534th MEETING, NOTTINCHAM
3 29
at least a substantial proportion of the 'red shift' has now been accounted for as an
artifact arising from light-scattering and other optical effects. It must be concluded
that there is not yet any very substantial experimental basis from which to predict the
nature and extent of lipid-protein interaction in biological membranes. More
quantitatively significant results are required.
Low-angle X-ray-diffraction patterns from dried membranes feature two groups of
reflexions : ones that are temperature-sensitive and readily affected by solvents such as
acetone and ether, and others that are relatively insensitive to temperature and to
acetone and ether but are modified by ethanol and by chloroform. The first group
have been suggested to be related to independent lipid phases and the second to
lipoprotein (Finean et al., 1968). Electron micrographs indicate that the lipoprotein is
a residual membrane framework that probably includes the membrane protein and the
more firmly bound lipid. In the native membrane a portion of the lipid may be only
loosely associated with protein or perhaps simply incorporated in the structure through
lipid-lipid associations with the more firmly bound lipid.
Treatment of erythrocyte membranes with Clostridium perfringens phospholipase C
before drying has been observed to eliminate the X-ray reflexions attributed to
independent phospholipid phases, but the reflexions attributed to residual lipoprotein
were not significantly affected (Finean & Coleman, 1970). It would thus appear that
most of the phosphatidylcholine and sphingomyelin and part of the phosphatidylethanolamine may be hydrolysed (Coleman et al., 1970) without destroying the general
membrane architecture.
Treatment of erythrocyte membranes with concentrated salt (1 M-KCI or -NaCI)
before drying has been shown to eliminate the reflexions attributed to residual lipoprotein in the diffraction patterns of the dried membrane preparation (Fig. l), but the
lipid reflexions were strengthened. These observations suggest that the membrane
structure is so modified by the salt that subsequent dehydration produces a complete
breakdown of lipid-protein interactions.
Chapman, D.,Kamat,V.B.,DeGier, J. &Penkett,S. A. (1968) J. Mol. Biol.31,101-114
Coleman, R., Finean, J. B., Knutton, S . & Limbrick, A. R. (1970) Biochim. Biophys. Actu 219,
81-92
Danielli, J. F. & Davson, H. (1935) J. Cell. Comp. Physiol. 5, 495-508
Finean, J. B. & Coleman, R. (1970) FEBS Symp. 20, 9-16
Finean, J. B., Coleman, R., Knutton, S., Limbrick, A. R. & Thompson, J. E. (1968) J. Gen.
Physiol. 5 1 , 1 9 ~ - 2 5 s
Lenard, J. & Singer, S. J. (1966) Proc. Nut. Acud. Sci. US.56, 1828-1835
Singer, S. J. & Nicolson, G. L. (1972) Science 175, 720-731
Tanford, C. (1972) J. Mol. Biol. 67, 59-74
Wallach, D. F. H. & Zahler, P. H. (1966) Proc. Nut. Acad. Sci. U.S. 56, 1552-1559
Magnetic-Resonance Studies of the Molecular Motion of Lipids in
Blayers and Membranes
J. C. METCALFE, N. J. M. BIRDSALL and A. G. LEE
National Institute for Medical Research, Mill Hill, London NW7 IAA, U.K.
Recent magnetic-resonance studies of phospholipids in bilayers and membranes have
emphasized the fluid state of a major part of the structure. The hydrocarbon chains
of the phospholipids form a highly fluid region at the centre of the hydrophobic
interior of many membranes, equivalent to a liquid hydrocarbon medium in its thermal
motion (Hubbell & McConnell, 1968, 1971 ; Levine et al., 1972). In addition, lateral
diffusion of the lipid molecules in bilayers and membranes occurs at a molecular
translocation rate of lo7 steps/s (Devaux & McConnell, 1972; Trauble & Sackmann,
1972). The overall order of the bilayer structure is maintained by orientation of the
Vol. I
330
BIOCHEMICAL SOCIETY TRANSACTIONS
phospholipids with their major axes preferentially directed perpendicular to the surface
of the bilayer, and by a very slow exchange rate of lipids between the two sides of the
bilayer with a half-time of at least several hours (Kornberg & McConnell, 1971).
However, only limited evidence is available about the molecular motion of membrane
proteins. The rotational rate of rhodopsin in the plane of the retinal-rod membrane
indicates a medium of low viscosity (approx. 2P) equivalent to a light oil (Cone, 1972).
Since the viscosity of the immediate environment of the rhodopsin molecules presumably determines their rotational diffusion, this very fluid environment is certainly
consistent with rapid lateral diffusion of the lipids. The biological significance of the
surface dynamics of membrane components is also indicated by the very rapid aggregation of immunoglobulins dispersed on the surface of the lymphocyte, these collecting
at one pole of the cell to form a cap when treated with multivalent anti-immunoglobulin
antibody or multivalent antigens (Taylor et al., 1971).
These observations suggest that static models of membrane structure, in which
spatial relationships between the various membrane components are regarded as
fixed, need to be recast. The dynamic properties of the structure need to be described in
terms of the persistence of interactions between individual components that determine
the short-range order, and the relative motions of groups of components maintaining
any long-range order that may exist.
The application of n.m.r. to structural studies of membranes has been severely
limited by the technical problems of low sensitivity, and of the overlap of many
resonances in broadened envelops of signals from the nuclei present at natural
abundance in the membrane. Until recently no attempts had been made to describe
more than qualitative changes in these broad and relatively featureless spectra from
intact membranes. The keys to detailed structural studies of individual membrane
components and their interactions in the membrane assembly lie in the introduction
of Fourier-transform n.m.r., which increases the effective instrumental sensitivity by
more than an order of magnitude, and in techniques of isotopic substitution to simplify
the spectra to assigned resonances from defined membrane components (Metcalfe
etal., 1972). The 13Cnucleus is particularly suitable, since its natural abundance is only
1.1 % and selective enrichment by up to 90-fold can effectively simplify the spectra
to single resonances from the enriched nuclei. The 13Cnucleus also has the technical
advantage that the line widths of the resonances are much narrower than the corresponding proton (or I9F) resonances in structures with severely restricted motion, and hence
the effective resolution and sensitivity are increased (Metcalfe et al., 1971;Levine et al.,
1972).
Quantitative information about the molecular motion of the lipids is obtained from
relaxation measurements. The pulse techniques used in Fourier-transform n.ni.r. are
readily adapted to measurement of the spin-lattice relaxation time (T,), which can
often be related to rotational correlation times for the individual I3C nuclei in the
lipid molecules. This provides a pattern of internal motions in the molecules that is
sensitive to steric interactions with other membrane components.
In recent n.m.r. studies of lipids in bilayers and membranes we have examined the
I3C spectra of the lipids and shown that their relaxation times are sensitive to the
structural organization of the lipids and to their chemical structure. We have also
shown that the permeability properties of phosphatidylcholine (lecithin) vesicles of
different chain structure can be accounted for in terms of the packing of the chains,
which results in systematic changes in the relaxation times. The effects of cholesterol
on permeability and lipid-chain motion can be correlated in the same way. In separate
experiments with 19F-labelledphosphatidylcholines it was found that highly sonicated
lipids fused with each other with little leak of external solvent into the vesicles.
These findings for lipids in model membranes provide a starting point for comparison
with lipids in biological membranes, and the relaxation times of the lipids in sarcoplasmic-reticulum membranes have been compared with the same lipids in sonicated
vesicles after extraction from the membranes (Robinson et al., 1972). Techniques for
introducing 13C-labelledlipids into membranes by fusion, by biosynthetic incorporation
1973
534th MEETING, NOTTINGHAM
33 1
(Metcalfe et al., 1972) and by reconstitution of functional membrane systems have been
developed, which allow the effects of membrane proteins on the organization of the
lipids to be examined in detail.
Cone, R. A. (1972) Nature (London) New Biol. 236, 39-43
Devaux, P. & McConnell, H. M. (1972) J. Amer. Chem. SOC.
94,4475-4481
Hubbell, W. L. & McConnell, H. M. (1968) Proc. Nut. Acad. Sci. US.61, 12-16
Hubbell, W. L. & McConnell, H. M. (1971) J. Amer. Chem. SOC.
93, 314-326
Kornberg, R . D. & McConnell, H. M. (1971) Biochemistry 10, 1111-1120
Levine, Y .K., Birdsall, N. J. M., Lee,A. G . &Metcalfe,J. C. (1972)Biochemistry 11,1416-1421
Metcalfe, J. C., Birdsall, N. J. M., Feeney, J., Lee, A. G., Levine, Y . K. & Partington, P. (1971)
Nature (London)233,199-201
Metcalfe, J. C., Birdsall, N. J. M. & Lee, A. G. (1972) FEBSLett. 21, 335-340
Robinson, J. D., Birdsall, N. J. M., Lee, A. G. & Metcalfe, J. C. (1972) Biochemistry 11,29032909
Taylor, R. B., Duffus, W. P. H., Raff, M. C. & de Petris, S. (1971) Nature (London) New Biol.
233,225-229
Trauble, H. & Sackmann, E. (1972) J. Amer. Cheni. SOC.
94, 4499-4506
Comparative Permeability Studies on Liposomal and Biological
Membranes with various Lipid Compositions
J. DE GIER
Biochemisch Laboratorium, Rijksuiziversiteit Utrecht, Vondeltaan 26, Utrecht,
The Netherlands
The rapidly increasing interest in liposomes as membrane models clearly indicates the
usefulness of the system. Two experimental forms are widely used. The classical liposomes (Bangham et at., 1965) are a heterogeneous population of multicompartmentalized
structures that are easily obtained by gentle shaking of thin layers of suitable lipids with
a water phase. More recently various investigators (Johnson & Bangham, 1969; Huang,
1969) have used sonicated versions consisting of very small vesicles with one lipid bilayer
membrane only. Permeability experiments on these sonicated liposomes demonstrated
less complicated kinetics, and the data can be related to an approximate surface area
so that permeability constants can be calculated. A disadvantage is that the prolonged
sonication and the subsequent gel filtration allow dangerous peroxidation and hydrolysis
of sensitive lipids. On the other hand the multilayered systems are very suitable for
comparative studies, as by standardization of the dispersion satisfactory reproducibility
can be reached. By careful manipulation of the surface charge comparable liposome
preparations can often be obtained from different lipids.
In our experiments we used the multilayered systems to evaluate relations between
permeability of membranes and the chemical composition of the lipid constituents. In
liposomes the chemical composition could be varied systematically and simple comparative permeability tests could be applied on the different systems. The significance
of conclusions that can be obtained from such model studies is, of course, dependent
on the extensibility of the results to biological interfaces. Therefore we discuss the
experiments on liposomes in direct relation to observations on biological cells with
variable lipid composition.
In Nature chemical variations in structural lipids occur both in the polar head-groups
and in the hydrophobic regions. The importance of the polar groups for the permeability
properties of membranes can be illustrated by discussing the properties of liposomes
prepared from phosphatidylglycerol and lysylphosphatidylglycerol, the two main structural lipids in the membranes of Staphylococcusaureus (Haest et al., 1972~).By dispersion
in ~ O ~ M - KatCpH
I 5.5 comparable multilayered liposome systems with opposite charge
Vol. 1