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Response of the Regulatory Oscillatory Behavior of CopperII - Morre 2008

Response of the Regulatory Oscillatory Behavior of CopperII - Morre 2008

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Response of the regulatory oscillatory behavior of copper
II
-containingECTO-NOX proteins and of Cu
II
Cl
2
in solution to electromagnetic fields
D. James Morré
a,
*
, Ziying Jiang
b
, Milorad Marjanovic
a
, John Orczyk
a
, Dorothy M. Morré
b
a
Department of Medicinal Chemistry and Molecular Pharmacology, Hansen Life Sciences Research Building, Purdue University, 201 S. University Street,West Lafayette, IN 47907-2064, United States
b
Department of Foods and Nutrition, Purdue University, 700 W. State Street, West Lafayette, IN 47907, United States
a r t i c l e i n f o
Article history:
Received 1 March 2008Received in revised form 30 May 2008Accepted 3 June 2008Available online 12 June 2008
Keywords:
Copper
II
Hydroquinone (NADH) oxidaseCNOXMolecular and biological time keepingRedox potentialLow frequency electromagnetic fieldsGrowth
a b s t r a c t
A family of cell surface and growth-related proteins, designated ECTO-NOX proteins, carry out both cop-per-dependentNADHandhydroquinoneoxidationandproteindisulfide–thiolinterchange.Thetwoactiv-ities they catalyze alternate to generate a regular period of 24min in length for the constitutive CNOX.Unexpectedly,Cu
II
saltsaloneinsolutioncatalyzeNADH(orhydroquinone)oxidationwithasimilaroscil-latory pattern. Both patterns consist of five maxima, two of which at physiological temperatures are sep-aratedbyanintervalof6minandthreeofwhichareseparatedbyintervalsof4.5min[6min+4(4.5min)].EXAFS and infrared spectroscopic measurements on pure water have shown previously that the ratios of ortho andparaisomers of thehydrogenatoms of water occur onasimilar time scaleandproduce regularpatterns of unequally spaced oscillations similar to those observed with ECTO-NOX proteins and Cu
II
Cl
2
solutions.Here,weprovideresultsfromCu
II
Cl
2
solutionsthatdemonstratethatECTO-NOX-/Cu
II
-catalyzedoscillations in NADH oxidation are phased by exposure to low frequency electromagnetic fields.
Ó
2008 Elsevier Inc. All rights reserved.
1. Introduction
Our work has described a family of growth and redox-relatedECTO-NOX (ENOX) proteins some of which appear to function ascore oscillators of the cells’ biological clock[1]. The unique featureof the ENOX proteins is that the two enzymatic activities they cat-alyze, hydroquinone (NADH) oxidation and disulfide–thiol inter-change, alternate with a regular period length[2]. Theconstitutive ENOX protein designated CNOX or ENOX1 carriesout hydroquinone (NADH) oxidation for 12min and then thatactivity rests. While the hydroquinone (NADH) oxidative activityrests, the proteins engage in disulfide–thiol interchange activityfor 12min. That activity then rests as the 24min cycle repeats.Within the hydroquinone oxidation part of the cycle, there aretwo maxima separated by 6min and within the protein disul-fide–thiol interchangeportionof the cycle, there are three maximaseparated by intervals of 4.5min. The periodic behavior is exhib-ited by pure recombinant ENOX protein and is accompanied by arecurring pattern of amide I/amide II FTIR and CD spectral changessuggestive of 
a
-helix-
b
-sheet transformations[2].Theperiodicoscillationsassociatedwiththetimekeepingactiv-ities of ENOX proteins both require copper and appear to be inher-ent in the molecular structure of the Cu
II
aqua ion[3]itself. Solvated Cu
II
as the chloride or other salts alone exhibit the prop-erty of catalysis of NADH oxidation. With Cu
II
Cl
2
, the pattern alsoconsists of five maxima, two of which are separated by an intervalof6minandthreeofwhichareseparatedbyanintervalof4.5min.The total period lengthis 24min and the period lengthis indepen-dent of temperature and pH[3]as observed with ENOX1. In a pre-vious report[4], a possible underlying pattern of distortion in thefour equatorial oxygens of the Cu
II
aqua ion at a close distance rel-ative to the two axial oxygen atoms at a longer distance wassought from extended X-ray absorption fine structure (EXAFS)studies that might correlate with changes in redox potential suffi-cient to catalyze NADH oxidation in solution. The findings wereconsistent with changes in the coordinated water molecules of the Cu
II
aqua ion possibly related to a metastable equilibrium con-dition in the ratio of ortho to para nuclear spin orientations of thewater-associated hydrogen atoms[5]. In this report we demon-strate that the periodicity of the ENOX/copper clock is phased byexposure to low frequency electromagnetic fields.
2. Methods
2.1. NADH oxidase purification from plasma membranes of soybeanhypocotyls
Plasma membrane of >90% purity were prepared frometiolatedhypocotyls of soybean (
Glycine max
) by aqueous two-phase parti-
0162-0134/$ - see front matter
Ó
2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jinorgbio.2008.06.001
*
Corresponding author. Tel.: +1 765 494 1388; fax: +1 765 494 4007.
E-mail address:
morre@pharmacy.purdue.edu(D. James Morré).Journal of Inorganic Biochemistry 102 (2008) 1812–1818
Contents lists available atScienceDirect
Journal of Inorganic Biochemistry
journal homepage:www.elsevier.com/locate/jinorgbio
 
tion as described[6]were thawed and resuspended in 50mMTris-MES [Tris-2-(4-morpholino) ethanesulfonic acid], pH 7.0 forassay.
2.2. NADH oxidation with copper (II) chloride in water 
NADHoxidationwithcopper chloride was measured as the dis-appearance of NADH determined at 340nm, using two HitachiU3210 spectrophotomers in parallel[7]. Unless stated otherwise,the buffer was 2.5mL 50mM Tris-MES, pH value adjusted to 6with MES plus 20
l
L 15mM NADH. Cu
II
Cl
2
was added (2.5–20
l
froma100mMstock)toprovidethefinalconcentrationsindicatedwith the figures. Period length was independent of copper concen-tration whereas amplitude was proportional to copper concentra-tion. NADH oxidation without copper was the blank. The reactionrates were determined from the slopes of absorbance traces usu-ally recorded at 37
°
C. The traces were recorded for 1min at inter-vals of 1.5min. Continuous traces of 48min were also recorded toobserve the oscillatory pattern. In each determination, the timeswhen the reactants were added and when the recordings werestarted and stopped were recorded. Blank values from reactionscontaining only NADH and buffer were subtracted.
Fig. 1.
Rateof NADHoxidationbyHeLa(humancervical carcinoma)cells(a), NOXproteinsarereleasedinsolubleformfromHeLacellsbytreatmentwithsodiumacetatepH4.5[8],(b)andrecombinanttNOXand(c)measuredafter(opensymbols,dottedlines)andwithout(solidsymbols,solidlines)2minofa50Hzelectromagneticfield(EMF)at50
l
T. In each example, the EMF reset the period maxima to rephase the oscillatory cycle. The cycle length was unchanged.
D. James Morré et al./Journal of Inorganic Biochemistry 102 (2008) 1812–1818
1813
 
2.3. Generation of non-thermal low frequency electromagnetic fields
The copper
II
chloride or ECTO-NOX-containing preparationswere added into buffer and placed in the low frequency electro-magnetic field (EMF) at room temperature for 2min. A solutionprepared in parallel but not in the field was considered as the con-trol. NADH was added to start the reaction at the same time withtwo parallel instruments. A 50Hz magnetic field with a magneticflux density of 50
l
T was used. The source was a single axis Helm-holtzcoil systemconsistingof twoidenticallywound,layeredcoilswired in series designed to use a 60Hz, 120 v AC power source.
2.4. Determination of copper 
Copper concentrations weredeterminedbyinductivelycoupledplasma mass spectroscopy. Calibration was with a commerciallyavailable copper standard (Fischer Scientific).
2.5. ESR measurements
A Brucker ESP 300E spectrometer operatingat 9.3GHz was em-ployed. Spectral parameters were: microwave power 12mW, fieldstrength 5000G, sweep width 2000G, sweep time 21s, modula-
Fig. 2.
The NADH oxidation activities of plasma membranes isolated from dark grown soybean hypocotyls (a,b) and an aqueous, unbuffered solution of 100
l
M CuCl
2
(c)measuredwithout(solidsymbols,solidlines)andafter(dashedlines,opensymbols)EMFtreatmentasinFig. 1. Ineachexample,theEMFphasedtheperiodtoestablishasetof maxima different from that originally present as evidence by the pattern expressed in the control preparation.1814
D. James Morré et al./Journal of Inorganic Biochemistry 102 (2008) 1812–1818

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