Effect of LED photobiomodulation on fluorescent light induced changes in cellular ATPases and Cytochrome c oxidase activity in Wistar rat.

BACKGROUND: Fluorescent light exposure at night alters cellular enzyme activities resulting in health defects. Studies have demonstrated that light emitting diode photobiomodulation enhances cellular enzyme activities. OBJECTIVE: The objectives of this study are to evaluate the effects of fluorescent light induced changes in cellular enzymes and to assess the protective role of pre exposure to 670 nm LED in rat model. METHODS: Male Wistar albino rats were divided into 10 groups of 6 animals each based on duration of exposure (1, 15, and 30 days) and exposure regimen (cage control, exposure to fluorescent light [1800 lx], LED preexposure followed by fluorescent light exposure and only LED exposure). Na+-K+ ATPase, Ca2+ ATPase, and cytochrome c oxidase of the brain, heart, kidney, liver, and skeletal muscle were assayed. RESULTS: Animals of the fluorescent light exposure group showed a significant reduction in Na+-K+ ATPase and Ca2+ ATPase activities in 1 and 15 days and their increase in animals of 30-day group in most of the regions studied. Cytochrome c oxidase showed increase in their level at all the time points assessed in most of the tissues. LED light preexposure showed a significant enhancement in the degree of increase in the enzyme activities in almost all the tissues and at all the time points assessed. CONCLUSIONS: This study demonstrates the protective effect of 670 nm LED pre exposure on cellular enzymes against fluorescent light induced change.

Kinetics of Ca2+ binding to the SR Ca-ATPase in the E1 state.

The time-resolved kinetics of Ca2+ binding to the SR Ca-ATPase in the E1 state was investigated by Ca(2+)-concentration jump experiments. Ca2+ was released by an ultraviolet-light flash from caged calcium, and charge movements in the membrane domain of the ion pumps were detected by the fluorescent styryl dye 2BITC. The partial reaction (H3E1 <-->) E1 <--> CaE1 <--> Ca2E1 can be characterized by two time constants, tau1 and tau2, both of which are not significantly Ca(2+)-concentration-dependent and only weakly pH-dependent at pH < 7.5. Both time constants differ by a factor of approximately 50 (4.7 vs. 200 ms). The weak substrate-dependence indicates that the rate-limiting process is not related to Ca2+ migration through the access channel and ion binding to the binding sites but to conformational rearrangements preceding the ion movements. The high activation energy obtained for both processes, 42.3 kJ mol(-1) and 60.3 kJ mol(-1) at pH 7.2, support this concept. Transient binding of Ca ions to the loop L67 and a movement of the Ca-loaded loop are discussed as a mechanism that facilitates the entrance of both Ca ions into the access channel to the ion-binding sites.

Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N3-SL-ATP.

2-N3-SL-ATP [2-azido-2',3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl-pyrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotide-binding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spin-spin interactions between two radicals corresponding to a distance of about 15 A between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca2+-ATPase is a dimer with the nucleotide-binding sites in close proximity.

Distinct topologies of mono- and decavanadate binding and photo-oxidative cleavage in the sarcoplasmic reticulum ATPase.

UV irradiation of the sarcoplasmic reticulum (SR) ATPase in the presence of vanadate cleaves the enzyme at either of two different sites. Under conditions favoring the presence of monovanadate, and in the presence of Ca(2+), ADP, and Mg(2+), cleavage results in two fragments of 71- and 38-kDa electrophoretic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca(2+) and ADP, cleavage results in two fragments of 88- and 21-kDa electrophoretic mobility. The amino terminus resulting from cleavage is blocked and resistant to Edman degradation. However, the initial photo-oxidation product can be reduced with NaB(3)H(4,) resulting in incorporation of radioactive (3)H label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specific for the each of the photo-oxidation conditions, and can be sequenced after purification. Collection of the Edman reaction fractional products reveals the radioactive label and demonstrates that Thr(353) is the residue oxidized by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct positioning of monovanadate at the phosphorylation site requires binding of Mg(2+) and ADP to the Ca(2+)-dependent conformation of the enzyme. Subsequent hydrolytic cleavage is likely assisted by the neighboring Asp(601), and yields the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly the following three residues: Val(187), Ile(188), and Lys(189)) is the residue that is photo-oxidized by decavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighboring acidic residues, and yields the 88- and 21-kDa fragments. The bound decavanadate, which we find to produce steric interference with TNP-AMP binding, must therefore extend to the A domain (i.e. small cytosolic loop) in order to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca(2+).

On the use of the phase memory time T2 for the quantitative characterization of the rotational motions of proteins in lipid bilayer systems.

Numerical simulations of the echo responses from a nitroxide label rigidly attached to a large protein undergoing ultraslow rotational motions in a lipid bilayer are presented. The echoes are formed by the application of Hahn, COSY, and 2D-ELDOR sequences utilizing both soft and hard microwave pulses. The simulations address the question of whether the echo responses elicited by these sequences are affected by restricted angular excursions of the long axis of the protein relative to the normal to the bilayer plane. The results indicate that all three pulse sequences yield the same quantitative motional information regardless of the nature of the microwave pulses and there is no theoretical reason for preferring one sequence above the others.

Effects of photo-oxidizing analogs of fluorescein on the sarcoplasmic reticulum Ca2+-ATPase. Functional consequences for substrate hydrolysis and effects on the partial reactions of the hydrolytic cycle.

Erythrosin B was used to photo-oxidize the sarcoplasmic reticulum Ca2+-ATPase. The ATPase activity is rapidly and irreversibly inhibited by photo-oxidation with erythrosin. This inhibition is protected by the presence of ATP during the photo-oxidation period. After photo-oxidation, the steady-state phosphorylation by ATP remains almost unchanged, whereas phosphorylation by inorganic phosphate is impaired. The pseudo-first order rate constants for phosphorylation by 15 microM ATP at 25 degrees C are strongly inhibited when starting from either a Ca2+-bound or a Ca2+-free enzyme form, decreasing from 145 to 23 s-1 for the Ca2+-bound form and from 50 to 18 s-1 for the Ca2+-free form. Concurrently, the rate constants for dephosphorylation are also severely inhibited, changing from a fast double exponential to a very slow single exponential decay in the reverse direction and from a moderately slow single to a very slow single exponential decay in the forward direction. Ca2+ binding data show that the phosphorylated intermediate formed by the photo-oxidized enzyme contains two occluded Ca2+, and TNP-ATP fluorescence measurements indicate that it accumulates in a E1-P.Ca2-like conformation. Protection by ADP against glutaraldehyde-induced cross-linking indicates that ADP binding to Ca2+-ATPase is not impaired by photo-oxidation nor by free erythrosin. These data support the view that an ADP-insensitive, Ca2+-bound, slowly interconverting phosphoenzyme is formed. Thus, photo-oxidation with erythrosin B leads to impairment of phosphoryl transfer reactions and related conformational changes.

Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.

The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluorescence intensity was converted into calcium concentration, which in turn was used to derive the kinetic parameters of the calcium pump, the Michaelis-Menten constant Km, and the maximal transport rate vmax. Km and vmax values derived in this manner were 24 +/- 14 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol A, and 4 +/- 3 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol B, respectively. The difference between A and B is presumably caused by calmodulin, which is inactive in the experiments with protocol A. The possibilities to extend the new method to living nucleus-containing cells transiently transfected with mutants of the plasma membrane calcium pump are discussed.

Irradiation with ultraviolet light in the presence of vanadate increases Ca2+ permeability of the sarcoplasmic reticulum membrane via Ca(2+)-ATPase.

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was irradiated with ultraviolet light (UV) in the presence of vanadate plus 2 mM EGTA, 10 mM MgCl2, 20% DMSO, and 50 mM PIPES (pH 6.5) at room temperature. In the presence of 100 microM vanadate, the Ca(2+)-uptake activity of SR rapidly decreased and was almost lost in 20 min. The activity was inhibited as a function of vanadate concentration with an apparent Ki of about 20 microM. On the other hand, Ca(2+)-dependent ATP hydrolytic activity as well as phosphoenzyme (EP) formation activity decreased very slowly, and more than 50% of these activities remained 20 min after initiation of the vanadate-UV treatment. Half inhibition of these activities required about 100 microM vanadate. The loss of the relationship between Ca(2+)-uptake and ATPase reaction was found to be mainly caused by an increase in the Ca2+ permeability of the SR membrane, which was raised by increasing the vanadate concentration or UV irradiation time in a manner similar to that observed for the Ca2+ uptake. No rise in Ca2+ permeability occurred in liposomes reconstituted from SR lipid when they were irradiated with UV in the presence of 100 microM vanadate. When the vanadate-UV-treated SR was allowed to react with fluoral-P (4-amino-3-penten-2-one), an indicator of aldehyde, and the membrane proteins were separated by HPLC in the presence of SDS, the fluorescent probe was found to be closely associated with the Ca(2+)-ATPase fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of static magnetic field on specific adenosine-5'- triphosphatase activities and bioelectrical and biomechanical properties in the rat diaphragm muscle.

In this study, we aimed to clarify the effects of chronically applied static magnetic field (200 Gauss) on specific ATPase activities and bioelectrical and biomechanical responses in isolated rat diaphragm muscle. The mean activities of Na(+)-K+ ATPase and Ca2+ ATPase determined from the diaphragm homogenates were significantly higher in the magnetic field exposed group (n = 20), but that of Mg2+ ATPase was nonsignificantly lower compared to the control group (n = 13). Resting membrane potential, amplitude of muscle action potential, and overshoot values (mean +/- SE) in the control group were found to be -76.5 +/- 0.6, 100 +/- 0.8, and 23.5 +/- 0.6 mV, respectively; these values were determined to be -72.8 +/- 0.4, 90.3 +/- 0.5, and 17.2 +/- 0.4 mV in the magnetic field-exposed group, respectively. The latency was determined to increase in the experimental group, and all the above-mentioned bioelectrical differences between the groups were significant statistically. Force of muscle twitch was found to decrease significantly in the magnetic field-exposed group, and this finding was attributed to the augmenting effect of magnetic field on Ca2+ ATPase activity. These results suggest that magnetic field exposure changes specific ATPase activities and, thence, bioelectrical and biomechanical properties in the rat diaphragm muscle.

Catalysis of an ATP analogue untethered and tethered to lysine 492 of sarcoplasmic reticulum Ca(2+)-ATPase.

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-AMP (TNP-8N3-AMP) and -ATP photolabel Lys-492 at the active site of the Ca(2+)-ATPase of sarcoplasmic reticulum (McIntosh, D. B., Woolley, D. G., and Berman, M. C. (1992) J. Biol. Chem. 267, 5301-5309). We now find that the hydrolysis of the gamma-phosphate of both TNP-8N3-ATP and the TNP-nucleotide tethered to Lys-492 is stimulated by Ca2+ (kcat = 0.02 s-1, Km = 1.6 microM; k(obs) = 0.08 s-1, respectively, pH 6.0) and exhibits acidic pH optima with shifted pH dependences (pKa = 5.7 and 7.0, respectively). TNP-8N3-ATP supports Ca2+ transport with a coupling stoichiometry of 2:1 in the pH range 5.0-7.5. Hydrolysis of the tethered substrate is largely uncoupled from transport; a small, substoichiometric amount of transport is observable at acidic pH. Ca(2+)-dependent phosphorylation of the ATPase with TNP-8N3-ATP is demonstrable under select conditions but with the tethered substrate is too low to be measured with confidence. Neither ADP nor ATP has any effect on the Ca(2+)-dependent catalysis of the tethered nucleotide. Tethering does not appear to affect formation of phosphoenzyme as shown by P(i)-dependent superfluorescence of the tethered nucleotide. The results indicate that Lys-492 is located at the catalytic site within approximately 14 A of Asp-351, which is phosphorylated, and Lys-492 and the adenyl moiety of the nucleotide are closely associated during phosphorylation. Evidently, Lys-492 is not essential for catalysis of phosphoryl transfer, but its movement, specifically separation from the nucleotide, may be critical for coupling with the transport sites.

Changes in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ ATPase enzyme in the presence of terbium: a time-resolved x-ray diffraction study.

The design of the time-resolved x-ray diffraction experiments reported in this and an accompanying paper was based on direct measurements of enzyme phosphorylation using [gamma-32P]ATP that were employed to determine the extent to which the lanthanides La3+ and Tb3+ activate phosphorylation of the Ca2+ATPase and their effect on the kinetics of phosphoenzyme formation and decay. We found that, under the conditions of our experiments, the two lanthanides are capable of activating phosphorylation of the ATPase, resulting in substantial levels of phosphoenzyme formation and they slow the formation and dramatically extend the lifetime of the phosphorylated enzyme conformation, as compared with calcium activation. The results from the time-resolved, nonresonance x-ray diffraction work reported in this paper are consistent with the enzyme phosphorylation experiments; they indicate that the changes in the profile structure of the SR membrane induced by terbium-activated phosphorylation of the ATPase enzyme are persistent over the much longer lifetime of the phosphorylated enzyme and are qualitatively similar to the changes induced by calcium-activated phosphorylation, but smaller in magnitude. These results made possible the time-resolved, resonance x-ray diffraction studies reported in an accompanying paper utilizing the resonance x-ray scattering from terbium, replacing calcium, to determine not only the location of high-affinity metal-binding sites in the SR membrane profile, but also the redistribution of metal density among those sites upon phosphorylation of the Ca2+ATPase protein, as facilitated by the greatly extended lifetime of the phosphoenzyme.

[The role of ligands in the effect of exposure to ionizing radiation on Ca2(+)-ATPase and Mg2(+)-ATPase]. / Rol' ligandov pri vozdeistvii ioniziruiushchego izlucheniia na Ca2(+)-ATFazu i Mg2(+)-ATFazu.

The change of Ca(2+)-ATPase and Mg(2+)-ATPase activity in plasma membranes of thymocytes irradiated with doses of 10(2), 10(3) and 10(4) Gy in the presence of Ca2+, Mg2+ and ATP was studied. Stabilizing effect of Ca2+ and Mg2+ on Ca(2+)-ATPase and ATP on Mg(2+)-ATPase under irradiation was established.

[The effect of the blood plasma from irradiated animals on the Ca2(+)- and Mg2(+)-ATPase activity in thymocyte plasma membranes]. / Vliianie plazmy krovi obluchennykh zhivotnykh na aktivnost' Ca2(+)- i Mg2(+)-ATFazy plazmaticheskikh membran timotsitov.

Rats were irradiated at doses 1.5, 4.0, 7.0 and 10 Gy. After 1, 8, 15, 22 and 30 days the effect of blood plasma on activity of Ca(2+)-ATPase and Mg(2+)-ATPase in plasma membrane of thymocytes was investigated. It was found that the raise of irradiation dose leads to increasing of blood plasma effect on membrane-bound enzymes.

[The effect of the repeated irradiation of rats on the Ca2(+)- and Mg2(+)-ATPase activity of thymocyte plasma membranes]. / Vliianie mnogokratnogo oblucheniia krys na aktivnost' Ca2(+)- i Mg2(+)-ATFazy plazmaticheskikh membran timotsitov.

Rats were daily irradiated at doses 0.5 Gy in period of two weeks. The activity of Ca(2+)-ATPase, Mg(2+)-ATPase and the extent of lipid peroxidation in thymus were determined. The peculiarity of changing of enzymes activity demonstrates the dependence of Mg(2+)-ATPase on lipid peroxidation.

[Photoreactivation of Ca2+-ATPase and Mg2+-ATPase after exposure to ionizing radiation]. / Fotoreaktivatsiia Ca2+-ATFazy i Mg2+-ATFazy posle vozdeistviia ioniziruiushchego izlucheniia.

Plasma membranes of thymocytes in doses 10(2), 10(3) and 10(4) Gr were irradiated and subsequently illuminated by lambda = 445,5 nm or lambda = 518 nm light. Then the activity of Ca(2+)-ATPase and Mg(2+)-ATPase was determined. The photomodulation of the electron shell of calcium and magnesium atoms is a factor decreasing the effect of radiation on Ca(2+)-ATPase or Mg(2+)-ATPase.

[The effect of D2O in irradiation of Ca2+-ATPase]. / Effekt D2O pri obluchenii Ca2+-ATFazy.

Ca(2+)-ATPase from the meat cattle thymocytes plasma membranes in solutions 70% D2O, pH 4.0, 7.0 and 10.0 was irradiated with the doses of 10-10(4)Gr. It was shown that D2O produced stabilizing action on protein macromolecules. The activity of Ca(2+)-ATPase was changed under the influence of ionizing radiation in the presence of D2O.

[Effect of staphylococcal peptidoglycan on the activity of Ca2+-ATP-ase from the sarcoplasmic reticulum in the early stages of ionizing radiation effect]. / Vplyv stafilokokovoho peptydohlikany na aktyvnist' Ca2+-ATP-azi sarkoplazmatychnoho retykulumu hnoho rannikh etapakh promenevoï diï.

It is established that peptidoglycan of Staphylococcus aureus possess superficial activity and can render influence on the functional activity of biomembranes. It tends to increase Ca(2+)-ATP-ase activity in the sarcoplasmic reticulum membranes, but no changes have been observed in case of the enzyme solubilization. The effect of peptidoglycan was more expressed, if it was used in the early period of acute radiation injury (1 and 24 h) in a dose of 0.21 Cl/kg for samples with irradiation-induced decrease of Ca(2+)-ATP-ase activity.

[Effect of ionizing radiation on ATPase activity of thymocyte plasma membranes]. / Vliianie ioniziruiushchevo izlucheniia na ATP-aznye aktivnosti plazmaticheskikh membran timotsitov.

Thymocytes were irradiated by the doses of 4-1000 Gr. Activity of Ca(2+)-ATPase and Mg(2+)-ATP-ase, and lipid peroxidation were determined. It is supposed that changes in Ca(2+)-ATPase activity is connected with the adsorption of irradiation energy, while changes in Mg(2+)-ATPase activity are determined by effect of lipid peroxidation.

[Postradiation changes in Ca2+-ATPase and Mg2+-ATPase in thymocyte plasma membranes]. / Poradiiatsiini zminy aktyvnosty Ca2+-ATPazy i Mg2+-ATPazy pliazmatycnykh membran tymotsytiv.

The rats were irradiated in the doses 1, 5, 4, 7 and 10 Gr and on the 1, 8, 15, 22 and 30 day after the irradiation activity of Ca(2+)-ATPase and Mg(2+)-ATPase and peroxidation lipids in the thymocytes was determined. It was found that postradiation changes in activity of Mg(2+)-ATPase were characterized by a higher sensitivity to the processes of lipids peroxidation as compared to Ca(2+)-ATPase.

[The effect of ionizing radiation on the Ca2+, Mg2(+)-ATPase activity of plasma membranes]. / Vliianie ioniziruiushchego izlucheniia na aktivnost' Ca2+, Mg2(+)-ATFazy plazmaticheskikh membran.

A study was made of a change in Ca2+, Mg(2+)-ATPase activity induced by the effect of ionizing radiation (5-10(4) Gy) on a thymocyte plasma membrane suspension. The Michaelis' constant and maximum rate of enzymic reactions were determined. With a dose of 10(3) Gy the structural changes in Ca2+, Mg(2+)-ATPase were shown to reduce the affinity of the substrate to an active enzyme center and to decrease the rate of the enzyme/substrate complex degradation.