The phenomenon of electro-optic orientation was discovered by John Kerr in 1875 and has been used extensively for determining the optical polarizability anisotropy of small molecules and for high-speed transmission of optical signals. Measurements on biopolymers have been made at least since 1950, but only in the last decade have these yielded definitive structural and physical information. In the course of this review, it should become obvious that among the reasons for this late development is the inherent difficulty of analysing optical data that depend simultaneously on intrinsic optical-structural properties of the molecules, and on their degree of orientation under the conditions of the experiment. The problem has been particularly difficult far biopolymers such as the nucleic acids, whose polarization in an electric field is dependent on their special polyelectrolyte properties. These unique electrostatic properties are an important feature in the interpretation of the experimental observations.

Molybednum-containing enzymes (Coughlan, 1980; Spiro, 1985) occupy a significant place in the development of the field now termed inorganic biochemistry. The importance of the metal as a biological trace element depends on its involvement in the known, and perhaps other as yet unknown, molybdoenzymes. That it plays a role in biological nitrogen fixation, the process whereby the enzyme nitrogenase in the root nodules of plants converts atmospheric nitrogen into ammonia, was recognized in the 1930s. The metal is also a constituent of a variety of other enzymes, having first been found in a mammalian enzyme, xanthine oxidase, in the 1950s.

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

Cryo-electron microscopy of vitrified specimens was just emerging as a practical method when Richard Henderson proposed that we should teach an EMBO course on the new technique. The request seemed to come too early because at that moment the method looked more like a laboratory game than a useful tool. However, during the months which ellapsed before the start of the course, several of the major difficulties associated with electron microscopy of vitrified specimens found surprisingly elegant solutions or simply became non-existent. The course could therefore take place under favourable circumstances in the summer of 1983. It was repeated the following years and cryo-electron microscopy spread rapidly. Since that time, water, which was once the arch enemy of all electronmicroscopists, became what it always was in nature – an integral part of biological matter and a beautiful substance.

The major elements of membranes, such as proteins, lipids and polysaccharides, are in dynamic interaction with each other (Alberts et al. 1983). Protein diffusion in the lipid matrix of the membrane, the lipid diffusion and dynamic domain formation below and above their transition temperature from gel to fluid state, have many functional implications. This type of behaviour of membranes is often summarized in one frequently used word membrane fluidity (coined by Shinitzky & Henkart, 1979). The dynamic behaviour of the cell membrane includes rotational, translational and segmental movements of membrane elements (or their domain-like associations) in the plane of, and perpendicular to the membrane. The ever changing proximity relationships form a dynamic pattern of lipids, proteins and saccharide moieties and are usually described as ‘cell-surface dynamics’ (Damjanovich et al. 1981). The knowledge about the above defined behaviour originates from experiments performed mostly on cytoplasmic membranes of eukaryotic cells. Nevertheless numerous data are available also on the mitochondrial and nuclear membranes, as well as endo (sarco-)plasmic reticulum (Martonosi, 1982; Slater, 1981; Siekevitz, 1981).

Ice formation in aqueous solutions and suspensions involves a number of significant changes and processes in the residual liquid. The resulting effects were described concerning the redistribution of dissolved salts, the behaviour of gaseous solutes and bubble formation, the rejection and entrapment of secondphase particles. This set of conditions is also experienced by biological cells subjected to freezing. The influences of ice formation in that respect and their relevance for cryopreservation were considered as well.

A model of transient heat conduction and solute diffusion with a planar ice front, propagating through a system of finite length was found to be in good agreement with measured salt concentration profiles. The spacing of the subsequently developing columnar solidification pattern was of the same order of magnitude as the pertubation wavelengths predicted from the stability criterion. Non-planar solidification of binary salt solutions was described by a pure heat transfer model under the assumption of local thermodynamic equilibrium.

The rejection of gaseous solutes and the resulting gas concentration profile ahead of a planar ice front has been estimated by means of a test bubble method, yielding a distribution coefficient of 0·05 for oxygen. The nucleation of gas bubbles has been observed to occur at slightly less than 20-fold supersaturation. The subsequent radial growth of the bubbles obeys a square-root time dependence as expected from a diffusion controlled model until the still expanding bubbles become engulfed by the advancing ice-liquid interface. The maximum bubble radii decrease for increasing ice front velocities.

The transition between repulsion and entrapment of spherical latex particles by an advancing planar ice-front has been characterized by a critical value of the velocity of the solidification interface. The critical velocity is inversely proportional to the particle radius as suggested by models assuming an undisturbed ice front. The increase of the critical velocity for increasing thermal gradients shows good agreement with a theoretically predicted square-root type of dependence. Critical velocities have also been measured for yeast and red blood cells.

The effect of freezing on biological cells has been analyzed for human lymphocytes and erythrocytes. The reduction of cell volume observed during non-planar freezing agrees reasonably well with shrinkage curves calculated from a water transport model. The probability of intracellular ice formation has been characterized by threshold cooling rates above which the amount of water remaining within the cell is sufficient for crystallization. The cooling rate dependence of viability exhibits largely different maxima for lymphocytes and erythrocytes at about 30 and 4700 K/min, respectively. The decrease of viability above these values has been attributed to the damaging effect of intracellular ice formation.

This paper consists of three parts. The first describes theoretically a general strategy for fragmentation and separation of membranes which can be used in the elucidation of their structure and function. The second part describes a practical separation method, partition in liquid aqueous polymer two-phase systems, which can be used for separation of macromolecules and membrane particles of biological origin. The third part gives examples of the application of this method to membrane vesicles, and how this separation in combination with the strategy described in the first part can be used for analysis of the structure of biological membranes.

Biological cells are defined by the membrane that shields their vital molecules from the environment. The lipid bilayer of the membrane is an effective dielectric shield (Parsegian, 1969; Andersen, 1978; Honig et al. 1986), preventing penetration by charged molecules: the lipid presents a large electrostatic energy barrier because it cannot neutralize the charge of solute molecules nearly as well as water. This energy barrier inhibits the permeation of solutes with local charge, even metabolites. Of course, metabolites do enter cells, and so physiologists have suspected (for a very long time, Hille, 1984, ch. 8) that the membrane shield is pierced by aqueous channels, through which solutes diffuse (with their local charge substantially neutralized) as they cross the membrane. These aqueous pores now have molecular reality (e.g. Noda et al. 1984; Miller, 1986). Each is formed by a specialized protein, integral to membranes, perhaps shaped like a thick-walled pipe, called ionic channels. Channels control the movement of many important molecules in and out of cells by the ‘gating’ mechanism that controls their opening and closing and by the selective properties of their open channel.

Excitation and conduction in the majority of excitable cells, as originally described in the squid axon, are initiated by a transient and highly selective increase of the membrane Na conductance, which allows this ion to move passively down its electrochemical gradient (Hodgkin & Katz, 1949; Hodgkin & Huxley, 1952). The term ‘Na channel’ was introduced to describe the mechanism involved in this conductance change (Hodgkin & Keynes, 1955).

The brain is one of the most highly organized structures in the known universe. It is a biological computer which has evolved over a billion years to program, monitor and control all bodily functions. It is also the organ of knowing, feeling and thinking. To understand how the brain works is perhaps the most difficult of all scientific problems. A scientific theory of the brain would provide a comprehensive understanding of a substantial body of facts on the basis of a few fundamental principles. In this sense, there is no theory of the brain.