John Mowbray

Evidence for the role of a specific monocarboxylate transporter in the control of pyruvate oxidation by rat liver mitochondria.

The most convincing evidence for the existence of mitochondrial transporters is the ability shown by certain respiratory poisons to prevent specific exchange ‘diffusion’ reactions across the inner mitochondrial membrane [ 1,2]. The absence of such a transport inhibitor for monocarboxylates has cast doubt that the ready penetration and exchange of pyruvate across the mitochondrial inner membrane [3-61 is mediated by a carrier; indeed it has been argued [2] that sufficient undissociated monocarboxylic acid exists in the cell to render redundant such a transporter. Very recently some data on the influence of (Ycyano-4-hydroxycinnimate on red cell and mitochondrial pyruvate content have been interpreted to mean that this compound is a specific inhbitor of pyruvate transport in these vesicles [7]. This report presents some studies of the effect of certain 2-oxo-acids on pyruvate content, pyruvate exchange reactions and pyruvate oxidation in rat liver mitochondria. The results are interpreted as evidence for the existence of a monocarboxylate transporter. Palmityl carnitine has been found to be a very powerful counter-transporter of pyruvate. The consequences of such an exchange in the control of the pyruvate dehydrogenase complex are discussed.

The influence of thyroid hormone on the degree of control of oxidative phosphorylation exerted by the adenine nucleotide translocator

Adenine nucleotide translocator Control strength Liver mitochondria Oxidative phosphorylation Triiodothyronine

Evidence against the incorporation into protein of amino acids directly from the membrane transport system in rat heart.

The possibility suggested recently [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433; and Adamson, L.F., Herington, A.C. and Bornstein, J. (1972) Biochim. Biophys. Acta 282, 352-365] that protein synthesis takes place using amino acids directly from the membrane transport system and not from an intracellular pool has been investigated in rat heart. The tissue was perfused first for 30 min with either [14C]glycine or [14C]leucine and then for a further 30 min with identical medium containing [3H]glycine or [3H]leucine, respectively. After an initial lag, [14C]glycine was incorporated into protein at a linear rate up to 60 min. The [3H]glycine was accumulated into tissue water and incorporated just as readily as the [14C]glycine had been. The rate of total protein synthesis agrees with literature values only if intracellular and not extracellular specific activity values are used in the calculation. Some glycine was converted to serine or threonine. Leucine influx and efflux were very rapid in contrast to the relatively slow exchange reported for incubated tissues [Hider, R.C., Fern, E.B. and London, D.R. (1969) Biochem. J. 114, 171-178; Hider, R.C., Fern, E.B. and London, D.R. (1971) Biochem. J. 121, 817-827; van Venrooij, W.J., Poort, C., Kramer, M.F. and Jansen, M.T. (1972) Eur. J. Biochem. 30, 427-433]. The results are consistent with the existence of an intracellular precursor pool for glycine. Some possible reasons for the discrepancies between this and the other studies are discussed.

Direct thyroid hormone signalling via ADP-ribosylation controls mitochondrial nucleotide transport and membrane leakiness by changing the conformation of the adenine nucleotide transporter

Addition of triiodothyronine at 10 pM in vitro to hypothyroid rat liver mitochondria doubles the rate of the adenine nucleotide transporter at low ADP concentrations. Nicotinamide abolishes this effect in parallel with its inhibition of the ADP‐ribosylation of an inner membrane protein identical in size to the transporter. Nicotinamide also renders euthyroid preparations indistinguishable from hypothyroid ones. A mechanism is offered to explain these findings in which it is proposed that the adenine nucleotide transporter is a true allosteric protein and that its covalent modification by ADP‐ribosylation increases the stability of the less favoured externally‐facing C‐conformation and thus increases the proportion of transporters in this orientation: although the C‐conformation is significantly more leaky to cations than the tight matrix‐facing M‐conformation, this enhances ADP import. This model is shown to offer an explanation not only for the transport effects of T3 but also for those of oxidative stress and ADP‐ribosylation inhibitors on Ca2+, H+ and K+ transfer across the mitochondrial inner membrane. Ca2+ at 30 nM appears to stabilize the M‐conformation of the transporter by a mechanism other than ADP‐ribosylation.

Inversely related oscillations in the contents of cyclic GMP and the total adenine nucleotides in steady-state perfused rat hearts

While investigating amino acid incorporation into protein in Langendorf perfused hearts we made the surprising observation that the tissue contents of cyclic AMP and cyclic GMP showed very significant variations when measured at time points 20 rain apart during a 2 h perfusion period [1]. Despite these large temp oral variations in the cyclic nucleotides, the heart preparation is apparently in a steady-state for up to 2 h as judged by oxygen uptake, glucose utilization, nicotinamide nucleotide ratio, glycogen turnover, lactate turnover, alanine output [2,3] protein-synthesis rate [1,4], beat rate and contraction amplitude [5]. It had been suggested that cellular feedback-regulation systems might give rise to stable oscillations on which metabolic steady-states could rely [6] and moreover oscillations which appeared to be related to glycolytic oscillations had been observed in the concentrations of the adenine nucleotides and IMP in particle-free rat skeletal-muscle extracts [7]. The possibility that such oscillatory phenomena were responsible for the cyclic nucleotide variations was investigated by measuring in both hearts and perfusate all purine nucleotides, nucleosides and their degradation products at selected time points over 80 min of perfusion. Highly significant differences were found in the contents of ATP, ADP, GTP, cyclic AMP, cyclic GMP in the ATP/ADP ratio and in the sum of the adenine nucleotides at time points 10 or 20 min apart [5]. The ATP/ADP ratio and the contents of GTP and cyclic AMP showed an identical pattern of variation which was distinct from that of the total adenine nucleotide content. The very large nett increases and decreases in the total adenine nucleotides extracted could neither be explained as operation of Lowensteins purine nucleotide cycle [7] nor as metabolism of any known nucleotide precursor, derivative or polymer and it was suggested that an unsuspected substantial storage form of purine nucleotide might exist in mammalian heart [5]. This paper reports that when hearts are sampled more frequently very regular statistically significant temporal variations in total adenine nucleotide content are discerned. Furthermore the changing adenine nucleotide content is mirrored by inverse alterations in the cyclic GMP content. Since the cyclic GMP content also appears to respond to the non-synchronous variations in total guanine nucleotide, it is argued that guanylate cyclase activity may be regulated by the level of free adenine nucleotide in the cell.

In perfused rat hearts ischaemia promotes the reversible conversion of appreciable quantities of soluble adenine nucleotides to a stable trichloroacetic acid-precipitable form

Radioactivity from [14C] adenosine was linearly incorporated into tissue nucleotides in perfused rat hearts. All the TCA-extractable 14C was confined to the purine nucleoside phosphates for up to 1 h of perfusion. Radioactivity was also incorporated linearly into the TCA-insoluble fraction, which by 40 min accounted for 24% of the tissue 14 C. Estimates based on precursor specific radioactivity suggest that at least 0.6 micro mol/g of the mononucleotide is in this stable insoluble form. Following 2 min total ischaemia, the tissue nucleotide content and soluble radioactivity decreased while the insoluble radioactivity showed a corresponding increase to account now for 35% of the tissue radiolabel. This redistribution was rapidly reversed by post-ischaemic reperfusion. A possible function for the rapid reversible sequestration of adenine nucleotides in ischaemia is proposed.

Evidence for ADP-ribosylation in the mechanism of rapid thyroid hormone control of mitochondria.

Triiodothyronine in vitro at concentrations between 10−13 and 1O−11M very rapidly activates oxidative phosphorylation in hypothyroid rat liver mitochondria. Comparing the concentrations of hormone with estimates of the amounts of respiratory chain components present suggests that this activation may involve an amplification mechanism. Here we present evidence that while no changes in phosphorylation were detected following hormone administration, nicotinamide, an inhibitor of mono ADP‐ribosylation reported to occur rapidly and reversibly in mitochondria, prevented activation by hormone. Moreover incubation with nicotinamide of euthyroid mitochondria and derived intact inner membrane vesicles revealed lowered ADP/O ratios under the same conditions as shown by hypothyroid preparations. While this lesion could be reversed simply by washing the intact mitochondria, the membrane vesicles required triiodothyronine addition.

Post-ischaemic synchronous purine nucleotide oscillations in perfused rat heart

Langendorff perfused rat hearts show synchronous, statistically significant, systematic variations in ATP and ADP. Here we show that AMP and IMP also vary in register with ATP and ADP and we suggest that the synchronizing trigger for these oscillations may be ischaemia. Oscillations in the ATP/ADP ratio were found to be significantly correlated with creatine phosphate content but by contrast these quantities vary quite differently from the GTP/GDP ratio. Cyclic GMP oscillations showed a significant negative correlation with variations in ADP. Epinephrine raised mean cyclic AMP content and stabilized cyclic GMP oscillations, but had little other effect on the purine nucleotide variations.

The rapid transient stimulation of both cytoplasmic and mitochondrial ornithine decarboxylases in the liver of thyroidectomized rats by 3,5,3'-triiodo-L-thyronine.

Evidence is presented that liver from thyroidectomized rats has ornithine decarboxylase (ODC) in both mitochondrial and soluble fractions. The cytosolic activity was stimulated 7-fold and the mitochondrial activity 3-fold 15 min after the administration of triiodothyronine (T3) . While the rapid transient stimulation of ODC could represent a direct intracellular response to T3, an incidental effect on a very-rapidly-turning-over enzyme seems more likely; this suggests thatin vivo ODC may be controlled by a demand for polyamines.

A comparison of rapid adenine nucleotide incorporation into phosphoglyceroyl-ATP and into RNA-like species in perfused rat heart

Four main species of rapidly synthesised trichloroacetic acid-insoluble derivatives of adenylate can be separated in extracts from rat hearts. The major species, accounting for more than 70% of the total, is phosphoglyceroyl-ATP; two others (16% of the total) are closely related to it. Around 10% of incorporated radioactivity is in a high-molecular-weight form with a phosphate/purine ratio of 0.8; comparison of [14C]uridylate and [14C]adenylate incorporation supports the suggestion that this is a rapidly synthesised species of RNA which represents about 4% of total RNA in heart. Values are given for the contents of UTP, UDP and UDPglucose in adult rat hearts.