Bioelectromagnetics 21:499^507 (2000)
Effect of Lo w F reque ncy, Lo w Ampl itude
M ag netic F i elds on the Permeab ility of Cati o n ic
Liposomes Entrapp i ng Carbon ic Anhydr ase
II. No evi dence for su rface enzyme invo lv em ent
Alfonsina Ramundo-Orlando ,
1
Francesca Mattia,
2
Alessandro Pal ombo,
2
and Gugl iel mo D'Inzeo
2
*
1
I nstitute of E xper imental M ed ic ine , CNR , Rom e, Italy
2
Department of Electron ic E ngi neering, Univ ers ity of Rom e``La Sapi en za'', R o me, Italy
Observations recently reported by our group indicate that combined 7 Hz sinusoidal (B
acpeak
50 mT) and parallel static (B
dc
50 mT) magnetic fields can induce a significant increase in dif-
fusion rate of substrate across carbonic anhydrase (CA)-loaded liposomes (DPPC:Chol:SA). A
direct involvement of charges of stearylamine (SA) on the lipid membrane surface was also
demonstrated. Kinetic studies showed that CA was mainly entrapped in liposomes at 5:3:2 molar
ratio, although a small amount (17%) of enzyme was also located on the external surface of these
cationic liposomes. In this paper we report steady state kinetic studies on this latter CA after ELF-
EMFs exposure. No difference in the apparent
K
m
between exposed and sham samples was
observed. On the contrary the apparent
V
max
was increased by approximately a factor of 2 after
field exposure. In spite of the proteolytic digestion of this external CA, a significant increase of
enzymatic activity, as a function of increase in the diffusion rate of substrate across the lipid bilayer,
was observed in the exposed samples. Based on these results, a conformational change induced by
the field on the CA located on the external surface of 5:3:2 liposomes is excluded as an explanation
for our previous observations, supporting the primary role of bilayer SA in the interaction with ELF.
A model of ELF interaction, based on the Larmor precession theory, explaining the physical
phenomenon induced on the dipole of SA has been developed. Bioelectromagnetics 21:499±507,
2000.
ß 2000 Wiley-Liss, Inc.
Key words: ELF-EMFs; Larmor precession; enzyme; kinetic constants; lipid membrane
INTRODUCTION
Cationic liposomes entrapping carbonic anhy-
drase (CA) have been recently used in our laboratory to
investigate effects of extremely low frequency electro-
magnetic ®elds (ELF-EMFs) on membrane perme-
ability. These studies indicated that CA-loaded
liposomes (DPPC:Chol:SA at 5:3:2 molar ratio)
exhibit an enhanced esterase activity, as a function of
increase in the diffusion rate of the substrate p-
nitrophenyl acetate ( p-NPA) across the lipid bilayer,
after exposure to 7 Hz sinusoidal B
acpeak
50 mT and
parallel static B
dc
50 mT magnetic ®elds. In addition,
the 7 Hz effect occurred at a temperature well below
the phase transition where the lipid bilayer was not
leaky.
This 7 Hz ®eld effect was potentiated by
increasing the concentration of stearylamine (SA),
and decreasing the concentration of dipalmitoylpho-
sphatidylcholine (DPPC) in the lipid bilayer. Further-
more, the deprotonation of SA, induced by increasing
the pH of the reaction medium to 8.95, nulli®ed the
magnetic ®eld effect. Taken together, these observa-
tions link the 7 Hz ®eld effect to the presence of
charges of SA on the lipid membrane surface
[Ramundo-Orlando et al., 2000].
Since previous kinetic experiments showed that
CA also partially stick onto the external surface of
these cationic liposomes [Ramundo-Orlando et al.,
1994], a possible role of this enzyme in the ®eld
interaction should be taken into account to obtain
unambiguous information on the primary target of
interaction for the applied ®eld.
CA is a potent catalyst for a fundamental reaction
in living processes, the reversible hydration of CO
2
ß 2000 Wiley-Liss,Inc.
ÐÐÐÐÐÐ
*Correspondence to: G. D'Inzeo, Department of Electronic
Engineering, University of Rome ``La Sapienza'', Via Eudossiana,
18, 00184 Rome, Italy. E-mail: dinzeo@tce.ing.uniroma1.it
Received for review 4 October 1998; Final revision received 22
November 1999.