Tbilisi, Metsniereba, 1993, pp 229

ABSTRACT: The book represents an analytical review of the world literature about the monomolecular technique and own experimental findings on the changes in protein and lipid physico-chemical properties under the influence of biologically active substances, hard metals, ionizing X-ray, etc. The book provides theoretical bases of an effective use of monomolecular technique in medico-biological investigations and modeling of biological membrane by means of Lengmuir-Adams thin layers, formed on the plane surface of division phase of liquid-air and liquid-liquid as well as on the spheric one - drops and bubbles of different types are given. The book is of interest to membranologists and other specialists studying the action mechanisms of various physical and chemical factors on the biological objects.

SUMMARY

The book is the authors’ endeavour to make a generalization of available data in the field of biology and medicine obtained by the so-called monolayers methods.It is an attempt to show that the formation and development of methodology of mono-molecular technique and its practical application in various fields of science are linked with theoretical and applied problems of superficial interaction or interaction on the level of phase boundary being a fundamental vital process. It has been noted in introduction that the investigations related to study of the structure and function of biological membranes are at present of primary importance in the world science; this tendency distinctly manifests itself at the treatment of information stream accumulated for the last quarter of this century. Methodical arsenal employed for this reason appears to be exclusively diverse and reflects a general tendency to the development of biological and medical science as a whole. One of the most common approaches used in these investigations is modelling of the structure and function of biological membranes, which enables analysis of the cell viability as a process of structural and functional interaction of membranes having a diverse level of biological organization from subcellular to integral organism, including intercellular interaction and interaction with the enviroment.

The first model of a biological membrane is a thin lipid film formed on the surface of liquid. In spite of an extremely sketchy character of this model which is far from the native structure by both the shape and essence, when used together with lipids and proteins, it proved to be a fairly efficient approach in the evolvement of physico-chemical biology and in the development of membranology, in particular. This has been promoted also by the possibility of formation of monomolecular layers of biomolecules not only on the plane surfaces but also on spheric ones, drops and vesicles of various types that are contiguous with the air embedded in organic or inorganic solutions.

The present-day achievements in the field of the structure and function of biological membranes in the in situ and in vivo studies are so significant that some experts question a further efficiency to use monomolecular technique in modelling or resolving any novel problem in membranology. This is bound, primarily, with that the conformational state of functional protein molecules in the cellular membrane and monolayer is principally diverse: in the first case the entire ierarchy of three dimensional structural organization of a protein molecule is maintained, whereas in monolayer there occurs the so-called superficial denaturation and the molecule is "untfolded", being actually in two-dimensional state. The last notice is mainly true for the protein molecules, when on the liquid-air phase boundary they undergo conformational changes with the hydrophobic nucleus of the molecule going out into the air medium. However, when an investigator wishes to preserve the structure of proteins near to the native state in monolayer the initial pressure of film is to be taken below or equal to 5 mN/m.

According to the sources available in the literature, some molecules are in the unfolded state on the surface of a living cell either as a temporary pattern for synthesis,or permanently, for example, as lipolytic enzymes whose activity is associated just with such a conformational state. It is also known that the process of breathing in the lungs proceeds on the air-liquid phase boundary. Therefore, it is reasonable to use the method of monolayer formed on the surface of pulsatile vesicle as an adequate model for superficial processes occurring in alveoli of the lungs. Yet there are some other open questions and principal controversies in evaluating the monomolecular technique. Nevertheless, the method of monolayers is unique in that it affords to solve a number of questions related to the structure and function of both the fragments of native membranes (microsomes, erythrocyte ghosts) and of individual molecular components that are inaccessible when employing other methods.

The very process of unfolding of molecules on the surface on the phase boundary and formation of monomolecular film, as well as the process of renaturation of unfolded in monolayer biomolecules appears to be an effective "functional" test for estimation of a number of physico-chemical parameters of biological molecules, including thermodynamic ones, as dependent upon external conditions for the formation of film or conformational changes in the structure of the molecule itself resulting from the influence of various physical and chemical factors or at superficial interaction with the already formed monolayers. This is a very important aspect of the use of monomolecular technique,since all biological processes are associated with the superficial interaction on the phase boundary beginning with the hydration cover of individual molecules to complex membranes performing a barrier function,including the polycellular ones.

Particular interest deserve investigations of main and minor muscular proteins as being most universal ones. Contractile-like proteins are encountered in many types of cells of nonmuscular origin and play an important role in their functioning. These proteins are similar to those of a muscular cell by the amino acid content, molecular weight, enzyme activity, capacity of some of them to associate with each other and again dissociate as dependent on the processes occurring in living cells. Thus, for example, in nerve tissue they are involved in axocurrent, in the secretion of transmitters in the synaptic terminals, contraction of glial cells, etc. In addition to these proteins, for the sake of comparative analysis of the results obtained by other methods, we have used a wide spectrum of serum molecules and a veriety of enzymes. As a lipid component, a comprehensive study was made of cholesterin, playing a crucial role in the maintenance of the so-called hydrocrystal state of the cell surface, as well as of phosphatidylcholine (lecithin) and phosphatidylethanolamine (PhEA), which play an important role in the process of functional renovation and involution of the membrane structure in the process of aging.

In chapter 2 dealing with the general principles of monomolecular technique there is a confirmation of the hypothesis on a special order of disposition of polar and nonpolar groups of proteins on the phase boundary resulting in the rise of electrical potentials.It has been experimentally proved that during the formation of a monomolecular layer the compound molecules like actomyosin and fibrillar actin are, as a rule, dissociated into subunits. Moreover, the monomolecular technique enabled to decipher the mechanism responsible for the diffusion of protein molecules through the lipid layer of membranes. Experiments carried out on the phase boundary of liquid-liquid enabled us to trace a direct transfer of chemical energy into mechanical occurring in a living cell as well.The theoretical prerequisites and mathematical apparatus, considered in chapter 3, lend support to the conclusion that with the use of a complex of monomolecular techniques it is possible to make quite correct quantitative measurements of basic physico-chemical parameters as in a monolayer as a whole, so in individual molecules being in its composition, to determine their elastic-viscous and thermodynamic characteristics. Presented in this chapter equations describe the state of molecules in a film with the help of which the molecular mass, molecule shape, the degree of elasticity of the whole polypeptide chain of protein, the compressibility coefficient, total thickness of the film, etc. are determined. Considered in the same chapter are also the causes of hysteresis loop formation in the process of compression and discompression of the monolayer and possibilities of calculation with the use of this approach of the values of the energy absorbed or released in the compression-expansion cycle of the monomolecular layer. This provides a thorough description of a wide spectrum of properties of monomolecular films and their components, revelation of some earlier unknown mechanisms of the cell membrane functioning in both the animal and plant world. An attempt to reconstruct the functional membrane system from the enzyme galactose and phospholipid has shown that the molecular disposition of components in a monolayer is identical to their disposition in the biomembrane. Such a reconstruction of the entire system requires a special order of addition of separate components. For example, lipopolysacharide penetrating into the lipid monolayer fails to permeat the lipid-enzyme layer. Thus, the enzyme blocks the access of lipopolysacharide (LPS) to the binding site of phospholipid monolayer. In the interaction of the enzyme with LPS+PhEA Mg ions, facilitating the binding of the enzyme with the substrate (LPS), play an important role. They act as a ligand inducing a comformational change of an enzyme or components of the binary system.Thus, it has been demonstrated that the reconstruction of three molecular layers of the functional membrane system is possible by the monomolecular technique and what is more, the enzyme maintains its activity. In the next chapter (4) are presented the results of experimental studies of properties of monolayer of proteins, lipids and their mixtures both on the boundary of division of phases liquid-air and liquid-liquid. The peculiarities of the formation and conformational changes in the proteins of blood, muscular cell, lipids and their mixtures are considered. It has been shown that cholesterine has a condensing role in monolayers and this may play a certain role in regulating the permeability of the cellular membranes. A considerable difference has been revealed in the values of energy of biomolecule adsorption in monolayer depending on the type of binary system lipid-protein, which may vary within the ranges of 34 to 34,2 cal/mol. The protein diffusion velocity in monolayers of various lipids was shown to depend upon the values of electrical charge of the latter,experimentally measured by the radionuclide plutonium electrode or the method of so called electrodynamic condenser. In addition to the kinetic and energetic parameters of the protein- lipid interactions, the number of nonpolar aminoacids diffused in the lipid monolayer was calculated. The possibility was proved to reproduce by means of monomolecular technique the immunological reactions of the phospholipid-antibody type.

Physiologically active substances were shown to have an identical effect on the cell of living tissue and biomolecule monolayer (chapter 5). This served as a basis for a broad-scale investigation of the mechanisms subserving the processes ongoing in monolayers of proteins and lipids in the presence of anesthetics of the cocaine group. This contributed to the understanding of the role of separate components of biomembranes while using narcotic or other drugs. Studies with polyene antibiotics revealed that they act but on the membrane of cells containing cholesterine. The following peculiarities of the action of physiologically active substances on various components of membrane were also revealed: 1) antagonistic effect of acetylcholine and tubocurarine on proteins and lipids; 2) possibility of penetration of inflammation enzymes deep into the cells and tissues including the eye crystal; 3) a different effect of microelements on the properties of monolayers of albumins and muscular proteins. Many conclusions drawn on the basis of studies on the effect of physiologically active substances on monolayers of proteins and lipids were confirmed by electron microscopical and microcalorimetric studies.

Chapter VI deals largely with original factual material, indicating that the exposure of radiation at long intervals of doses and dose power causes changes in the area, viscosity and molecular weight of proteins. Based on these findings hypotheses are proposed on the mechanism of realization of radiobiological effect.Study has been also made of the effect of radiation on the protein-lipid interaction. A protecting role of some classic radioprotectors has been shown on the example of molecules of BSA and phosphatidylcholine. It has been demonstrated, for example, that mexamin at a definite concentration and dose of radiation is able to completely restore virtually all the parameters of biomolecules, both of proteins and lipids determined by the method of monolayer. Dynamic changes were shown in physico-chemical properties of monolayers of complex objects - microsomes of the rat brain as a function of radiation dose and concentration of adenylic acids administered to the animals prior to radiation, as also in the process of postradiation observation from 1 h to the animals' death. A hypothesis is proposed about the postradiation course of changes in the microsomes. Many a classic commonly accepted radiobiological statement established by other methods have been confirmed. This permits to recommend the method of monolayer for screening radioprotectors and a wide spectrum of pharmacological drugs.

The materials considered in the present book support the conclusion that the monomolecular technique affords not only to investigate the problems of membranology, including radiobiology of cellular membranes, but also to study a wide spectrum of physico-chemical processes related to the structural changes in biomolecules. At the same time, the structural reorganization of molecules when they are unfolding on the phase boundary may mimick the fundamental biological processes, since the processes of vital importance are realized namely on the boundary of phases at various levels of molecular organization.