On the dielectrically observable consequences of the diffusional motions of lipids and proteins in membranes. 2. Experiments with microbial cells, protoplasts and membrane vesicles.

The dielectric properties of suspensions of intact cells of Methylophilus methylotrophus, Paracoccus denitrificans and Bacillus subtilis have been measured in the frequency range 1 kHz to 13 MHz. All possess a pronounced dispersion corresponding in magnitude and relaxation time to the "beta-dispersion" in a terminology defined by Schwan [Adv. Biol. Med. Phys. 5:147-209 (1957)]. The latter two strains, but not M. methylotrophus, also possess a substantial alpha-dispersion. The relaxation time of the beta-dispersion of B. subtilis is significantly lower than that of the other two strains, due to the higher internal K+ content of this Gram-positive organism. Treatment of P. denitrificans or B. subtilis with lysozyme greatly reduces the magnitude of the alpha-dispersion; in the latter case it is virtually abolished. The magnitude of both the alpha- and beta-dispersions of protoplasts of these organisms is significantly decreased by treatment with the cross-linking reagent glutaraldehyde, indicating that diffusional motions of the lipids and/or proteins in the protoplast membranes contribute to the dielectric relaxations observed in this frequency range. Such motions cannot be unrestricted, as in the "fluid mosaic" model, since the relaxation times of the lipids and proteins, if restricted by hydrodynamic forces alone, should then correspond, in protoplasts of this radius (0.4-0.5 micron), to approximately 10 Hz. Even after treatment of the (spherical) protoplasts with glutaraldehyde, the breadth of the remaining beta-dispersion is still significantly greater than (a) that of a pure Debye dispersion and (b) that to be expected solely from a classical Maxwell-Wagner-type mechanism. It is recognised that the surfaces of the protein complexes in such membranes extend significantly beyond the membrane surface as delineated by the phospholipid head-groups; such molecular granularity can in principle account for the broadened dielectric relaxations in the frequency range above 1 kHz, in terms of the impediment to genuinely tangential counterion relaxation caused by the protruding proteins themselves. The relaxation time of a previously observed, novel, low-frequency, glutaraldehyde-sensitive (mu-) dispersion in bacterial chromatophore suspensions, as well as that of their alpha-dispersion, is significantly increased by increasing the aqueous viscosity with glycerol. This finding is consistent with the view that, from a dielectric standpoint, the motions of charged proteins (and lipids) in biological membranes are rather tightly coupled to those of the adjacent ions and dipoles in the electric double layer.(ABSTRACT TRUNCATED AT 400 WORDS)