Giro questo importante documento del prof. A. Morelli dell'Università di Genova.
Antonio Gagliardi
----- Original Message -----
From: Alessandro Morelli
To: pinceti@die.unige.it ; rconti@cesi.it ; Acustica@ambientescrl.com ; giancarlo.martarelli@unifi.it ; elettrosmogvolturino@interfree.it
Sent: Sunday, August 07, 2005 3:27 PM
Subject: interazioni tra sistemi biologici ed onde elettromagnetiche

A tutti gli interessati,
ho il piacere di comunicare che l'importante rivista Archives of Biochemistry and Biophysics ha accettato la pubblicazione, di cui sono primo Autore, che allego per conoscenza. 
Qui di seguito riporto i commenti assai lusinghieri dell'editore.
La pubblicazione dovrebbe avvenire intorno alla metà del mese di settembre pv e questa che allego è la forma definitiva visto che l'Editore non l'ha sottoposta a revisione  ritenendola estremamente interessante.
Degno di nota che  queste indagini di laboratorio portino alla conoscenza della comunità scientifica internazionale e della popolazione interessata alcuni bersagli molecolari certi, per giunta estremamente importanti (p.e. l'enzima Acetilcolinesterasi), dei campi elettromagnetici a bassa frequenza.
Senza tema di essere smentito credo che i nostri dati costituiranno un riferimento internazionale obbligato nella valutazione dell'influenza dei Campi Elettromagnetici con Frequenza estremamente bassa (ELF EMF) sui sistemi biologici.
Affatto eloquenti sono le Fig. 2 e 4.
Con viva cordialità,
            Alessandro Morelli
 
 
Copia della lettera dell'Editore di Archives of Biochemistry and Biophysics:
 
 
 
Ms. No.: ABBI-05-384
Title: Effects of extremely low frequency electromagnetic fields on
membrane-associated enzymes.
Archives of Biochemistry and Biophysics

Dear Prof. Pepe,

Thank you for submitting your paper to Archives of Biochemistry and
Biophysics. The review process has concluded, and I am pleased to inform
you that your manuscript has been accepted for publication in Archives of
Biochemistry and Biophysics. Although there is little mechanistic data, it
was felt that the observations are extremely interesting.

The publisher has been notified of this decision, and you will receive
confirmation from them shortly.

Congratulations on being one of very few authors to have your submission
accepted without any revisions requested, which accounts for just about 2
percent of all our submissions per year.

Sincerely,

Antonio Scarpa
Executive Editor
Archives of Biochemistry and Biophysics

Effects of extremely low frequency electromagnetic fields on membrane-associated enzymes.

 

A. Morelli,1 S. Ravera,1 I. Panfoli,1 and I.M.Pepe,2*

Departments 1DIBISAA, 2DISTBIMO of the University of Genoa , viale Benedetto XV, 3 Genova, 16131 Italy .

 

*Correspondence to: I.M.Pepe, DISTBIMO, Faculty of Medicine, University of Genoa, Padiglione 4 Ospedale S. Martino, Genova 16132 , Italy .  E-mail: mario.pepe@unige.it

 

 

Keywords: ELF electromagnetic fields; membrane enzyme; enzyme inactivation.

 

 

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 % to 61 % with field amplitudes above a threshold of 73 mTesla to 151 mTesla 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 periferal 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.

 

INTRODUCTION

It has been recently reported that extremely low frequency electromagnetic fields (ELF EMFs) of 75 Hz and amplitudes above a 125 mTesla threshold, caused a 54 % decrease in the enzymatic activity of the adenylate kinase of the rod disk membranes of bovine retina [Ravera et al., 2004]. This enzyme is presumably lipid-linked through a fatty acylation of the protein [Notari et al., 2003], as it reacts with a polyclonal antiserum against the first 15 N-terminal amino acids of AK1b, a membrane-associated isoform of adenylate kinase [Collavin et al., 1999] belonging to the N-terminus myristoylated proteins family.

The applied field was effective on the activity of the membrane-bound enzyme but gave a negligible effect on Triton solubilized disk membranes or on soluble isoforms of adenylate kinase. Small effects of ELF EMFs on the activities of soluble enzymes [Zhang and Berg, 1992; Dutta et al., 1994; Litovitz et al., 1991; Thumm et al., 1999] have been already reported. This suggests that the membrane could have an important role for mediating the effect of the field on the enzymatic activity. In fact, interesting results involving biological membranes exposed to ELF electromagnetic fields were reported [Miller, 1991; Paradisi et al., 1993; Astumian et al., 1995; Volpe et al., 1998; Bersani et al., 1997; Baureus-Koch et al., 2003; ]. In particular, measurements by fluorescent probes showed changes in lipid molecular dynamics of the cell membrane [Volpe et al., 1998], while electron microscope images of freeze-fractured membranes exposed to the field indicated a significant clustering of the distribution of the intramembrane proteins [Bersani et al., 1997]. Therefore, in this paper the hypothesis that ELF EMFs could affect membrane-associated enzymes activities was tested on enzymes with different degree of membrane association. The choise was done among integral membrane proteins such as CaATPase, Na/K,ATPase, succinic dehydrogenase, periferal membrane proteins such as photoreceptor phosphodiesterase 6 (PDE), lipid-linked proteins such as alkaline phosphatase, acetylcholinesterase. Phosphoglycerate kinase. was also assayed, although its binding to membranes is still not clear. When exposed to ELF-EMFs of 75 Hz , only three enzymes lowered dramatically their activities of about 54% to 61% . Moreover, no effects of the field on the activity of the same enzymes solubilized with Triton was detected. In order to give an interpretation to these results, the importance of both the membrane structure and the kind of linkage of the enzyme to the membrane is discussed.

 

MATERIALS AND METHODS

Erythrocyte membranes (ghost) preparation

Erythrocytes, completely free of leucocytes and platelets, were obtained following a method already reported [Pontremoli et al., 1979] with minor modifications, starting from 25 mls human blood treated with 1mg/ml EDTA to avoid coagulation. The sample was centrifuged at 3,500 rpm for 15 min at 4 °C and the pellet containing erythrocytes was collected and resuspended in 130 mM KCl and 20 mM Tris-HCl pH 7.4 (1:1 v/v) and centrifuged thrice at 3,500 rpm for 15 min. In order to obtain the erythrocytes membranes the last pellet was resuspended in hemolysis buffer (1:5 v/v) containing 1 mM EDTA-Na and 10 mM Tris-HCl pH 7.4 and centrifuged at 17,000 rpm for 40 min. The pellet containing erythrocyte membranes was resuspended in few mls of distilled water. Samples were stored at -80°C.

 

 Synaptosomal preparation

For synaptosomal preparation, the method published by Rawls [1999] was followed with minor modifications. For each preparation, six dorsal striata were removed from decapitated Mus musculus mouse and pooled in ice-cold 100 mM Tris buffer pH 7.4. The striata were transferred to 0.32 M unbuffered sucrose and manually disrupted in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 3,000 g for 2 min. The supernatant was centrifuged at 14,000 g for 12 min. The soft pellet was resuspended in 0.32 M sucrose containing 10 mM N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES, pH 7.4), and 4-ml aliquots were loaded onto discontinuous gradients consisting of three layers of Ficoll (Sigma) in sucrose (wt/vol ; 12%, 4 ml ; 9%, 1 ml ; 6%, 4 ml). The gradients were centrifuged at 62,483 g for 35 min. Synaptosomes were harvested from the 9% layer, diluted with 3 volumes of 10 mM TES buffer containing 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM Na2HPO4, 1 mM MgCl2, and 10 mM glucose (pH 7.4), and centrifuged at 14,000 g for 12 min. Synaptosomes were resuspended in 0.5 ml of 10 mM TES buffer (pH 7.4). The preparation was divided into aliquots containing 0.8 mg of protein and centrifuged at 10,000 g for 2 min. Synaptosomes were overlaid with 200 µl of 0.25 M sucrose containing 5 mM TES (pH 7.4) and stored as pellets at -80°C.

 

Acetylcholinesterase activity 

Acetylcholinesterase activity was measured by using acetylthiocholine chloride (Sigma) as substrate and monitoring the thiocoline production as already described [Bergmeyer et al., 1983]. Synaptosomal (100 ?g of protein) or erythrocyte membranes preparations (200 ?g of protein) were added to a reaction mixture containing 0.4 mM acetylthiocholine in 50 mM phosphate buffer pH 7.2. The final volume of 1 ml was kept in the electromagnetic field 75 Hz, 2.5 mT. At different time intervals, aliquots of 100 ml were withdrawn and added to 50 ml of 25% PCA. Control samples were run in the same experimental conditions as above but in the absence of field.

Each sample was centrifuged for 3min at 14,000 rpm and then 100 ?l of supernatant was withdrawn and neutralized with 50 ?l 2 M K2CO3. Centrifugation was repeated to remove potassium perchlorate. Aliquots of 100 ?l of each neutralized extract were used for thiocoline assay by addind 0.5 mM DTNB in phosphate buffer pH 7.2 (1 ml final volume). Thiocoline reaction with DTNB gave thionitrobenzoate formation (?405 = 1.33 mM-1  cm-1) which was monitored spectrophotometrically by following the rise in absorbance.

 

Phosphoglycerate kinase activity

Phosphoglycerate kinase activity was measured in red blood cell ghosts by following a method already described [Bergmeyer et al., 1983] with minor modifications. Ghosts (100 ?g) were added to a solution containing 6.5mM glycerate-3-phosphate and 1.2mM ATP in 40 mM Tris HCl pH 8, 120 mM KCl, 10 mM MgCl2 . The final volume of 1 ml was kept in the electromagnetic field of 75 Hz and 2.5 mT. At different time intervals, aliquots of 100 mls were withdrawn and added to 50 ml of 25% PCA. Control samples were run in the same experimental conditions as above but in the absence of the field.

Each sample was centrifuged for 3min at 14,000 rpm and then 100 ?l of supernatant was withdrawn and neutralized with 50 ?l 2 M K2CO3. Centrifugation was repeated to remove potassium perchlorate. Aliquots of 100 ?l of each neutralized extract were used for the enzymatic assay by addind 2?l of glycerate-3-phosphate dehydrogenase, 0.2 mM NADH, 10 mM MgCl2, 50mM Tris HCl buffer pH 8 (1 ml final volume). The corresponding NADH oxidation was monitored spectrophotometrically by following the decrease in absorbance at 340 nm (?340 = 6.22 mM-1  cm-1).

 

CaATPase activity

Plasma membrane Ca-ATPase activity was measured in erythrocyte membranes preparation through inorganic phosphate release. The standard reaction mixture contained 30 mM HEPES, pH 7.6, 140 mM KCl, 0.2 mM ouabain, 2 ?g/ml calmodulin, 2 mM ATP and 0.005 mM free Ca2+ (obtained with a Ca2+-EGTA buffer [Fabiato and Fabiato, 1979]; EGTA was 2mM). 2mM EGTA) in a final volume of 1 ml. Aliquots of erythrocyte membranes preparation (about 70 ?g proteins) were added to the reaction mixture and incubated at room temperature for 30 min in the presence or in the absence of the field of 75 Hz and 2.5 mT. The final volume of 1 ml was kept in the electromagnetic field. At different time intervals, aliquots of 100 mls were withdrawn and added to 50 ml of 25% PCA. Each sample was centrifuged for 3min at 14,000 rpm and then 100 ?l of supernatant was withdrawn and neutralized with 50 ?l 2 M K2CO3. Centrifugation was repeated to remove potassium perchlorate. Control samples were run in the same experimental conditions as above but in the absence of field. The released inorganic phosphate was determined colorimetrically as described by Martin and Doty (1949). Blank subtraction was obtained by considering the amount of inorganic phosphate released by parallel samples incubated in the absence of Ca2+ and in the presence of 2 mM EGTA.

 

(Na, K)ATPase activity

ATPase activity of erythrocyte membranes was assayed by the pyruvate kinase, lactate dehydrogenase system in which hydrolysis of ATP is coupled to the oxidation of NADH [Bucher and Pfleiderer, 1955]. ATP hydrolisis was measured in the presence or in the absence of the field of 75 Hz and 2.5 mT for 20 min. An aliquot of 0.145 mg/ml erythrocyte membranes preparation was added to the reaction mixture containing 100 mM Tris-HCl pH 8, 2 mM MgCl2, 150 mM NaCl, 50 mM KCl, 1 mM ATP [Balestrino et al., 1998]. Aliquots of 100 ?l of the reaction mixture were withdrawn and added to 50 ?l of 25% PCA. Each sample was centrifuged shortly at 14,000 rpm and then 100 ?l of supernatant was withdrawn and neutralized with 50 ?l of 2M K2CO3. The centrifugation was repeated to remove potassium perchlorate. In order to measure the amount of ATP hydrolized, 100 ?l aliquots of each neutralized extract were added to 1 ml final vol of reaction mixture containing 0.16 mM NADH, 1.5 mM phosphoenolpyruvate, 8 ?g/ml pyruvate kinase, 8 ?g/ml lactate dehydrogenase, 100 mM Tris-HCl pH 8.0; 5 mM MgCl2. ATP hydrolysis coupled to oxidation of NADH was followed at 340 nm (rounded small epsilon, Greek340 for NADH=6.22×103M-1cm-1) in spectrophotometers. Ouabain-sensitive (Na+,K+)ATPase was determined as the difference between the enzyme activities in the presence and in the absence of 0.1 mM ouabain [Yoda and Hokin, 1970].

 

Microsome preparation

Bovine liver was chopped and homogenized in 20 mM Tris-HCl pH 7.5 containing 0.25 M sucrose in a Potter apparatus. The homogenate was centrifuged for 10 min at 900 g. The supernatant was collected and centrifuged for 20 min at 20,000 g. The resulting supernatant containing cytosol and microsomes was collected and centrifuged at 100,000 g for 1 hr. The pellet containing microsomes was stored at -80°C.

 

Alkaline phosphatase activity

Alkaline phosphatase activity was measured by using 4-nitrophenylphosphate (Sigma) as substrate and monitoring 4-nitrophenol formation as already described [Bergmeyer et al., 1983]. Microsomes from liver (100 ?g of protein/ml) were added to a reaction mixture containing 4-nitrophenylphosphate (12 mM) 0.1 M glycine, 0.1 mM ZnCl2, 1mM MgCl2 (glycine buffer pH 10.5). The final volume of 1 ml was kept in the electromagnetic field of 75 Hz and 2.5 mT for 20 min. At different time intervals, aliquots of 100 mls were withdrawn and added to 50 ml of 25% PCA. Control samples were run in the same experimental conditions as above but in the absence of field.

Each sample was centrifuged 3 min at 14,000 rpm and then 100 ?l of supernatant was withdrawn and neutralized with 50 ?l 2 M K2CO3. Centrifugation was repeated to remove potassium perchlorate formed by the reaction between PCA and K2CO3. Aliquots of 100 ?l of each neutralized extract were used for 4-nitrophenol assay by adding 0.1 M glycine buffer pH 10.5 (1 ml final volume) and measuring spectrophotometrically the increase in absorbance at 405 nm (? = 1.85 mmol-1 x cm-1) which is proportional to the nitrophenol produced.

 

Mitochondria preparation

Pieces of rat liver (3 g) were homogenated at ice temperature by Potter-Elvehjem system in 20 ml Buffer containing 0.25M Sucrose, 5 mM HEPES pH 7.2, 1mM EDTA. The liver homogenate was centrifuged for 10 min at 500g, at 4°C. After centrifugation, precipitate was discarded and supernatant was centrifuged again for 20 min at 20,000g. Then the pellet was collected and dissolved in 2 ml of homogenizing Buffer.

 

Succinic dehydrogenase activity

Succinic dehydrogenase activity was assayed by the oxidation of succinic acid to fumaric acid coupled with K3Fe(CN)6 reduction, following the method of Bonner [1955] with minor modifications. The enzymatic activity was measured in the presence or in the absence of the field of 75 Hz and 2.5 mT for 20 min after addition of mitochondria preparation (0.1 mg of proteins) to 1 ml final volume of the reaction mixture containing 100 mM Phosphate buffer pH 7.6, 70 mM Na succinate, 2 mM K3Fe(CN)6 . At different time intervals, aliquots of 100 mls were withdrawn and the reaction was stopped with 0.25 ml Aceton. Then the samples were centrifuged for 3 min at 14,000 rpm, and supernatant was used for the succinic acid oxidation coupled to reduction of K3Fe(CN)6 , which was followed spectrophotometrically at 400 nm (rounded small epsilon, Greek400 for K3Fe(CN)6 = 920 M-1cm-1).

 

Rod outer segments and disk membranes preparations

            In a typical experiment, rod outer segments (ROS) were isolated from 30 bovine retina in dim red light by following the method of Schnetkamp and Daemen [1982] by sucrose gradient centrifugation. Intact ROS were first washed in an isotonic medium containing 40 mM Tris-Maleate pH 7, 120 mM KCl and then centrifuged for 10 min at 1,500 g.

Osmotically intact disks were obtained after bursting intact ROS for 3 h in 30 ml of 5% Ficoll (Sigma) in distilled water containing 5 mM DTT and 70 mg/ml leupeptin and then by collecting them in dim red light at the 5% Ficoll surface after centrifuging for 2 h at 25,000 rpm in a Beckman FW-27 rotor [Smith et al., 1985]. The disk membranes were washed in distilled water and centrifuged for 10 min at 1,500 g. The pellet (purified disk membrane preparation) was resuspended in the initial volume of distilled water and stored in the dark at  - 80° C.

 

Rod disk phosphodiesterase activity

cGMP Phosphodiesterase was assayed as follows. The reaction mixture contained 100 mM MOPS (pH 7.1), 140 mM KCl, 20 mM NaCl, 5 mM MgCl2, 2mM GTP and 5 mM cGMP. The reaction was started by adding 50 ?l of reaction mixture containing 4 ?Ci [8-3H] cGMP (specific activity: 12.7 Ci/mmol) to 50 ?l of purified disk membrane preparation previously bleached in the room light (final concentration: 1 - 2 mg/ml protein). The reaction was run in the presence or absence of ELF-EMF of 75 Hz and 2.5 mT, at room temperature and stopped after 1, 3, 5, 10 and 20 min by adding 5 ?l of 100 mM EDTA to each 20 ?l withdrawn sample and boiling for 2 min. After centrifuging, 10 ?l of the supernatants were analysed by TLC on polyethyleneimine-cellulose developed in one dimension with 1M LiCl. 5´-GMP was added as internal standard. The separed spots corresponding to cGMP and 5´-GMP, visualized under UV light, were cut into scintillation vials, eluted in 0.5 ml of 0.7M MgCl2/1M TrisHCl pH 7.4 (10/2 v/v) , added to 3 ml of Instagel, left overnight and then counted in a LKB liquid scintillation counter.

 

Protein concentration

Protein concentrations were determined using the Bradford method [1976].

 

Electromagnetic field production

ELF EMFs were produced by an equipment (Biostim Igea, Modena, Italy) mainly used for clinical application such as that to accelerate the healing of bone fractures. The generator system supplies a square wave with a maximal applied tension of 180 V, a period of 13.3 ms (75 Hz of frequency), a duty cycle of 10%, to a couple of Helmoltz coils (each with 1000 turns of copper wire of 0.2 mm of diameter) with internal and external diameter of 72.5 mm and 82.5 mm respectively. Measurements of the maximal intensity of the magnetic field B with a gaussmeter, showed that it was fairly constant midway between the coils, giving values of about 240 ?T when the distance between the coils were 12 cm or a value of about 2.5 mT at a distance of 3 cm. The time course of the magnetic field as well as that of the induced electric field were measured, as already described [Ravera et al., 2004; De Mattei et al., 2003], at the center of the distance between the two coils where the sample for the enzymatic activity measurements was always placed. The temperature variation of the sample in the presence of the applied field was found constant within the experimental error of ±0.1 ºK.

 

RESULTS

When membrane associated enzymes were exposed to ELF - EMFs of 75 Hz frequency and 2.5 mT amplitude for 20 min and their activity were measured immediately after the withdrawal of the field, no effect was detected. Instead, when the enzymatic activity was measured during the exposure to the field, a dramatic decrease in enzymatic activity was found for three enzymes out of seven membrane-associated enzymes. As an example, Figure 1 shows the activity of alkaline phosphatase of bovine liver microsomes in the absence or in the presence of the field. It can easily be seen that the enzymatic activity appears reduced by the alternate field. In fact, the least-square linear regression of the process gave slopes of about 58 nmoles of substrate transformed/min/mg protein for the control sample and of about 29 nmoles /min/mg protein for the sample in the presence of the field with a decrease of 50%.

Table 1 shows the results of the activities of different membrane associated enzymes exposed to the same field. The three enzymes of the first four lines, that is alkaline phosphatase, acetylcholinesterase from ghosts or from synaptosomes and phosphoglycerate kinase lowered their activities of about 54% , 60%, 58% and 61% respectively, while either integral membrane enzymes such as CaATPase, Na/K,ATPase and succinic dehydrogenase, or periferal membrane enzymes such as photoreceptor PDE were insensitive to the applied field. The Table shows also the 54% decrease of adenylate kinase activity previously described [Ravera et al., 2004].

The decrease of the enzymatic activities of the three enzymes was independent of the time of permanence in the field as shown in Fig 2. In these experiments, the reaction mixture containing the membrane enzyme but not the substrate was kept in the field for different time intervals of 0, 2, 5, 10 min and immediately after the substrate was added to the reaction mixture and the enzymatic activity was measured for one min in the presence of the field. Control samples were run in the same experimental conditions as above but in the absence of the field. The enzymatic activities exposed to the field showed a dramatic mean decrease of about 54 % ± 4 % for alkaline phosphatase, 58 % ± 5 % for acetylcholinesterase from synaptosomes, 61% ± 3% for phosphoglycerate kinase and 60 % ± 4% for acetylcholinesterase from blood cells (the latter is not shown in Fig 2 because it was not statistically different from the activity decrease of acetylcholinesterase from symaptosomes). The decrease of the enzymatic activities appears constant within the experimental errors and independent of the time of permanence in the field.

The activities of the field-sensitive enzymes were measured in function of the concentration of their substrates and the resulting Michaelis-Menten kinetics showed a behaviour of a pure noncompetitive inhibition where only Vmax was affected by a field exposure. Fig 3 shows the Lineweaver-Burk plot of the data obtained for acetylcholinesterase from blood cell ghosts: while KM remained constant with a value of 0.18 mM, Vmax instead dropped of about 60% from 3.5 to 1.4 U/mg protein when exposed to the field.

The decrease of the enzymatic activities was studied at different amplitudes of the applied field. Fig 4 shows that an amplitude of about 151 ± 7 mT was the lowest value which produced a supra-threshold response for alkaline phosphatase while amplitudes of 50 to 137 mT gave results of enzymatic activities statistically indistinguishable from those of the controls. Similar data were found for the other field-sensitive enzymes (not shown in Fig 4 for simplicity) and the corresponding suprathreshold field values were 73 ± 3 mT, 87 ± 4 mT and 95 ± 5 mT for phosphoglycerate kinase, acetylcholinesterase from blood cells and from synaptosomes respectively. The effect of the field on enzymatic activities seems independent of its amplitude above the threshold up to 240 mT and for the amplitude of 2.5 mT reported in Table 1.

In order to verify that the membrane structure is crucial in determining the field effect on the enzymatic activity, experiments were run on the field-sensitive enzymes solubilized by 0.1 % Triton. The results showed that the activities of the solubilized enzymes were not affected by the field (see Table 2) with a statistical significance of P < 0.001. The solubilization with Triton in itself did not affect the activity of alkaline phosphatase or acetylcholinesterase from blood cells but decreased of about 7.5 % the activity of phosphoglycerate kinase while enhanced of about 10% the activity of acetylcholinesterase from synaptosomes. Such activity differences are usually observed when membrane bound enzymes are solubilized in low concentration of a mild detergent [Helenius and Simons, 1975].

 

DISCUSSION

The data reported in this paper show that ELF-EMFs of 75 Hz with amplitudes above a threshold produces a decrease of about 54% to 61 % of the enzymatic activities of three membrane-bound enzymes: alkaline phosphatase, phosphoglycerate kinase and acetylcholinesterase from blood cell or from synaptosomes. Considering also the adenylate kinase activity decrease found in rod disk membranes exposed to the same field [Ravera et al., 2004], the so called field-sensitive enzymes become four out of eight under consideration. The effect of the field was highly reproducible and reversible at least for 20 min field exposure. The same decrease in the enzymatic activity was measured independently of the exposure time, indicating that the effect of the field starts within the first min exposure and remains constant during the experiment. The action of the field seems that to switch the enzyme to a state of a reduced activity, which is independent on the field amplitude, at least at amplitudes above the different thresholds up to 240 ?T and at 2.5 mT. The full activity is rapidly restored when the enzyme is removed from the field.

                                        However, the field had no effect on Triton solubilized enzymes. No effect of the same field was also found on the enzymatic activity of a soluble isoform of adenylate kinase present in rod outer segment preparations [Ravera et al., 2004; Notari et al., 2001] or of the soluble adenylate kinase purified from rabbit muscle. These results strongly suggest that the field action is mediated by the membrane organization and structure which is crucial in determining the conditions of the enzyme inactivation. However, the effect of the field on the membrane is not sufficient alone to explain the decrease in enzymatic activity: in the case of acetylcholinesterase, it was independent of the source of the membrane, as the activity decrease for the enzyme coming from blood cells was not statistically different from that prepared from synaptosomes. On the other hand, integral membrane enzymes such as Ca,ATPase and Na/K,ATPase or periferal membrane enzymes such as PDE were not affected by the field suggesting that the linkage to the membrane is an important cofactor. Three enzymes such as alkaline phosphatase, acetylcholinesterase from blood cell or from synaptosomes and adenylate kinase from rod disk membranes are lipid-linked membrane enzymes: alkaline phosphatase as well as acetylcholinesterase are known to be anchored to the membrane through a glycosylphosphatidylinositol while adenylate kinase is presumably bound to rod disk membranes through a myristic fatty acid [Notari et al., 2003]. Instead, the kind of attachment to red blood cell membranes of the field-sensitive enzyme phosphoglycerate kinase is not yet known, although a covalent linkage to hydrophobic group such as myristylate could be hypothized. Indeed, the sequence of phosphoglycerate kinase in epimastigotes of Trypanosoma cruzi has been shown to contain a number of potential N-myristoylation sites [Concepcion et al., 2001].

                                        The lipid moyety linked to the enzyme is embedded into the lipid bilayer and allows the protein to move along the membrane surface to search for the substrate molecules. A recent report [Caseli et al., 2005] on glycosylphosphatidylinositol-anchored alkaline phosphatase incorporated into artificial phospholipid monolayers showed that the enzyme activity dropped of about 40 % following a lipid phase transition from liquid expanded to liquid condensed state, when surface pressure increased above 18 mN/m. Fluorescence microscopy revealed that above this pressure, proteins aggregated and formed clusters which would affect the substrate accessibility to the catalytic site and therefore decrease the enzymatic activity. Changing the conditions of the lipid matrix where the enzyme is inserted through its anchor may have profound influence on the protein flexibility, a requisite for its functional activity. It has been reported that a pulsed field of 50 Hz induced a decrease in lipid molecular dynamics of cell membranes [Volpe et al., 1998] and significant clustering of the distribution of the membrane proteins [Bersani et al., 1997]. Therefore, we can speculate that the field of 75 Hz would be able to induce a transition of the lipid bilayer to a more liquid ordered phase, as suggested by the sharp threshold behaviour shown in Fig 4, followed by the partition of the lipid-anchored enzymes in clusters with a subsequent activity decrease. However, despite the large number of reports on the effects on biomembranes by ELF-EMFs [Glaser, 1992; Walleczek, 1995; Barnes, 1996; Kaiser, 1996; Lacy-Hulbert, 1998; Neumann, 2000; Weaver, 2002; Volpe, 2003], an unified physical-chemical explanation has not been provided yet. The field could modify the membrane organization and structure by acting directly on the strong anisotropy of diamagnetic susceptibility of membrane phospholipids [Del Moral et al., 2002]. An interesting result on the effects of ELF EMFs on carbonic anhydrase entrapped in liposomes seems to exclude the role of the enzyme molecule in favour of the direct action of the field on charged lipids of the membrane [Ramundo-Orlando et al., 2000].

            However, the results presented in this paper are important for the interpretation at molecular level of macroscopic effects produced by ELF- EMFs on biological systems. Several studies indicate that ELF-EMFs affect cell division timing and embryos development of many organisms [Delgado et al., 1982; Dixey and Rein, 1982; Falugi et al., 1987; Koch et al., 1993]. In particular, the effects of low-intensity pulsed electromagnetic fields on the early development of the sea urchin Paracentrotus lividus. [Falugi et al., 1987] could be interpreted as due to acetylcholinesterase inhibition. In fact, acetylcholinesterase is thought to regulate the embryonic first developmental events of the sea urchin, from gamete interaction to cleavages [Shmukler et al., 1998].

On the other hand, cholinesterase inhibitors are currently used for treatment of Alzheimer's disease [Pacheco et al., 1995]. Moss and colleagues [1999] reported that methanesulfonyl fluoride, a very selective CNS acetylcholinesterase inhibitor, improves cognitive performance in patients with senile dementia of the Alzheimer type. Therefore, ELF-EMFs could be a mild tool for the treatment of Alzheimer desease.

Another example of field effect which needs a molecular interpretation is that of the clinical application of weak EMFs of 75 Hz frequency currently used to accelerate the healing of bone fractures. The molecular mechanisms underlying these empirical observations could originate from alkaline phosphatase inhibition by the pulsed field. As a matter of fact, alkaline phosphatase is commonly used as an osteoblast differentiation marker, although its role in bone formation is still controversial as it could be involved in the process of bone mineralization or in the organic matrix synthesis as well. However, a decrease in its activity was found in concomitance with an increase of the number of human bone cells in culture following dynamic strain [Kaspar et al., 2000].

 


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Table 1  Effect of a field of 75 Hz and 2.5 mT on the activities of different membrane associated enzymes

 

Membrane-associated enzyme

Control sample

Sample + field

alkaline phosphatase

61 ± 2 U/mg   

28 ± 1 U/mg

acetylcholinesterase

from blood cell membranes

3.5 ± 0.1 U/mg   

1.4 ± 0.1 U/mg

acetylcholinesterase

from synaptosomes

13.7 ± 0.2 U/mg   

5.8 ± 0.1 U/mg

phosphoglycerate kinase

0.465 ± 0.005  U/mg  

0.18 ± 0.01 U/mg

adenylate kinase*

177 ± 10 U/mg  

81 ± 5 U/mg

CaATPase

21 ± 1 U/mg

20 ± 1 U/mg

Na/K,ATPase

78 ± 5 U/mg   

80 ± 5 U/mg

succinic dehydrogenase

100 ± 5 U/mg 

101 ± 5 U/mg 

rod outer segment PDE

50 ± 5 U/nmol of Rhodopsin

48 ± 5 U/nmol of Rhodopsin

 

Each value represents the mean ± SD of ten (first four enzymes) or five measurements. The enzymatic activity is expressed as Units/mg (nmoles of substrate transformed /min/ mg protein).

* already reported [Ravera et al., 2004]

 

 

 

 

 

Table 2. Effect of the field on the activity of lipid-linked enzymes solubilized in 0.1 % Triton

 

Enzymes in 0.1 % Triton

Control sample

Sample + field

alkaline phosphatase

58 ± 2 U/mg

56 ± 2 U/mg

acetylcholinesterase

from blood cell membranes

3.3 ± 0.2  U/mg

3.0 ± 0.2 U/mg

acetylcholinesterase

from synaptosomes

15.1 ± 0.2 U/mg

14.5 ± 0.2  U/mg

phosphoglycerate kinase

0.43 ± 0.01 U/mg

0.43 ± 0.01 U/mg

 

The enzymatic activity was measured during 20 min exposure of a field of 75 Hz and 2.5 mT. Control samples were run in the same way but in the absence of the field. Each value represents the mean ± SD of ten measurements.

 

 

 

FIGURE LEGENDS

 

Fig 1. Alkaline phosphatase activity in the absence ( ? ) or in the presence ( ? ) of an alternate magnetic field of 75 Hz and 2.5 mT. Each point represents the mean of two measurements.

 

Fig 2. Enzymatic activities of phosphoglycerate kinase, alkaline phosphatase, acetylcholinesterase from synaptosomes respectively in the absence ( ? ? ? ) or in the presence ( ? ? ? ) of an alternate magnetic field of 75 Hz and 2.5 mTesla. The enzymatic activity was measured for one min in the absence or in the presence of the field, immediately after the exposure time. Each point represents the mean of ten measurements. Standard deviations of the activity measurements were not drawn to avoid superpositions of the error bars. However, they varied from 2 to 6 % of the mean values.

 

Fig 3. Lineweaver-Burk plot from the Michaelis-Menten kinetics of acetylcholinesterase from blood cells in the absence (? ) or in the presence ( ? ) of a field of 75 Hz frequency and 2.5 mT amplitude. Each value represents the mean ± SD of five measurements.

 

Fig 4. Alkaline phosphatase activity measured during 10 min exposure to 75 Hz magnetic field of different amplitudes. Each value represents the mean ± SD of five measurements.

 

 

 

 

 

 

 

 

 

Fig 1

 

 

 

 

 

 

 

 

 

Fig 2

 

 

 

 

 

Fig 3

 

 

 

Fig 4