Nutrition, Digestion, Metabolism: Proceedings of the 28th International Congress of Physiological Sciences, Budapest, 1980

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HORMONAL RECEPTORS IN THE CELL REGULATION OF DIGESTIVE FUNCTIONS

Serge Bonfils,     Unité de Recherches de Gastroentérologie, INSERM U. 10, Hôpital Bichat, F-75877 PARIS CEDEX 18, France

Publisher Summary

This chapter describes hormonal receptors in the cell regulation of digestive functions. The main digestive functions can be activated or inhibited by hormones secreted by the GEP system. Strong evidence has been presented for action of these hormones on: (1) exocrine secretions, (2) motor activities (tonic and propulsive), and (3) function of survival, that is, tissue metabolism and cell renewal. The contribution of receptor studies to the knowledge of digestive physiology is impressive. This can be exemplified in the field of general concepts. The ability to compete for the same receptor makes it possible to explain the common behavior of structurally related hormones (gastrin family, secretin family). The knowledge of receptor structural requirements is a crucial clue in the synthesis of competitive inhibitor such as modified gastrin. Pharmacological and physiological interactions of hormones are better understood taking into account receptor events such as cooperativity and spareness phenomena. Speculation on abnormal digestive functions in pathological conditions could be more fruitful in hypothesizing possible change in the number or the sensitivity of the receptors.

The main digestive functions can be activated or inhibited by hormones secreted by the GEP system. Strong evidence has been presented for action of these hormones on: 1) exocrine secretions (water and electrolytes, proteins and glycoproteins); 2) motor activities (tonic and propulsive), 3) function of survival, i.e. tissue metabolism and cell renewal. Less documented and still controversial are the possible effects on other endocrine secretions and on absorption, mainly by transcellular pathway.

But as indicated in table I, there is so far no strict identities between tissues undergoing hormonal influences and tissues (or cells) evidencing receptor activation and/or inhibition by the same hormones.

HORMONAL DEPENDENCE OF DIGESTIVE FUNCTIONS

Comparison between evidences for hormonal receptor and for physiological actions at the organ level (-: no evidence so far; ?: no direct evidence).

The concept of hormonal receptor is however highly stimulating; it particularly implies the cellular events resulting from hormone-tissue interactions, that could be considered as representative of function(s) (3). This could lead to controversial results when compared with organ physiology, all the more that in experimental approaches concerned with these effects, problems arise from the multiplicity of the GEP hormones that may interfere on a same target organ and the capability for one hormone of triggering simultaneously various physiological activities. A prerequisit for optimizing reliability and reproducibility of studies appears thus to minimize or suppress the other parameters of regulation, i.e. nervous influx, feed-back phenomena driven by the digestive secretions, blood supply.

I FUNCTIONAL MODELS AT THE CELL LEVELS

Ideally, receptor studies should not only encompass binding parameters, but extend to specific functional activities: technically the methods used taken alone, are well defined and rather sophisticated; however, for an ideal demonstration, four criteria should be fulfilled. 1) Homogeneity of the tissue, i.e. constituted of one cell type (eventually equipped with more than one receptor type). 2) Specificity of hormone binding to receptor; this technically needs radiolabed hormones with full biological activities. 3) Characterization and measurement of intracellular messenger activities, in connection with 2 and 4. 4) Determination of the most relevant activity of the cell for assessing the functional response. These criteria which cover the succession of events intervening in the cell regulation of digestive functions are not often simultaneously obtained (table II).

Table II

CRITERIA FOR RECEPTOR STUDIES

Isolated Cell Models

They are largely used as intact and living cells derived from various tissues: stomach fundic mucosa with isolation and separation of parietal and non-parietal cells (8, 13); pancreas acinar cells (3); enterocytes (5). Two of the four above criteria may be parallely obtained, i.e. homogeneity of the tissue and specificity of hormone binding; this specificity, not only derives from the tissue homogeneity but also from the presence of the hormone in its native condition, at a precisely known concentration and without interferences of blood supply and/or nervous influx.

On the other hand, assessing relevant activity(ies) of the cell is often difficult due to the decrease and/or to the change in cell activity when separated from normal tissue structures. Furthermore, often the biological signal is not easily characterized in the survival medium where the cells are tested: for instance acid secretion of the parietal cells can be only indirectly evidenced with the trapping of the C labelled weak basis aminopyrine. Pancreatic acinar cell activity is easier to assess by measuring amylase output. O2 consumption (12) is a usefull tool when the specific functions of the cells have close relation with oxydative metabolism. On the whole, the sensitivity of these models is poor (table III).

Table III

ISOLATED CELL MODELS

Membrane models, such as vesicles, cannot be studied by reference to a specific biological response. Measurement of intracellular messenger activities is usually considered as representative of the biological response, but cannot ascertain the physiological interest of the results. Even if some kinds of biological activity are characterized (e.g. change in intravesicular pH or ion transports), their relevance to physiology may remain disputable.

From tissue or cells one can obtain rough homogenates and further purifications increase the specificity of binding and/or messenger studies. However, the homogeneity of the original material remains the most determinant factor for specific hormone receptor studies.

Isolated cells models have evidenced a number of cell-hormone relationships that could be interpreted in respect to receptor:

- affinity of one hormone for one cell type, considering the presence of one or multiple receptor type(s) (e.g. high and low affinity);

- variety of hormone receptors for one cell type; site to site interactions;

- determination of the number of sites per cell;

- ability for one receptor-type to recognize various molecular forms of one hormone or various hormones;

- internalization of receptors suggesting intracellular hormone transport.

Isolated cell models have been particularly fruitful in the following studies:

- parietal cell receptors (7): binding and stimulation with gastrin (9, 13) and their inhibition by gastrin analogues (NPS gastrin and pentagastrin) (2); binding of somatostatin and subsequent intracellular events (6);

- pancreatic acinar cell receptors: comparison and interferences between secretin and VIP; caerulein activation and inhibition; comparative activity of the various molecular forms of CCK;

- enterocytes: VIP receptor and adenylate cyclase stimulation (5);

- colonic carcinoma cells: priviledged activity of VIP on adennylate cyclase with persistence of functional receptors (11).

Supra cellular models

They are made of group of cells separated from the original tissue; but these cells are keeping connections between them that could favor permanence of specific functional activities, such as ion transport (e.g. gastric H+ secretion) or enzyme secretion (e.g. pancreatic amylase). Current works have used isolated gastric glands (1), pancreatic acini, pancreatic duct fragments, clusters of enterocytes or colonic mucosal cells.

On these models, characterization and measurement of intracellular messenger activities can be simultaneously obtained, but specificity of hormone binding to receptor could be more disputable in respect to cell heterogeneity. For the physiologist, evidence for messenger-metabolism coupling is more significant that for binding-messenger (recognition-activation) coupling.

Conclusively, one can stated that isolated cell models and supra cellular models are complementary: increasing the number of cells, i.e. the size of the sample from the original tissue results in an approach more and more approximating organ physiology. On the other hand, pure homogeneous cell preparation appears to be mainly an extension of biochemical research on membranes. On both extremities of this range, improvement should be obtained from a) chemical isolation of receptors and determination of their location on or in the cells, b) isolated organ preparations allowing to control parameters such as blood flow, nervous influx, veinous arterial gradients of hormones or nutrients.

II FUNCTIONAL MODELS AT THE ORGAN LEVEL

These models have been largely used, in the past in in vivo or ex vivo studies, without refering to receptor theory.

More recently, and particularly under M. I. Grossman’s influence functional responses to GEP hormones have been analyzed according to the Michaelis-Menten enzymatic model. It is generally considered that this model may correctly describe the stimulus-secretion coupling.

However, kinetics of organ responses have been sometimes abusively linearized by mathematical transformations. Contrariwise to the case of receptor models, the hormone concentrations at the cell level is not actually known, blood hormone concentration being not necessarily representative for this parameter. But, in respect to physiological mechanisms, organ studies on the one hand and biochemical studies of receptors on the other hand have provided rather complementary than controversial results (4).

III CONCLUSIONS

The contribution of receptor studies to the knowledge of digestive physiology is impressive. This can be examplified in the field of general concepts: 1) the ability to compete for the same receptor makes it possible to explain the common behaviour of structurally related hormones (gastrin family, secretin family) (10). Furthermore, the knowledge of receptor structural requirements is a crucial clue in the synthesis of competitive inhibitor such as modified (nitro phenyl sulfenyl tryptophan) gastrin; 2) pharmacological and physiological interactions of hormones are better understood taking into account receptor events such as cooperativity and spareness phenomena (3); 3) speculation on abnormal digestive functions in pathological conditions could be more fruitful in hypothesizing possible change in the number or the sensitivity of the receptors. Basic works have documented this hypothesis in colonic carcinoma (11); 4) interferences of systemic hormone supply may be overcome in direct receptor studies and these could moreover inform on the hormone catabolism at the target cell level. These benefits are especially valuable for hormone analogues; 5) a cell response different in nature from that anticipated from organ studies is sometimes observed: e.g. amylase secretion under secretin stimulation of pancreatic acinar cells. A new approach to the potential functional activities of the cell is thus suggested.

However, for the physiologist as well as for the gastroenterologist gathering physiological or pathophysiological information at the organ level, transposition or adaptation of basic science progresses to their own field is a hard work. Many factors are hindering this inavoidable extension. Without numbering all of them, one could stress on 3 major points. 1) There is no common language between men interested in organ functions and those working at the cell level: even more, the same words are often used with a different meaning by both. 2) Pathophysiology is often poorly understood by the basic scientist, because it is associated with a large number of non-controlled parameters: this feature is strongly rejected whatever the value of the remaining information. 3) An organ is not a simple cluster of cells but an association of cells monitored by vessels, nerves, energetic supply, etc.; this association simultaneously assums specific functions (i.e. secretion, transport, ect.) and controls its own cell renewal.

Cell physiology does not account for all these interferences which are included in (and not easily substracted from) organ responses.

REFERENCES

1. Berglindh, T., Obrink, K.J. A method for preparing isolated glandsfrom the rabbit gastric mucosa. Acta Physiol Scand. 1976; 96:150–159.

2. Dubrasquet, M., Ory-Lavollée, L., Van Chuong, Pham P., Morgat, J.L., Fromageot, P., Bonfils, S. Effet inhibiteur d’un analogue de la pentagastrine sur la sécrétion gastrique acide du rat. Gastroenterol Clin Biol. 1980. [(in press)].

3. Gardner, J.D. Receptors for gastrointestinal hormones. Gastroenterology. 1979; 76:202–214.

4. Grossman, M.I. Neural and hormonal regulation of gastrointestinal function: an overview. Annu Rev Physiol. 1979; 41:27–33.

5. Laburthe, M., Besson, J., Hui Bon Hoa, D., Rosselin, G. Récepteurs du peptide intestinal vasoactif (VIP) dans les entérocytes: liaison spécifique et stimulation de l’AMP cyclique. C R Acad Sci (Paris). 1977; 284:D2139–D2142.

6. Lewin, M.J.M. Hormonal receptor control of electrolyte secretion in the gastrointestinal tract. In: Jerzy Glass George B., ed. Gastrointestinal Hormones. New York: Raven Press; 1980:477–504.

7. Lewin, M.J.M. Parietal cell receptors. In: Holtermüller K.H., ed. Pathogenesis and Therapy of Ulcer Disease. Amsterdam: Excerpta Medica, 1980. [(in press)].

8. Lewin, M.J.M., Cheret, A.M., Soumarmon, A., Girodet, J. Méthode pour l’isolement et le tri des cellules de la muqueuse fundique de rat. Biol Gastroenterol (Paris). 1974; 7:139–144.

9. Lewin, M.J.M., Soumarmon, A., Bonfils, S. Gastrin receptor sites in rat gastric mucosa. In: Bonfils S., Fromageot P., Rosselin G., eds. Hormonal Receptors in Digestive Tract Physiology. Amsterdam: Elsevier/North Holland; 1977:379–387.

10. Morley, J.S. Information about peptide hormone receptors from structure activity studies. In: Bonfils S., Fromageot P., Rosselin G., eds. Hormonal Receptors in Digestive Tract Physiology. Amsterdam: Elsevier/North Holland; 1977:3–12.

11. René, E., Vissuzaine, C., Dupont, C., Lewin, M.J.M., Bonfils, S. VIP stimulation of adenylate cyclase in a population of 11 human colorectal adenocarcinomae: evidence for two classes of responsiveness. Gastroenterology. 1980; 78:1243. [(abst)].

12. Soll, A.H. The actions of secretagogues on oxygen uptake by isolated mammalian parietal cells. J Clin Invest. 1978; 61:370–380.

13. Soumarmon, A., Cheret, A.M., Lewin, M.J.M. Localization of gastrin receptors in intact isolated and separated rat fundic cells. Gastroenterology. 1977; 73:900–903.

REVISED CONCEPT OF FUNCTIONAL AND STRUCTURAL ORGANIZATION OF THE ENZYME-TRANSPORT SYSTEMS OF THE APICAL MEMBRANE OF THE ENTEROCYTES

A.M. Ugolev,     I. P. Pavlov Institute of Physiology, The Academy of Sciences of the USSR, Leningrad, USSR

Publisher Summary

This chapter describes revised concept of functional and structural organization of the enzyme-transport systems of the apical membrane of the enterocytes. It highlights that the mechanism of coupling the enzyme and transport systems is a central problem not only of gastroenterology but also of the biology of the cells and membranes. The chapter discusses a study that succeeded in a separation of the apical glycocalyx by the technique of agar replica. The apical glycocalyx is separated from the enterocyte plasmic membrane without disturbing the latter. This fact was confirmed not only electronmicroscopically but also by means of membrane markers. It turned out that the apical glycocalyx is practically free of invertase but contains about 60% of pancreatic amylase adsorbed onto the structures of the intestinal mucosa. Besides, some 85% trypsin and less than 20% chymotrypsin activities were found in the glycocalyx zone. Finally, it was shown that intrinsic intestinal enzymes such as γ-amylase, aminopeptidase, and some dipeptidases are largely bound with the lipoprotein membrane fraction.

Introduction

The mechanism of coupling the enzyme and transport systems is a central problem not only of gastroenterology, but also of the biology of the cells and membranes. Indeed, complex metabolic chains may ultimately be represented as a combination of the (1) transformational and (2) transport links. The means of integration of these links is a third fundamental element of metabolism in the living systems. This problem will be discussed below and several fundamental and accepted concepts (see Fig. 1) will be revised in the light of the new evidence.

Fig. 1 Enterocyte structure and functions. A – structure, B – substance fluxes, C – water flows.

The glycocalyx (7, 20, 22)

. The glycocalyx performs some vital functions such as adhesion, recognition, intercellular interactions, defense, separation of different types of molecules according to their sizes and charge etc.

Until very recently ideas on this mechanism remained uncertain since the glycocalyx was not preparatively separated from the other cellular structures. However, we have recently succeeded in a separation of the apical glycocalyx by the technique of agar replica, proposed by us and schematically represented in Fig. 2. As can be seen, the apical glycocalyx is separated from the enterocyte plasmic membrane without disturbing the latter. This fact was confirmed not only electronmicroscopically, but also by means of membrane markers.

Fig. 2 A scheme of the separation of the apical glycocalyx from the lipoprotein membrane.

The results of our work were quite unexpected. It turned out that the apical glycocalyx is practically free of invertase, but contains about 60% of pancreatic amylase adsorbed onto the structures of the intestinal mucosa. Besides, some 85% trypsin and less than 20% chymotrypsin activities were found in the glycocalyx zone. Finally, it has been shown that intrinsic intestinal enzymes, such as γ‐amylase, aminopeptidase, a some dipeptidases are largely bound with the lipoprotein membrane fraction.

The analysis of the results obtained suggests that a passage of nutrients from the small intestinal lumen to the surface of the lipoprotein membrane of the enterocytes through the glycocalyx space is under the control and selectively facilitated for those substrates, for which in the glycocalyx layer there are corresponding enzymes and, probably, binding proteins. This mechanism effectively prevents the entry of many molecules retaining antigenic and toxic properties to the plasmic membrane.

We have made an attempt to characterize the role of the glycocalyx enzymes and found out that for soluble starch, a desorption of pancreatic amylase from the intestinal mucosal surface causes a 8–10 fold decrease in the uptake of this substrate.

N.N. Iezuitova, P. De Laey and myself have demonstrated that in every segment of the rat small intestine there exists a certain correlation between adsorbed enzymes and the activity of the intrinsic enzyme and transport systems of the lipoprotein membrane. This circumstance gives reasons to suggest a definite relationship between various links of the certain enzyme‐transport chains, in particular, between the functional spectrum of the enzymes and binding proteins of the glycocalyx, on the one hand, and between enzymes, binding proteins and the transport systems of the lipoprotein membrane, on the other hand. This rule must be taken into account to evaluate the effective work of intestinal uptake system and other spatially distributed systems.

The characteristics of the enzyme-transport interactions. Phenomenology (11, 16–19, 21)

Membrane digestion was discovered in 1958. It is accomplished both by the intrinsic intestinal enzymes being an integral part of the lipoprotein membrane and the enzymes adsorbed onto the intestinal mucosal structures.

With a discovery of membrane digestion it became clear that the apical membrane of the enterocytes is a structure where the final stages of hydrolysis and initial stages of transport occurs. The proximity of the final enzymes and the entrances into the transport systems should enhance efficiency of a coupling of these two processes. However, as can be seen from Fig. 3, for monomers (for example, glucose and amino acids) formed during the hydrolysis of di‐ and oligomers the possibility of a loss of substances due to its diffusion through the water phase markedly increases in the region of transition from the enzyme onto the entrance to the transport system. Besides, the transport of enzyme‐released monomers and that of free monomers should be identical in many properties. Finally, the transport of free monomers should be a more rapid process than that of monomers formed during the hydrolysis of oligomers, since in the latter case a dissipation of the reaction products due to diffusion takes place.

Fig. 3 Different types of enzyme‐transport interactions.

In reality, the enzyme‐dependent transport has many, for a long time remained unexplicable, advantages over the transport of free monomers:(1) Monomers formed during oligomer hydrolysis are absorbed more rapidly than free monomers; (2) In contrast to free monomers, the monomers released from membrane hydrolysis do not compete for a common entrance to the transport system; (3) A competition between oligomers and monomers is absent or reduced. At the same time, within each group, a marked competition between different monomers and di‐ and oligomers takes place; (4) During the transport of monomers formed by hydrolysis of oligomers Kt is often less, and V is higher than during the transport of free monomers; (5) The active transport of glucose and amino acids (at least in vitro) is Na+‐dependent process, while the transport of the same substances released during membrane hydrolysis of respective oligomers, in many cases (although not always) is independent on the presence of Na+.

In order to explain all the characteristics of the enzyme‐dependent transport, it is necessary to assume that it is accomplished by another way distinguished from the transport of free monomers. Prom this point of view, the hypothesis on the existence of specific transport systems for di‐ and oligomers seems especially attractive. Our alternative hypothesis postulating the presence of the enzyme‐transport ensembles or complexes on the membrane surface of the intestinal cells appears to be less probable, since such a complex should possess a number of properties seemed, at least initially, unlikely.

However, at present it is known that the catalytic part of membrane enzymes, as shown on the example of carbohydrases, aminopeptidase etc., is projected over the apical membrane and therefore faces the small intestinal lumen.

The molecular structure of membrane enzymes (2, 3, 5, 12)

Under natural conditions, membrane enzymes appear to be oligomers consisting of two, three and even more subunits.

Most intestinal membrane enzymes are transmembrane integral proteins (namely, glycoproteins) and possess amphipatic structure, composed of a hydrophobic and hydrophilic parts. The catalytic sites of the enzymes are concentrated in the hydrophilic head making up more than 90% of the total mass of the enzymes. The remaining part is attributed to the hydrophobic one (molecular mass between 8000–10000 daltons) spanning the phospholipid bilayer of the membrane. In some cases, the hydrophobic part of the enzyme is terminated by a small hydrophilic peptide, exposed on the internal membrane surface.

It has been demonstrated that the hydrophobic part of the enzyme is necessary for realization of the anchor functions. A suggestion has been made that the hydrophobic part of the enzyme may be involved into the transport processes. However, this hypothesis has not yet been experimentally confirmed. We have demonstrated such a vital function of the hydrophobic part of enzymes as a maintenance of the optimal conformation of the hydrophilic catalytic part. This function has been found when comparing the kinetic characteristics of the triton and trypsin forms of carbohydrases and alkaline phosphatase pf the enterocytes. The hydrophobic part tends also to stabilize structure of the enzyme under the action of different factors. Finally, it has been established that the hydrophobic part is of importance for a manifestation of allosteric functions and regulation of the enzyme activity.

Cooperative interactions between the enzyme and transport parts of the complex (8, 9, 12, 21)

The final stages of hydrolysis of most nutrients are accomplished on the external membrane surface and hence, a transmembrane transfer follows hydrolysis rather than preceds it. This conclusion is consistent with data of the kinetic analysis of the transport of free monomers and ones formed by di‐ and oligomer hydrolysis as well as with the fine localization of final enzymes.

In order to describe the evidence obtained by the properties of a single model, an assumption should be made on the existence of the enzyme‐transport complex characterized by the following properties: (1) the hydrolysis products should be transferred directly from the active site of the enzyme onto the entrance to the transport system without dissipation into water phase; (2) the transport system should not interact with the monomers present in the water medium. The only way to explain this, is to postulate that a carrier and the final enzyme form a cooperative system. In this case there should be kinetic signs of such cooperativity. It is true for an exact structure of the enzyme‐transport system irrespective of whether it is (1) the enzyme‐transport system is a constant heterological quaternary complex consisting of particular periodically dissociating subunits, (2) one complex molecule where the hydrophilic part carries out catalytic functions and the hydrophobic part – transport ones, or (3) the transport part represents a channel in an oligomer.

It is known that the allosteric enzymes and cooperative systems are characterized by sigmoid kinetics. On the contrary, the functioning of non‐cooperative systems, including enzymes and carriers, may be described by Michaelis kinetics.

The typical hyperbolic curves have been drawn in our and other laboratories when studying the accumulation of free glucose in the rat jejunal mucosa. The behaviour of highly purified preparations of maltase, γ‐amylase and invertase of the rat intestinal mucosa also fits well Michaelis kinetics.

We have recently suggested and later demonstrated that a pronounced cooperativity of the enzyme‐transport systems is possible only under the conditions of undisturbed interactions between the catalytic and transport parts of the complex. Indeed, in co‐author with E.G. Gurman, E.E. Nurks and G.G. Koltushkina it has been shown that the tissue accumulation of glucose formed during maltose, sucrose and starch hydrolysis in actively functioning intestinal preparations is described by a sigmoid curve. Furthemore, it is important that not only the kinetics of transport but also that of sugar hydrolysis is described by a sigmoid curve if an interaction between the enzyme and transport systems takes place.

An exclusion of the active transport of glucose by a substitution of oxygen for nitrogen in the experiment, the sigmoid curves of carbohydrate hydrolysis are transformed into hyperbolic ones. Of greater interest is a possibility to transform sigmoid curves into hyperbolic ones by a specific blockage of the glucose transport system by phlorizin (Fig. 4).

Fig. 4 Maltose hydrolysis by perfusion of the isolated jejunum segment of the rat in the presence and in absence of phlorizin (chronic experiments). Abscissa – substrate concentrations (in glucose equivalents); ordinate – glucose concentration (in mM).

These results may serve as a direct proof of the existence in the apical membrane of the enterocytes of the enzyme‐transport interactions of allosteric type which are expressed in positive cooperativities.

Enzyme and energetization of transport processes in the enzyme‐transport complexes (4, 9, 14, 21)

It is important that having recognized a positive cooperativity of the enzyme and transport parts of the complex, we can compare the elementary cycles in the enzyme, carrier and the whole complex which we called permeome. It involves an energy source. The permeome hypothesis can account for some physiological processes, such as a transfer of substances through the membrane including their transformation and energetization.

As can be seen, the Equation (1) describes a sequence of states caused by the interaction between an enzyme (E) and a substrate (S) and resulted in a formation of the enzyme‐substrate complex (ES).

In the given case a substrate induces some conformational changes in the enzyme being subjected to reverse effects from the active site. The enzyme‐substrate complex is converted into a next state which is associated with a transformation of a substrate into a product (P) and energy release that may lead to a further change in enzyme conformation.

At a definite stage of transformation of the enzyme‐substrate complex into the enzyme‐product complex an energy is released which may be accumulated fully or partly as the energy of changed enzyme conformation.

The interactions between a transported substance and the ;

Equation (1)

where X – state of substrate in the activated complex. The dashed line with arrow indicates a closing of a complete cycle.

Equation (2)

binding site of a carrier (Car) appear to follow the same laws. However, the active transport being endothermal reaction requires an external energy source.

The Equation (2) describes the hydrolase‐carrier system in which there exist positive cooperative interactions between the enzyme transforming an inpermeable substrate into a transportable product and a carrier which is responsible for the transmembrane transfer of such a product. In this case a substrate interacting with the active site of the enzyme forms a substrate‐enzyme‐carrier complex in which a fit is induced only in the enzyme but not in a carrier. The latter at that moment is uncapable of binding a reaction product, even if it is present in the solution.

While the transformation of initial substrate into a product, the energy released as a result of hydrolysis of intramolecular bonds may be transformed into conformational changes of the enzyme. The latter induces affinity for the product in a binding site of the carrier. It would be logical to characterize it as a transfer of the energy released during hydrolysis, from the enzyme onto the transport system.

EPCar complex is a complex in which the reaction products are transferred from the enzyme onto the entrance to the transport system. This system is unstable and appears to be characterized by a higher energy level both for the enzyme and carrier as compared to the energy level at rest. The reaction product may be transferred onto the internal surface of the membrane either through the channel in a carrier or as a result of changes in its conformation with a formation of ECarP complex which while dissociating produces ECar + P. This enables the system to repeat the cycle (Fig. 5).

Fig. 5 A scheme of sequential conformational interactions of the enzyme and transport parts in functioning of the enzyme-transport complex.

Thus, we have a typical cycle in which a transformation of substance and its transfer across the membrane occur. In this case, the energy released by hydrolysis doesn’t dissipate but is utilized for the functioning of the transport system. It becames understandable why in some instances (although not always) the enzyme‐dependent transport is simultaneously Na+‐independent. In this case, the function of energetization which in the transport systems is performed by a Na+ gradient, in the enzyme‐transport complexes is fulfilled by the energy of hydrolysed bonds. If this suggestion is correct, then the transport of free monomers will depend upon basolateral K+‐Na+‐ATP‐ase to a greater extent than the enzyme‐dependent transport.

The role of oxygenation in enzyme-transport processes and apical respiration (13–15)

The energetization of the active transport in the small intestine, nephron and some other tissues is accomplished by means of ionic gradients produced by K+‐Na+‐ATP‐ase of basolateral membrane. Let us consider the results suggesting a scheme of energetization in which a basolateral mechanism is not unique.

Recently, L.G. Ekkert, A.A. Bagiyan and myself obtained new evidence using a technique schematically shown in Fig. 6.During bilateral nitrogenation as after an administration of oxygen into the serouse lumen of everted sac of the small intestine one could observe only a passive transport of glucose. During mucosal oxygenation the distinctly expressed active transport of glucose takes place. However, bilateral oxygenation practically leads to a doubling of the active transport of glucose as compared to mucosal oxygenation (Fig. 7). Therefore, apart from mucosal mechanism there exists a serouse mechanism, though it is latent and requires induction of the rucosal