Microwaves and the cell membrane. III. Protein shedding is oxygen and temperature dependent: evidence for cation bridge involvement.

Microwaves (2450 MHz, 60 mW/g) are shown to result in the release or shedding of at least 11 low-molecular-weight proteins (less than or equal to 31,000 Da) from rabbit erythrocytes maintained in physiological buffer. Protein release was detected by gel electrophoresis of cell-free supernatants using sensitive silver staining. This release is oxygen dependent and occurs in 30 min for exposures conducted within the special temperature region of 17-21 degrees C, which is linked to a structural or conformational transition in the cell membrane. Shedding of 26,000 and 24,000 Da proteins is unique to microwave treatment, with enhanced release of 28,000 and less than or equal to 15,000 Da species during microwave compared to sham exposures. Two-dimensional isoelectric focusing further reveals that proteins of less than or equal to 14,000 Da shed during microwave treatment exhibit a pI of 6.8-7.3 not seen in sham-treated cells. Treatment of erythrocytes with a serine-directed protease inhibitor does not prevent release of proteins. However, when erythrocytes are maintained at 17-21 degrees C by conventional heating in the absence of divalent cations, release of 28,000-31,000 and less than or equal to 14,000 Da components is detected. This indicates that cation-bridge stability may be important for release of these proteins. The above results provide evidence that microwaves alter erythrocyte protein composition at temperatures linked to a transition in the cell membrane and that destabilization of salt bridges may play a role in an interaction mechanism for protein release.

Microwaves and the cell membrane. II. Temperature, plasma, and oxygen mediate microwave-induced membrane permeability in the erythrocyte.

Microwaves (2450 MHz) are shown to increase 22Na permeability of rabbit erythrocytes for exposures only within the narrow temperature range of 17.7 to 19.5 degrees C (Tc) which coincides with a nonlinearity in the Arrhenius plot reflecting an apparent membrane phase transition. Significantly, this response is not observed for cholesterol-loaded erythrocyte membranes which exhibit a linear Arrhenius plot and no apparent phase transition at Tc. The permeability increase at Tc is a nonlinear function of absorbed power but is a linear function of the internal electric field strength of the sample and saturates at approximately 400 mW/g and 600 V/m, respectively. The permeability increase was found to be reversible and transient in that immediately following termination of exposure sodium influx is significantly reduced but returns to normal within 60 min. Extracellular factors exert a significant influence on the microwave effect. The presence of plasma markedly potentiates the increase in 22Na permeability at Tc. Oxygen also modulates the microwave effect with relative hypoxia (5 mm Hg) and hyperoxia (760 mm Hg) enhancing the permeability increase. In contrast, the presence of two antioxidants, ascorbic acid or mercaptoethanol, inhibits the effect. These findings raise important questions about the physical and chemical nature of microwave interactions with cell membranes and also shed light on earlier studies reporting either positive or negative effects on membrane permeability.

Microwaves and the cell membrane. IV. Protein shedding in the human erythrocyte: quantitative analysis by high-performance liquid chromatography.

The erythrocyte responds to microwave fields by shedding at least 11 low-molecular-weight proteins of less than or equal to 31,000 Da, with components of 28,000-31,000 Da released during the destabilization of divalent calcium-protein bridges [R.P. Liburdy and P.F. Vanek, Radiat. Res. 109, 382-395 (1987)]. Significantly, protein shedding was shown to be restricted to exposure temperatures coinciding with the cell membrane phase/structural transition temperature, Tc, of 17-25 degrees C. We report here a further characterization of protein shedding at Tc using high-performance liquid chromatography and membrane-associated blood group antigen testing. Proteins shed from human erythrocytes in microwave fields (2450 MHz, CW) compared to sham-heating displayed a twofold increase in total protein mass released concomitant with the appearance of unique protein species during reverse-phase, hydrophobic interaction, and anion-exchange HPLC. These HPLC analyses indicate that microwaves result in the shedding of proteins which are relatively nonpolar and hydrophobic and which carry a net positive electrostatic charge compared to those released during sham-heat treatment. Assessment of 23 blood group antigens that represent integral protein markers on the erythrocyte cell surface indicates that microwave fields do not result in the exhaustive loss of these proteins. The class of proteins that is shed in response to microwave fields most likely is the loosely bound "peripheral" or extrinsic proteins associated with the exterior of the cell surface. Such proteins play a major role in the transduction of signals to integral membrane proteins which span the bilayer. That this class of proteins is susceptible to release by microwave fields is discussed in relation to microwave absorption at the cell surface by membrane-associated bound water, field interaction with dipolar side groups, and the disruption of divalent cation bridges known to stabilize peripheral membrane proteins.