Albert L. Lehninger

The enzymic oxidation of d- and l-β-hydroxybutyrate☆

Both dextrorotatory and levorotatory forms of β-hydroxybutyric acid are oxidized aerobically by suspensions of rat liver and kidney particles (mitochondria). However the data clearly indicate that the initial stages of oxidation of the two isomers are quite different in liver, although ultimately both isomers are oxidized via the tricarboxylic acid cycle. n nThe enzymic mechanisms involved in the primary dehydrogenation of the two isomers were examined in clear extracts of acetone-dried mitochondria. It was found that the l-isomer causes the reduction of DPN+, presumably by action of the already known l-specific DPN-linked β-hydroxybutyric dyhydrogenase, which of course does not attack the d-isomer. The d-isomer also causes reduction of DPN+ but only if the extracts are supplemented with ATP, Coenzyme A, and Mg++. Evidence is presented that d-BOH is capable of forming a CoA derivative at the expense of ATP: d-β-hydroxybutyrate + CoA ⤥ ATP d-β-hydroxybutyryl-CoA The extracts contain a dehydrogenase catalyzing reversibly the reduction of DPN+ by d-β-hydroxybutyryl-CoAd-β-hydroxybutyryl-CoA + DPN+ ⤥ acetoacetyl-CoA + DPNH + H+. Reaction (1) is not stereochemically specific; both isomers form a CoA derivative. However the dehydrogenase catalyzing reaction (2) appears to be specific for d-β-hydroxybutyryl-CoA. n nThe metabolic significance of the pathways taken by the two isomers of β-hydroxybutyrate are briefly discussed.

The role of aldonolactonase in the conversion of L-gulonate to L-ascorbate

Abstract Aldonolactonase of the soluble fraction of rat liver has been purified 110-fold in a yield of 34% by isoelectric precipitation and heat treatment in the presence of Mn ++ , fractionation with acetone, and finally chromatography on carboxymethylcellulose columns. During the purification of the enzyme, the aldonolactonase activity and the activity in stimulating conversion of L -gulonate to L -ascorbate by the L -gulonolactone oxidase of rat liver microsomes accompanied each other in an essentially constant ratio. The aldonolactonase thus was identified as catalyzing the lactonization of L -gulonate (reaction a) prior to the oxidation of the lactone (reaction b): L -gulonate ⇆ L -gulonolactone + H 2 O (a) L -gulonolactone + 1 2 O 2 → L -ascorbate + H 2 O (b) Aldonolactonase was shown to catalyze the net accumulation of equilibrium quantities of lactone from free L -gulonate. The tissue distribution and substrate specificity of the enzyme are discussed in relation to the ability of various species to synthesize L -ascorbic acid.

Functional equivalence of monomeric (shark) and dimeric (bovine) cytochrome c oxidase.

Cytochrome c oxidase isolated from hammerhead shark red muscle is monomeric in relation to the dimeric form of isolated bovine cytochrome c oxidase but in other ways bears a close resemblance to the enzyme isolated from mammalian tissue [1, 2]. Comparative studies of shark and bovine cytochrome c oxidase were extended to address the degree of functional similarity between the monomeric (shark) and dimeric (bovine) enzymes in the kinetics of peroxide binding and in the extent to which the catalytic action of the enzymes in vesicles can establish a proton gradient. Although the kinetics of peroxide binding and the proton pumping processes are complex, the dimeric and monomeric forms are quite similar with respect to these functional attributes. The kinetic heterogeneity of the process of peroxide binding is expressed in the shark enzyme as well as in the bovine enzyme, and both types of enzymes in vesicles can generate transmembrane proton gradients. On this basis we conclude that the dimeric state of isolated cytochrome c oxidase from mammalian sources is not essential for its function in vitro.

Effect of temperature on uptake and extrusion of water by isolated rat-liver mitochondria.

Abstract Respiration-dependent swelling of rat-liver mitochondria induced by phosphate, thyroxine, or reduced or oxidized glutathione has a rather high temperature coefficient, in the range 3.2–4.6. On the other hand, swelling induced by oleate, hypotonicity of the medium or that occurring spontaneously proceeds approximately as rapidly at 0° as at 21°. The latter types of swelling are not substantially inhibited by cyanide. Water uptake produced by the first group of agents is thus dependent on a chemical or enzymic event as the rate-limiting step, whereas a physical event, probably diffusion, is rate-limiting for the second type of water uptake. Reversal of mitochondrial swelling by ATP was also found to be dependent on temperature, in accordance with the fact that inhibitors of oxidative phosphorylation such as azide and oligomycin can block reversal by ATP. On the other hand, the reversal of swelling by making the medium hypertonic with sucrose proceeds as rapidly and completely at 0° as 21°; the rate of strictly osmotic reversal thus is probably dependent on a physical process such as diffusion. These findings provide additional evidence for the existence of two distinct modes of mitochondrial water uptake and extrusion: “passive” volume changes caused by changes in osmolality of the medium and “active” or respiration-dependent changes coupled to enzymic reactions.

THE ROLE OF ALDONOLACTONASE IN THE BIOSYNTHESIS OF L‐ASCORBIC ACID*

The three enzymes that catalyze the reactions shown above have been separated from one another and their properties studied. Our detailed studies of purified pig kidney TPN-L-gulonate dehydrogenase,? which catalyzes Reaction 1, have shown that the enzyme reduces only the glucuronate anion but does not reduce glucuronolactone or UDP-glucuronate. On the other hand, Mano el aZ.* claim that the liver dehydrogenase reduces glucuronolactone more readily than glucuronate even though the relative rates of oxidation of gulonate and gulonolactone by the two enzymes are identical. These differences appear to be due probably to an actual difference in specificity of the two enzymes, depending on the source, the manner in which the glucuronates used as substrates were prepared from glucuronolactone, or on the ratio of the rates of the nonenzymatic hydrolysis of the lactone to the rate of the dehydrogenase reaction, and to the pH. The failure of the enzyme to reduce UDP-glucuronate as well as other lines of evidence, such as the inability of preformed glucuronolactone or glucuronate to give rise to glucuronides in intact the failure to detect the UDP-glucuronate pyrophosphorylase reaction in animal tissues,I2 and the finding that in intact animals glucuronolactone is a more efficient precursor of ascorbate than glucose, exclude a pathway of ascorbate formation from UDP-glucuronate involving uridine nucleotide derivatives. Washed rat liver microsomes contain L-gulonolactone oxidase, which catalyzes Reaction 3, as first noted by Burns el aZ.I4 Ascorbic acid formed from labeled gulonolactone by this enzyme was identified by paper chromatography, chromatography on Dowex 1-formate, and recrystallization of the chromatographed ascorbic acid to constant specific activity.16 Rigorous identification of ascorbic acid is absolutely imperative in studies of biosynthesis, as is pointed out later. When limiting amounts of substrate are used, the reaction proceeds according to the stoichiometry shown in Reaction 3. The reaction occurs with gulonolactone but not with free gulonate as substrate.

Some Aspects of Energy Coupling by Mitochondria

This paper will review some recent developments and trends of thought on the energetics, mechanism, and dynamics of electron transport and oxidative phosphorylation, as well as some aspects of the interplay between the mitochondrial and cytosolic compartments of cells basic to an understanding of metabolic regulation.

The Transport Systems of Mitochondrial Membranes

The respiratory and phosphorylative functions of mitochondria are now widely known and much information is available regarding the nature and intramitochondrial location of the enzymes of the tricarboxylic acid cycle, the electron transport chain, and the enzymes concerned in oxidative phosphorylation. The concept of the respiratory assembly of flavoproteins and cytochromes as a structural and functional unit of the inner membrane and the successful reconstitution of oxidative phosphorylation from purified ATP-synthetase and inner membrane vesicles are land-marks in the progress of the last decade.

Role of C-factor in water uptake and extrusion by mitochondria and interference by various drugs

WATER movements through mitochondrial membranes are of some interest because they involve the enzymes of cxidative phosphorylation and they are strikingly affected by certain hormones and other chemical agents. Mitochondria isolated from rat liver or kidney can take up significant amounts of water from an in vitro incubation medium, leading to doubling or tripling of mitochondrial volume. This process of spontaneous swelling can be markedly enhanced by adding any one of a number of so-called swelling agents, which include thyroxine, phosphate, calcium, inorganic and organic mercurials, thiols, and disulfides, phlorizin and others. Such a change in mitochondrial volume is not a passive process but is geared to the action of the respiratory chains in the mitochondrial membrane since swelling (does not occur when respiration is blocked or under anaerobic conditionsly2. Mitochondrial swelling produced in this manner in vitro can be reversed again by the addition of ATP -IMgS-+, with easily measured extrusion of water. It is of interest that ATP-dependent water extrusion from thyroxine-swollen mitochondria takes place independently of the nature of the ionic environment and does not require or depend on movements of potassium or sodium ions.3. “Contraction” is specifically linked to ATP and is inhibited by uncoupling agents like azide or sucrose, but not by 2,4-dinitrophenol 4. However mitochondrial water uptake induced by reduced glutathione (GSH) is an exception, since it is not reversed on addition of ATP + Mg+-‘:. More recently Lehninger and Gotterers have demonstrated that mitochondrial swelling induced

Ca 2+ TRANSPORT BY MITOCHONDRIA: A SURVEY

This paper summarizes in broad strokes the properties of mitochondrial Ca 2+ influx and efflux systems in the possible role of mitochondria in maintaining cytosolic Ca 2+ homeostasis. Recent reviews (1-3) may be consulted for some details.

LIVING SYSTEMS AS ENERGY CONVERTERS : Energy in biological molecules: Report on session of Monday 18 October

Publisher Summary This chapter discusses energy in biological molecules. Living cells can maintain their complexity and order only at the expense of free energy from their environment. Cells utilize free or high-grade energy from the environment and then return to it an equal amount of low-grade energy, largely heat, which becomes randomized in the environment. Living organisms, thus, maintain their order at the expense of their environment, which they cause to become more and more random with time. Ultimately, the form of free energy that sustains all life, whether plant or animal, is solar light energy. This chapter describes the conversion of energy by the living systems. It presents a general introduction to the science of bioenergetics and discusses research on the evolution, structure, and function of some of the important biocatalysts involved in energy conversion by living cells. The chapter also discusses some practical implications of such basic research in approaching the energy problem.