes is different in the forward and reverse reactions. The thermostable protein both of adenylate kinase and of the pterin-protein complexes had identical activity in the ADP dismutation and the reverse reaction.