CAN CELL FREE MYOSIN PHOSPHORYLATION BE USED AS A TOOL FOR BIOLOGICAL DOSIMETRY?

Marko S. Markov
EMF Therapeutics, Inc., Chatanooga TN, USA
1200 Mountain Creek Rd, Suite 160, Chattanooga TN 37405, Email: msmarkov@emftherapy.com

Abstract:  Cell-free calcium-calmodulin dependent myosin phosphorylation has been used as a tool to investigate the response of a model system to various magnetic fields.  It has been shown that this method could be a plausible instrument for biological dosimetry of magnetic fields.

INTRODUCTION

Years of research in the area of bioelectromagnetics and hundreds of publications highlight the need for a complete temporal and spatial dosimetry at the exact location of the target site for a proper assessment of the effect of an electromagnetic field exposure. This is even more important when one takes into account that the field metric is basically not homogeneous throughout the target.  Several magnetic field studies report the existence of “window” effects or resonance-type responses of biological systems to the amplitude and/or frequency metrics of the electromagnetic field.   However, there is lacking a well-established and commonly accepted methods for biological dosimetry.

During the past decade evidence has accumulated and more than 15 papers have been published to show that cell-free myosin phosphorylation can be a plausible method for assessing the effect of magnetic fields. Research shows the phosphorylation of myosin light chain kinase (MLCK) strongly depends on Ca²⁺ as well as on the specific calcium binding protein calmodulin.  It is known that calcium ions appear to be essential in the first steps of transductive coupling of exogenous physical signals to biological tissues and in the ensuing steps of calcium-dependent signaling to intracellular enzyme systems. Calmodulin regulation of enzyme activity has generally been found to require the presence of calcium ions.  Calmodulin is capable of detecting micromolar concentrations of Ca²⁺ and once bound to calcium, calmodulin undertake a more helical conformation to become the active species. 

METHODS

The cell free calcium-calmodulin-dependent phosphorylation of myosin light chains (MLC) occurs in the following manner: Ca²⁺ binds to calmodulin, causing a conformational change in calmodulin; the calcium/calmodulin complex then interacts with the inactive catalytic subunit of MLCK to form a catalytically active holoenzyme complex, which further proceeds to phosphorylate MLC.  Calcium at micromolar concentrations is assumed to be obligatory for complex formation.  The experimental and theoretical analysis performed by us suggests that the most likely target for interaction of magnetic fields with this enzyme-substrate system is the binding of the calcium ion to binding sites already existing in the calmodulin molecule. Affecting the process of calcium binding, magnetic fields could modify the ability of calmodulin to undergo conformational changes and thus could regulate the phosphorylation process.

Myosin phosphorylation as a typical enzyme-substrate reaction in the modifications done in our Laboratory appears to be a plausible tool for biological dosimetry of magnetic fields.  The main advantage of the method is that the cell free solution of 100 µL which represents the “biological sensor” allows dosimetry to be performed in a small volume which makes such samples a very sensitive detector of the biological response within the target volume. The improved experimental conditions facilitate the application of the assay for studying the response to various by amplitude and frequency magnetic fields, such as 

¨       ambient range (up to 200 µT) static magnetic fields in a well controlled laboratory set up;

¨       PRF (pulsed radiofrequency fields) associated   with clinically approved 27.12 MHz magnetic field (200 µT peak value);

¨       static magnetic fields (both in gradient and homogeneous configurations) of the range from 5 mT to 100 mT;

¨       pulsating therapeutic electromagnetic fields  (TEMF) with 120 pps monophase pulsating mode (up to 20 mT and various frequencies of 80-180 pps).

In summary, this cell-free myosin phosphorylation approach allows

¨       Establishment of a frequency window at 100-120 pps for pulsating magnetic fields (TEMF signal) in the range of 80-180 pps

¨       Evaluation of the conditions at which a magnetic field can stimulate or inhibit the activity of various muscles using the ambient magnetic field range

¨       New evidence for 15-20 mT and 45-50 mT amplitude windows

¨       Optimization of clinical settings for static magnetic fields, PRF magnetic fields and TEMF  magnetic fields

The method suggested in this paper allows creation of reference standards and further comparing the response of an unknown magnetic field to the reference standards.