[Show abstract][Hide abstract] ABSTRACT: In the last years, the effect of extremely low-frequency electromagnetic fields (ELF-EMF) on the activity of different enzymes were investigated. Only the membrane-anchored enzymes did decrease their activity, up to 50%. In this work, the effect of ELF-EMF on bovine lung membrane carbonic anhydrase (CA) were studied. Carbonic anhydrases are a family of 14 zinc-containing isozymes catalyzing the reversible reaction: CO(2)+H(2)O = HCO(3)(- )+H(+). CA differ in catalytic activity and subcellular localization. CA IV, IX, XII, XIV, and XV are membrane bound. In particular, CA IV, which is expressed in the lung, is glycosyl phosphatidyl inositol-linked to the membrane, therefore it was a candidate to inhibition by ELF-EMF. Exposure to the membranes to a field of 75 Hz frequency and different amplitudes caused CA activity to a reproducible decrease in enzymatic activity by 17% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. When the source of enzyme was solubilized with Triton, the field lost its effect on CA enzymatic activity, suggesting a crucial role of the membrane, as well as of the particular linkage of the enzyme to it, in determining the conditions for CA inactivation. Results are discussed in terms of the possible physiologic effects of CA inhibition in target organs.
Electromagnetic Biology and Medicine 06/2011; 30(2):67-73. DOI:10.3109/15368378.2011.566770 · 0.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of extremely low frequency magnetic fields (ELF-MF) on acetylcholinesterase (AChE) activity of synaptosomal membranes were investigated. Sinusoidal fields with 50 Hz frequency and different amplitudes caused AChE activity to decrease about 27% with a threshold of about 0.74 mT. The decrease in enzymatic activity was independent of the time of permanence in the field and was completely reversible. Identical results were obtained with exposure to static MF of the same amplitudes. Moreover, the inhibitory effects on enzymatic activity are spread over frequency windows with different maximal values at 60, 200, 350, and 475 Hz. When synaptosomal membranes were solubilized with Triton, ELF-MF did not affect AChE activity, suggesting the crucial role of the membrane, as well as the lipid linkage of the enzyme, in determining the conditions for inactivation. The results are discussed in order to give an interpretation at molecular level of the macroscopic effects produced by ELF-MF on biological systems, in particular the alterations of embryo development in many organisms due to acetylcholine accumulation.
[Show abstract][Hide abstract] ABSTRACT: The disks of the vertebrate retinal rod Outer Segment (OS), devoid of mitochondria, are the site of visual transduction, a very energy demanding process. In a previous proteomic study we reported the expression of the respiratory chain complexes I-IV and the oxidative phosphorylation Complex V (F(1)F(0)-ATP synthase) in disks. In the present study, the functional localization of these proteins in disks was investigated by biochemical analyses, oxymetry, membrane potential measurements, and confocal laser scanning microscopy. Disk preparations, isolated by Ficoll flotation, were characterized for purity. An oxygen consumption, stimulated by NADH and Succinate and reverted by rotenone, antimycin A and KCN was measured in disks, either in coupled or uncoupled conditions. Rhodamine-123 fluorescence quenching kinetics showed the existence of a proton potential difference across the disk membranes. Citrate synthase activity was assayed and found enriched in disks with respect to ROS. ATP synthesis by disks (0.7 micromol ATP/min/mg), sensitive to the common mitochondrial ATP synthase inhibitors, would largely account for the rod ATP need in the light. Overall, data indicate that an oxidative phosphorylation occurs in rod OS, which do not contain mitochondria, thank to the presence of ectopically located mitochondrial proteins. These findings may provide important new insight into energy production in outer segments via aerobic metabolism and additional information about protein components in OS disk membranes.
The international journal of biochemistry & cell biology 09/2009; 41(12):2555-65. DOI:10.1016/j.biocel.2009.08.013 · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interaction of direct electric current (dc) and proteins is a little explored topic. We had reported that exposure of Crotalus atrox venom to dc caused irreversible inactivation of phospholipase A(2) and metalloprotease and that the eukaryote adenylate kinases (AK) precipitate in nondenaturing gel electrophoresis. AK1 displays an elevated percent difference of CHarged versus POlar amino acid content (CH-PO 14). Commercial AK1 and other 17 enzymes with various CH-PO values were exposed in solution to dc (0-0.7 mA) from low voltage (0-10 V), then enzymatic activity was assayed. The enzymes with CH-PO higher than 10.0 were irreversibly inactivated by current exposure; those with CH-PO between +3 and -5 were not. Inactivation was dependent on the ionic strength of the medium and not on the net charge of the protein. Circular dichroic spectroscopy showed a structural modification in some of the inactivated enzymes. CH-PO could be a crucial, although rough, parameter for predicting protein inactivation by low-voltage exposure. The observed effect seems due to the current density. Enzymatic activity maybe a more accurate sensor of conformational changes than circular dichroism spectroscopy. A better understanding of efficacy of many electrical devices utilized in medical practice may follow.
Journal of Biochemical and Molecular Toxicology 09/2009; 23(5):309-17. DOI:10.1002/jbt.20293 · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Red and near-infrared laser irradiation is reported to have a range of biological effects on cultured cells and different tissues, leading to the hypothesis that laser light can affect energy metabolism. Increased adenosine triphosphate (ATP) synthesis has been reported in cultured cells and rat brain tissue after irradiation at 632.8 nm and 830 nm, respectively. This study investigated whether diode pulsed laser irradiation enhances ATP production in lymphocytes.
Aliquots (500 microL) of an extract of cultured lymphocytes of the Molt-4 cell line were irradiated with diode laser light (lambda = 904 nm, pulsed mode, 6 kHz frequency) with an average emission power of 10 mW for 60 min. A Spectra Physics M404 power meter was used to measure light intensity. Controls were treated similarly but not irradiated. The amount of ATP was measured by the luciferin-luciferase bioluminescent assay.
The amount of ATP in irradiated cell cultures was 10.79 +/- 0.15 microg/L (SD; n = 10), and in non-irradiated cell cultures it was 8.81 +/- 0.13 microg/L (SD; n = 10). The average percentage increase of irradiated versus control cell cultures was about 22.4% +/- 0.56% SD (p < 0.001).
This significant increase is probably due to laser irradiation; it cannot be attributed to any thermal effect, as the temperature during irradiation was maintained at 37.0 degrees +/- 0.5 degrees C. Thus the therapeutic effects of the biostimulating power of this type of laser are identified and its indications may be expanded.
Photomedicine and laser surgery 11/2008; 26(5):451-3. DOI:10.1089/pho.2007.2218 · 1.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The vertebrate retina is an array of "narrow-capture" photoreceptive elements of diverse cellular types that allow the fine spatial resolution characteristic of vision. Imaging of photoreceptors and of the whole retina has been previously reported; however, both were achieved exclusively after fixation. We report our development of a new technique for imaging live bovine retinas ex vivo. Using this technique, we conducted fluorescence confocal laser scanning microscopic imaging of bovine retinas. Eyecups were incubated with conventional fluorescent mitochondrial probes (MitoTracker and JC-1). Unexpectedly, we found that, besides the retinal mitochondria, the rod outer segments that are devoid of mitochondria were also stained. No other neuron was stained. Both protonophores, which decrease mitochondrial membrane potential, or inhibit electron transport strongly inhibited the selective association of dyes with both retinal rod outer segments and mitochondria. This is the first time that living rod outer segments were visualized by this technique. This finding may shed light on previous reports of the existence of a proton potential across the disk membranes and on the mechanism of the adenosine tri-phosphate (ATP) supply for phototransduction, which still requires investigation.
[Show abstract][Hide abstract] ABSTRACT: The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segment consists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins that reside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separation of disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks were isolated from bovine retinal rod outer segments by a method that minimizes contamination from inner segment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additional set of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional (1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism were found, among which are the five complexes of oxidative phosphorylation. Results from biochemical, Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest that F 1F o-ATP synthase is located and catalytically active in ROS disk membranes. This study represents a step toward a global physiological characterization of the disk proteome and provides information necessary for future studies on energy supply for phototransduction.
Journal of Proteome Research 08/2008; 7(7):2654-69. DOI:10.1021/pr7006939 · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The honeybee retina contains the enzyme photoisomerase that converts all-trans-retinal to 11-cis-retinal in light within the visible range. The activity of the bee photoisomerase was studied on all-trans-retinal under pulses of laser light. The experimental data were fitted with a simple equation that describes the mechanism of the isomerization reaction based on three steps having different time constants.
[Show abstract][Hide abstract] ABSTRACT: Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated.
A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs.
cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy.
A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.
[Show abstract][Hide abstract] ABSTRACT: Adenylate kinases (AKs) are ubiquitous phosphotransferases that contribute to homeostasis of adenine nucleotide composition in cells. Six AK isoforms were found in vertebrates. We report that soluble AK isoform 1 is expressed in the cytosol of bovine retina consistently devoid of rod outer segments. Immunoblotting analysis with a polyclonal antibody raised against soluble adenylate kinase and subsequent sequencing of eluted peptide by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry allowed enzyme isolation by joining purification methods to two-dimensional electrophoresis. In this study, we found that cytosolic adenylate kinase isoform 1 is expressed in bovine retina. Cytoplasmic AK1 would physiologically contribute to retinal energy metabolism.
Current Eye Research 04/2007; 32(3):249-57. DOI:10.1080/02713680601161212 · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To achieve our aim of understanding the interactions between direct current and enzymes in solution, we exposed reconstituted Crotalus atrox venom to direct electric current by immersing two platinum thread electrodes connected to a voltage generator (between 0 and 8 V) into a reaction mixture for a few seconds. Then, we assayed the residual activity of phospholipases A(2) (PLA(2)),metalloproteinases, and phosphodiesterases, abundant in crotaline snake venoms and relevant in the pathophysiology of envenomation, characterized by hemorrhage, pain, and tissue damage. C. atrox venom phospholipase A(2) and metalloproteinases were consistently and irreversibly inactivated by direct current (between 0 and 0.7 mA) exposure. In contrast, C. atrox venom phosphodiesterases were not affected. Total protein content and temperature of the sample remained the same. Secretory pancreatic phospholipase A(2), homologue to snake venom phospholipases A(2), was also inactivated by direct current treatment. In order to understand the structural reasoning behind PLA(2) inactivation, circular dichroism measurements were conducted on homogeneous commercial pancreatic phospholipase A(2), and it was found that the enzyme undergoes structural alterations upon direct current exposure.
Journal of Biochemical and Molecular Toxicology 03/2007; 21(1):7-12. DOI:10.1002/jbt.20152 · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark-blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long-wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.
[Show abstract][Hide abstract] ABSTRACT: Exposure of fertilized eggs of the sea urchin Paracentrotus lividus to an electromagnetic field of 75-Hz frequency and low amplitudes (from 0.75 to 2.20 mT of magnetic component) leads to a dramatic loss of synchronization of the first cell cycle, with formation of anomalous embryos linked to irregular separation of chromatids during the mitotic events. Because acetylcholinesterase (ACHE) is thought to regulate the embryonic first developmental events of the sea urchin, its enzymatic activity was assayed in embryo homogenates and decreased by 48% when the homogenates were exposed to the same pulsed field. This enzymatic inactivation had a threshold of about 0.75 +/- 0.01 mT. The same field threshold was found for the effect on the formation of anomalous embryos of P. lividus. Moreover, ACHE inhibitors seem to induce the same teratological effects as those caused by the field, while blockers of acetylcholine (ACh) receptors are able to antagonize those effects. We conclude that one of the main causes of these dramatic effects on the early development of the sea urchin by field exposure could be the accumulation of ACh due to ACHE inactivation. The crucial role of the membrane in determining the conditions for enzyme inactivation is discussed.
Biology of Reproduction 01/2007; 75(6):948-53. DOI:10.1095/biolreprod.106.051227 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of extremely low frequency electromagnetic fields of 75 Hz were studied on different membrane-associated enzymes. Only the activities of three enzymes out of seven exposed to the field decreased approximately of about 54-61% with field amplitudes above a threshold of 73-151 microT depending on the enzyme. The same field had no effect on the activities of either integral membrane enzymes such as Ca,ATPase, Na/K,ATPase, and succinic dehydrogenase or peripheral membrane enzymes such as photoreceptor PDE. The decrease in enzymatic activity of the field-sensitive enzymes was independent of the time of permanence in the field and was completely reversible. When these enzymes were solubilized with Triton, no effect of the field was obtained on the enzymatic activity, suggesting the crucial role of the membrane in determining the conditions for enzyme inactivation. The role of the particular linkage of the field-sensitive enzymes to the membranes is also discussed.
Archives of Biochemistry and Biophysics 10/2005; 441(2):191-8. DOI:10.1016/j.abb.2005.07.011 · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Extremely low frequency electromagnetic fields (ELF-EMFs) of 75 Hz with amplitudes above a threshold of about 125 microT have a dramatic effect on the adenylate kinase (AK) activity of the rod outer segment (ROS) membranes. In fact, the ATP production by ROS membranes or by purified disk membranes placed in the field decreased by approximately 54%. The decrease in enzymatic activity was independent of the time of exposure to the field and was completely reversible. When disk membranes were solubilized with Triton or a soluble isoform of AK was used, negligible effects of the field were obtained on the enzymatic activity, suggesting that the membrane has an important role in determining the conditions for the enzyme inactivation.
[Show abstract][Hide abstract] ABSTRACT: Adenylate kinase activity in rod outer segment membranes of bovine retina decreased of about 55% when exposed to an extremely low frequency electromagnetic field of 75 Hz and 250 microT. The effect was independent of the time of permanence in the field. Negligible effects of the field were found on the enzymatic activity of a soluble isoform of adenylate kinase or of rod outer segment membranes solubilized with Triton, suggesting the importance of the membrane in determining the conditions of the enzyme inactivation.