Biological energy sources: the surface energy and the physical chemistry of water. Examples from studies on muscle contraction.

The physical chemistry of water at nanometre dimensions was used to explain the conformational changes and water breaking properties of the glucose transporter protein (GLUTI) in human erythrocytes more than ten years ago. The energy for this hidden work arises from cycles of evaporation and condensation of water within the cells but was several times larger than resting metabolism. Physical chemical principles can quantify the hidden work done and demonstrate that a significant source of energy is available, which is free of the metabolic energy derived from the hydrolysis of ATP. Therefore, a more widespread biological use of this "free" energy source was probable and a working hypothesis, which applied this energy to supplement the work derived from ATP hydrolysis in muscle, was proposed. The scheme gives a complete explanation for the unexpected and novel findings in skeletal muscle reported from Italy. The problem of using two energy sources and the novel properties of water at nanometer dimensions as they would apply in muscle are briefly discussed but they merit further interdisciplinary studies.

The surface energy of water: the largest but forgotten source of energy in biological systems.

Many functional proteins perform mechanical, structural or chemical work. Such proteins often use the energy from the hydrolysis of adenosine triphosphate (ATP). The role of ATP as an energy source and its production by metabolism was established in the middle of the twentieth century and replaced glycolysis as the focus of study. Before this time the surface energy of water, quantified in the middle of the nineteenth century, had been visualized as an important source of biological energy. Experimental and theoretical work has shown that the internal work done by this energy source may greatly exceed the energy derived from metabolism. Although the energy from ATP usually does the work external to the body, even this may be supplemented by the surface energy of water to give greater efficiency. The consideration of the principles by which proteins might employ this larger source of energy to do work is germane at this time.

The sequestration of hydroxyl ions by C2 in liquid water: useful physiological roles for a reversible complex formation in the presence of protein catalysts.

A second function of carbonic anhydrase (CA) isoforms has already been proposed. This involves the dispersal of complexes in which six carbon dioxide molecules sequester a hydroxyl ion when the gas reacts with liquid water. The semi-catalytic reaction does not require the formation of bicarbonate as an essential corollary. This function is, therefore, a likely activity of carbonic anhydrase related proteins that have recently been discovered and which lack the active zinc site essential for the hydration of carbon dioxide. Re-examination of possible functions for the complex of six CO2 molecules with a hydroxyl anion have brought to light several circumstances where the presence of fully reversible complexes could have physiological advantages. A catalytic synthesis and dissolution of the complexes could thus be the important function for the carbonic anhydrase-related proteins (CA-RP) molecules as well as of some CA isoforms. The possible mechanisms for this extended second catalytic function and examples are briefly discussed.

The sequestration of hydroxyl ions by CO2 in liquid water: the physiological implications and the second function of carbonic anhydrase.

The pH changes due to bubbling CO2 through water produced anomalies which were more readily explained by an hypothesis based on electrostatic attractions between the molecules. The present studies have suggested that an hexagonal array of six carbon dioxide molecules could bind and sequester a hydroxyl anion. The binding energy of the complex is estimated to be comparable with that of a covalent compound and its dissociation may only occur at the water interface with air or at the water/hydrophobic protein interface in a protein cleft. The physiological importance lies in the consequential release of an equal number of free hydrogen ions (H3O+) and the disruption of the normal action of buffer systems in regulating the cytoplasmic pH. The counteraction of this sequestration reaction and the acid-base disturbances which result, form the second important function of carbonic anhydrase isoforms, the mechanisms of which are briefly discussed.

Glucose transport inhibitors protect against 1,2-cyclohexanedione-produced potassium loss from human red blood cells.

It has been suggested that the glucose transport system of human erythrocytes contains an arginine shield to prevent the leak of potassium through the transporter. To investigate this suggestion we treated human erythrocytes with the specific arginine reagent 1,2-cyclohexanedione. Under conditions which produce a covalent reaction between arginine and the reagent, a steady leak of potassium occurs. If glucose, maltose or the inhibitor phloretin are present during the reaction the extent of the leak is reduced. These findings support the view that arginines have a role in preventing potassium loss through the glucose transporter.

The effect of ATP-depletion on the inhibition of glucose exits from human red cells.

The effect of ATP-depletion or its consequence, by metabolic inhibition, on the inhibition of glucose transport by various inhibitors was studied in human red cells. In cells depleted of ATP, glucose exit times were longer than in normal cells and the times increased with the duration of depletion. The Km for external glucose was higher in ATP-depleted cells than in normal undepleted cells (3.0 mM c.f. 2.5 mM at 30 degrees C). In contrast, the apparent Ki for cytochalasin B decreased from 0.85 microM in the normal cells to 0.5 microM after ATP-depletion. Half-maximal rates of glucose exit in the absence, and in the presence of 2 microM cytochalasin B were found at ATP concentrations of 0.43 and 0.68 microM, respectively. Although glucose exits from ATP-depleted cells exposed to the irreversible inhibitor of glucose transport, 1-fluoro-2,4-dinitrobenzene (FDNB) were slower than in normal cells, the relative degrees of inhibition were not significantly different. However, normal and ATP-depleted cells responded differently to treatment with 1,2-cyclohexanedione, a modifier of arginine residues which inhibits glucose exit. While normal cells were markedly inhibited, depleted cells were much less affected and the inhibitory effect of cytochalasin B seen in normal cells was reduced. These findings demonstrate that the glucose transport system of human red cells is affected by intracellular ATP and that ATP alters the affinity of the transporter for certain inhibitors. The implications of these findings are discussed.

The physiological properties of human red cells as derived from kinetic osmotic volume changes.

Volume changes were originally used for studying the dynamic properties of glucose transport in red cells. As an extension it has been found possible to examine the interplay of three functional proteins evolved for the physiological role of human erythrocytes in transporting carbon dioxide and bicarbonate. The proteins chiefly concerned in this investigation were the cytoplasmic carbonic anhydrase and the two membrane transporting proteins, namely the band 3 anion exchanger and the unique bicarbonate transporter, which are distinct from the anion exchanger. The rates of anion membrane transport measured and the volume changes may be more than two orders of magnitude faster than those which regulate cationic movement in red cells, but this may only be an adaptation for the physiological role of red cells. The new concepts derived from the studies and their possible wider applications to physiological mechanisms are briefly discussed.

Temperature dependence of the exchange of monovalent anions in human red blood cells.

The temperature dependence of anion exchange across the red cell membrane was studied between 5 degrees C and 55 degrees C by measuring the rate of shrinkage of cells when transferred from a medium of pH 7.6 to one of pH 9.3 (as measured at 22 degrees C). The rates of shrinkage varied with the anion studied, the order being F-> Cl-> Br-> I-> SCN- but were faster in the presence of trace amounts of carbon dioxide than in its absence. NO3- was as fast as Cl- when carbon dioxide was present but comparable with I- when it was removed. Arrhenius plots of the rates were linear for all anions over the temperature range studied and gave the following apparent activation energies in kJ mol-1; F-, 67.7; NO3-, 68.4; Cl-, 70.2; Br-, 79.6; SCN-, 87.4 and I-, 95.1 in the presence of carbon dioxide. Inhibition of carbonic anhydrase with 5 microns ethoxzolamide and the removal of the carbon dioxide by degassing raised the activation energies to; F-, 124.8; NO3-, 129.0; Cl-, 141.5: Br-, 159.4; SCN-, 150.0 and I-, 185.6 kJ. mol-1. With the exception of F-, the apparent activation energies of the anions were linearly related to their thermochemical (dehydrated) radii in both cases. The relationship between the ionic radii and the energy of transfer suggests that anion exchange involves transfer through a hydrophobic pathway and that additional energy is required to overcome restrictions experienced in passing through the pathway. It is proposed that this, rather than a conformational change in the protein determines the activation energy of the process.

The triphasic volume response of human red cells in low ionic strength media: demonstration of a special bicarbonate transport.

When human red cells are first placed in isotonic media of low ionic strength there is a triphasic volume change. The initial shrinkage (phase A) starting immediately is followed by a rapid reflation (phase B). The final shrinkage (phase C) is slower and has more variable properties. The first two phases need Cl- and HCO3- and they have been shown to involve both the band 3 anion exchanger and the red cell carbonic anhydrase. Phase C is only seen when phase B is present. Both phase B and phase C were accelerated by the lower monohydric alcohols. With methanol the acceleration was maximal near 2M, thereafter inhibition developed. The hypothesis is advanced that phase C involves a permeability for bicarbonate which is independent of the band 3 anion exchanger, and which may be a dimer of carbonic anhydrase. The unique kinetics, the physiological significance, and implications of the special bicarbonate transport system are discussed.

Effect of carbon dioxide on the temperature dependence of anion exchange in human red cells.

Anion exchange across the red cell membrane was studied by measuring the rate of shrinkage of cells when transferred from a medium of low pH to one of higher pH. Removal of trace amounts of CO2/bicarbonate from the media by degassing and the inhibition of carbonic anhydrase with 5 microM ethoxzolamide slowed the shrinkage rate. Arrhenius plots were linear over a temperature range of 40 degrees C, both in the presence of trace amounts of CO2/bicarbonate and in their absence, and gave an apparent activation energy for Cl- exchange of 73.9 +/- 8.9 kJ mole-1 when CO2 was present but this increased to 135.8 +/- 7.7 kJ mole-1 in its absence. From the results it is concluded that the lower value for the activation energy is determined by the rate of formation of bicarbonate ions while the high value represents hydroxyl:anion exchange and is a truer measure of the activation energy of the exchange process.

Clonal chromosome aberrations in a case of nodular fasciitis.

We report a case of nodular fasciitis with clonal chromosome aberrations including a reciprocal t(3;15)(q21;q22) and interstitial deletion (13)(q14q21).

The acceleration of pH volume changes in human red cells by bicarbonate and the role of carbonic anhydrase.

The red cell shrinkage rate due to bicarbonate in media of high pH (ca 9.4) has been compared with the hydroxyl shrinkage rate on a per mM basis. The shrinkage rate due to bicarbonate was only half that due to OH-/Cl- exchanges. It was therefore deduced that the Jacobs-Stewart cycle was limited by the carbonic anhydrase step and not by the rate of transport on the anion exchanger protein. To explain this and other anomalies the hypothesis is made that carbonic anhydrase has evolved as a pH-dependent catalyst with specific physiological functions in pH regulation and in other cellular mechanisms. The kinetic theory and some physiological implications of the hypothesis are discussed.

The physiological properties of the band 3 anion exchanger as derived from pH and other studies with human red cells.

The kinetics of the erythrocyte anion exchanger which have been studied at high pH values have confirmed the importance of hydroxyl ions in the overall physiological roles of the band 3 protein in human red cells. Although usually characterised as a one-for-one anion exchanger, conditions have been identified which give the appearance of net transport. The present findings have uncovered new functional features consistent with the properties of anion transport in other tissues. The molecular models proposed to interpret the results could have wider applications to other cellular mechanisms. Some of these are discussed to illustrate the physiological implications which can be deduced from the novel kinetics of this unique membrane transport protein.

An hypothesis for a contractile protein based on the principles of submicroscopic physiology.

The discovery of the polyguanidinium ring complex and its various properties has emphasised the versatility of arginines in functional proteins. In an extension of these studies the possibility of formulating a polypeptide which could be the basis for a contractile element in muscle presented itself. Although hypothetical, the deduced molecular properties, based on the unique chemistry of arginine guanido groups are in keeping with known functional aspects of some muscles, and could form the foundation for a more detailed theory of muscular contraction. The structural requirements and the theoretical principles involved are briefly described in this paper.

The pH volume changes of human red cells in vitro due to the exchange of chloride and hydroxyl anions.

In glucose exit experiments measured photoelectrically, the excursions on the chart recorder were found to be larger for exits in media of alkaline pH. This was shown to be due to the addition of a pH volume effect to that of the osmotic shrinkage resulting from the glucose efflux. The pH-dependent volume change also occurred in glucose-free cells and was a linear function of the pH of the medium between pH 6.8 and 9.0. The effect is consistent with the loss (or gain) of chloride in exchange for hydroxyl anions on the band 3 anion transporter and with the buffering of the hydroxyl anions by haemoglobin. The implications for the working of the anion exchanger and for respiratory physiology are discussed.

Infantile myofibromatosis.

Infantile myofibromatosis is part of a heterogeneous group of rare childhood fibromatoses characterized by the proliferation of myofibroblasts. It is not a common condition and is frequently misdiagnosed. We present an unusual patient who had small, depressed, atrophic, skin lesions uncharacteristic of infantile myofibromatosis.

The polyguanidinium complex of the glucose transporter: a possible basis for bistable cationic gates and anionic channels in biology.

The detailed molecular structure and mechanism of action of the red cell glucose transporter would endow it with the ability to transfer the less hydrated potassium ion. This is prevented by a polyguanidinium-ring-complex cation which provides an effective electrical energy barrier. The ring complex has structural features which could form a bistable state of ring charges; in one form the electrical repulsions would be fully effective (gate 'shut'), whereas in the other form they would be ineffective (gate 'open'). It is also suggested that the polyguanidinium linkages could form a linear complex which could be the basis for anionic channels in biology. The theoretical basis for both these hypotheses is described.

The role of the surface energy of water in the conformational changes of the human erythrocyte glucose transporter.

There is now strong experimental evidence that the red cell glucose transporter protein operates by a conformational change which has the effect of presenting a sugar binding site to the outside and inside medium of the cells in an alternating manner, but the way in which this is brought about is still unknown. Kinetic evidence that the conformational changes which create the inward and outward facing modes can occur in the absence of a sugar substrate has been put on a firmer experimental basis by Appleman and Lienhard (1989). Conformational changes of the magnitude envisaged are too large to be attributed only to spontaneous thermal agitation in the protein. Theoretical considerations show that the surface energy of water could be an alternative source of energy which, in a suitable reciprocating cycle of activity, could sustain the conformational changes throughout the existence of the transporter.