M. Tuena de Gómez-Puyou

ENZYMES IN LOW WATER SYSTEMS

Water is fundamental for enzyme action and for formation of the three-dimensional structure of proteins. Hence, it may be assumed that studies on the interplay between water and enzymes can yield insight into enzyme function and formation. This has proven correct, because the numerous studies that have been made on the behavior of water-soluble and membrane enzymes in systems with a low water content (reverse micelles or enzymes suspended in nonpolar organic solvents) have revealed properties of enzymes that are not easily appreciated in aqueous solutions. In the low water systems, it has been possible to probe the relation between solvent and enzyme kinetics, as well as some of the factors that affect enzyme thermostability and catalysis. Furthermore, the studies show that low water environments can be used to stabilize conformers that exhibit unsuspected catalytic properties, as well as intermediates of enzyme function and formation that in aqueous media have relatively short life-times. The structure of enzymes in these unnatural conditions is actively being explored.

Thermostability of membrane enzymes in organic solvents

ATPase Cytochrome oxidase Enzyme stability Organic solvent Thermostability Water content

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252-257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca2+ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca2+-ATP on the association of the ATPase inhibitor peptide to F1-ATPase. This action of Ca2+ may explain why mitochondria utilize respiratory energy for the transport of Ca2+ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.

Electrochemical gradient induced displacement of the natural ATPase inhibitor protein from mitochondrial ATPase as detected by antibodies against the inhibitor protein

Summary Antibodies against the natural ATPase inhibitor protein of bovine heart mitochondria (1) block the inhibitory action of the protein on ATP hydrolysis by soluble or particulate F 1 -ATPase. By immunodiffusion tests, inhibitor protein may be detected in Mg-ATP and State 3 submitochondrial particles. The binding of 121 I-labeled antibodies to submitochondrial particles that possess their ATPase in the inhibited state is several times lower than in particles that had been exposed to electrochemical gradients and which show higher rates of ATPase activity. The results indicate that electrochemical gradients induce a change in position of the inhibitor protein in relation to F 1 -ATPase which results in the appearance of the catalytic properties of the enzyme.

Increased conformational stability of mitochondrial soluble ATPase (F1) by substitution of H2O for D2O

Abstract Between 20 and 40 °C D 2 O inhibits the hydrolytic activity of soluble mitochondrial ATPase F 1 . The effect of D 2 O is proportional to its concentration in the incubation mixture and at nearly 100% D 2 O in the incubation mixture the ATPase activity is inhibited by 50–60%. The effect of D 2 O is mainly on the V of the reaction. At temperatures above 45 °C, D 2 O does not inhibit the activity. D 2 O protects against the denaturation of the enzyme that is observed at relatively high temperatures and against the cold-induced inactivation of F 1 . The intensity of fluorescence of 8-anilino-1-naphthalene sulfonate incubated with F 1 increases as the enzyme becomes inactivated by low temperatures; in D 2 O the changes of fluorescence are almost nil. These observations indicate that H (or D) bonding between the solvent and the protein as well as the strength of the hydrophobic interactions within the enzyme as determined by the solvent are of central importance in determining the overall activity of F 1 and the stability of the enzyme to denaturing conditions. Moreover, the data indicate that the enzyme may exist in two different conformations, each with a characteristic activation energy. It is also proposed that D 2 O may be employed with success in the isolation and purification of labile enzymes.

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

Abstract The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F1 were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F1 preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.

The interaction of mitochondrial F1-ATPase with the natural ATPase inhibitor protein

The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.

On the mechanism of action of alkylguanidines in oxidative phosphorylation: Their action on soluble F1

Abstract Octylguanidine inhibits the adenosine triphosphatase (ATPase) activity of bovine heart submitochondrial particles and soluble F 1 . The characteristics of the inhibition as a function of octylguanidine and Mg 2+ concentrations and pH are very similar in submitochondrial particles and soluble F 1 . Only those guanidines that possess an alkyl chain of more than six carbons inhibit the ATPase activity of submitochondrial particles and F 1 . The inhibiting action of octylguanidine on F 1 is fully reversible. Octylguanidine prevents the cold-induced inactivation of F 1 at concentrations similar to those that inhibit ATPase activity. Guanidines that inhibit ATPase activity also prevent the cold-induced inactivations of F 1 .

On the locus of action of Na+ at site I of oxidative phosphorylation

The effect of octylguanidine on the Na+ stimulated oxygen uptake of rat liver mitochondria and bovine heart submitochondrial particles and on the Na+ induced efflux of K from the mitochondria has been examined. The results indicate that the action of Na+ is inhibited by octylguanidine, but that the degree of inhibition depends on the concentration of the cation. Apparently, a competition exists between Na+ and octylguanidine for a common site. Octylguanidine, but not oligomycin, at certain concentrations restores in mitochondria incubated with Na+ the capacity to respond to uncouplers. A competitive effect between monovalent cations and octylguanidine has been observed in submitochondrial particles oxidizing NADH.

Monovalent cations in mitochondrial oxidative phosphorylation.

The purpose of this paper is to review work that relates to the effect of inorganic and organic monovalent cations on oxidative phosphorylation. The extensive research on the transport of cations across the mi tochondr ia l membrane , which undoubted ly effects oxidative phosphorylation, will not be discussed, except in those cases in which it is not clear whether modifications of oxidative phosphorylation are a consequence of monovalent ion transport or of a direct action of a cation on the reactions that lead to ATP formation.