Pyridine Nucleotide Transhydrogenase Research Articles

The dependence of the rate of transhydrogenase on the value of the protonmotive force in chromatophores from photosynthetic bacteria

In conditions where the pH gradient is negligible, the rate of the pyridine nucleotide transhydrogenase in chromatophores of Rhodobacter capsulatus has a threshold dependence on membrane potential. The relationship is similar when either antimycin or myxothiazal or carbonyl cyanide p-trifluoromethoxyphenylhydrazone is used to depress the membrane potential. The relationship is distorted when membrane potential is reduced by lowering the photosynthetic light intensity.

Nucleotide sequence of the pntA and pntB genes encoding the pyridine nucleotide transhydrogenase of Escherichia coli.

A 3240-base-pair DNA fragment spanning the pyridine nucleotide transhydrogenase (pnt) genes of Escherichia coli has been sequenced. The sequence contains two open-reading frames, pntA and pntB of 1506 and 1386 base pairs, coding for the transhydrogenase alpha and beta subunits, respectively. The coding sequences are preceded by a promoter-like structure and are most likely co-transcribed. Each coding sequence is preceded by a Shine-Dalgarno sequence. The amino-terminal amino acid sequences were determined from the purified alpha and beta subunits of the transhydrogenase. These sequences agree with those predicted from the nucleotide sequences of the pntA and pntB genes. The predicted relative molecular masses of 53906 (alpha) and 48667 (beta) are close to the values obtained by analysis of the subunits by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed in each subunit. These results indicate that transhydrogenase is an intrinsic membrane protein.

Expression of the cloned subunits of Escherichia coli transhydrogenase from separate replicons

The pntA and pntB genes of Escherichia coli, encoding the α- and β-subunits of the pyridine nucleotide transhydrogenase, were cloned individually in two different compatible plasmids into Escherichia coli mutants lacking transhydrogenase activity. Energy-linked and non-energy-linked transhydrogenase activities were produced only in cells carrying both plasmids thus showing that the products of both genes are required for the formation of an active enzyme. ATP-energized transhydrogenase activity was not increased in cells containing amplified levels of the transhydrogenase when the cell membrane ATPase was also amplified. It is suggested that the excess transhydrogenase is effectively uncoupled from the ATPase by compartmentalization in the cell.

The reaction mechanism of the mitochondrial pyridine nucleotide transhydrogenase. A study utilizing arylazido-pyridine nucleotide analogues.

The addition of a purified mitochondrial pyridine nucleotide transhydrogenase enzyme preparation to complex I (NADH-CoQ reductase) results in a significant increase in the NADPH-AcPyAD+ transhydrogenase activity of the complex without influencing the NADH-AcPyAD+ transhydrogenase activity. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of complex I, the purified transhydrogenase enzyme preparation was found to co-migrate with the Mr = 130,000 (130K) subunit of the NADH-CoQ reductase. Loss of the NADPH-NAD+ transhydrogenase activity of complex I following limited tryptic digestion was associated with a corresponding loss of the 130K subunit from the complex. These results suggest that the 130K subunit of complex I is the specific peptide responsible for the catalysis of the NADPH-NAD+ transhydrogenase activity observed in complex I. Studies have been carried out testing the influence of photoaffinity pyridine nucleotide probes on the NADPH-NAD+ transhydrogenase activity catalyzed at three levels of resolution, i.e. a homogeneous transhydrogenase preparation, a partially resolved membrane preparation (complex I), and an intact mitochondrial membrane preparation (EDTA particles). Such studies have revealed arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+) to be a potent inhibitor and an active site-directed reagent for NADPH-NAD+ transhydrogenation at all three levels of resolution. On the other hand, arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)-amino]propionyl)NAD+ does not produce a significant degree of inhibition of NADPH-NAD+ transhydrogenase activities prior to or following photoirradiation. Nevertheless, the NAD+ analogue has been found to specifically label, covalently, the transhydrogenase protein following photoirradiation of an enzyme-analogue mixture. Arylazido-beta-alanyl NAD+ can as well function as a substrate during transhydrogenation by virtue of being able to accept a hydride ion from NADPH. An interpretation of the observed nucleotide photoprobe specificity for interaction at the active site for transhydrogenation is advanced. In this interpretation, an ordered binding of substrate involves an initial NADP(H) (or NADP+ photoprobe) interaction with a hydrophobic region at the transhydrogenation site. This initial reactivity is followed by a positioning of NAD(H) (or the NAD+ photoprobe analogue) above or periphery to the NADP(H) nucleotide present at the active site region. Supportive evidence for this model for transhydrogenation is presented and discussed.

Why are two different types of pyridine nucleotide transhydrogenase found in living organisms?

Two types of pyridine nucleotide transhydrogenases have been reported in living organisms. The energy-linked transhydrogenase is found in mitochondria and in certain heterotrophic and photosynthesizing bacteria, while the non-energy-linked transhydrogenase is found in certain heterotrophic bacteria. The presence of a structurally similar non-energy-linked transhydrogenase in Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas fluorescens is readily shown in extracts from these bacteria with Western (protein) blotting. This non-energy-linked enzyme is lacking in Escherichia coli, while the presence of a structurally similar energy-linked enzyme in E. coli and in beef heart mitochondria is indicated with the Western blotting technique. Spinach (Spinacia oleracea) lacks the non-energy-linked transhydrogenase occurring in bacteria. The chloroplast enzyme ferredoxin:NADP+ oxidoreductase, which exhibits non-energy-linked transhydrogenase activity, is immunologically distinct from the bacterial transhydrogenases. In order to provide a rationale for the distribution of the two types of pyridine nucleotide transhydrogenases, the steady-state degrees of reduction of the NADP(H) and NAD(H) pools in A. vinelandii (R'NADP(H) and R'NAD(H)) have been measured for cells metabolizing sucrose at a variable oxygen flux (phi O2). It is found that the degree of reduction of the NADP(H) pool is always higher than that of the NAD(H) pool (R'NADP(H) greater than R'NAD(H)) except when phi O2 goes to zero (R'NADP(H) approximately equal to R'NAD(H)). Comparison of these results with literature values indicates that the inequality R'NADP(H) greater than R'NAD(H) is always found in a membrane-enclosed compartment, irrespective of the type of transhydrogenase present. This allows an understanding of the function of the two types of pyridine nucleotide transhydrogenases in vivo. The physiological role of non-energy-linked transhydrogenase is to catalyze the reaction NADPH + NAD+ leads to NADP+ + NADH, that of energy-linked transhydrogenase to catalyze the reaction NADH + NADP+ leads to NADPH + NAD+. Since at equilibrium R'NADP(H) approximately equal to R'NAD(H) the inequality R'NADP(H) greater than R'NAD(H) under steady-state conditions explains the energy requirement in the latter reaction. The dependence of the non-energy-linked transhydrogenase activity of ferredoxin:NADP+ oxidoreductase on R'NADP(H) is compared with that of A, vinelandii transhydrogenase. The results indicate that this activity is unlikely to be of physiological importance in plant chloroplasts.

Dissociation and assembly of pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

Thermal inactivation studies of pyridine nucleotide transhydrogenase (55°C, pH 7.5, ionic strength 0.1 M) show a biphasic loss of enzyme activity with time. Approximately half of the activity is lost in both the rapid and the slow phase of the inactivation reaction.With electron microscopy it is found that the quaternary structure of transhydrogenase does not change during thermal inactivation. Using Naja naja venom phosphodiesterase to monitor the release of FAD it is shown that dissociation of FAD and activity loss are proportional at every degree of inactivation. The biphasic loss of enzyme activity thus indicates non‐equivalent positions for the subunits (Mr 54000, 1 FAD per subunit) in the transhydrogenase polymer.The process of thermal inactivation is greatly accelerated when the enzyme‐bound flavin is reduced by addition of NADH. The apoenzyme, obtained by gel filtration of transhydrogenase in the presence of NADH at 37°C, can be partly (10–15%) reactivated by addition of excess FAD. The dissociation of FAD under non‐reducing conditions at 55°C is irreversible.In a guanidine hydrochloride denaturing solvent (cGuHCl > 2 M, 25°C, pH 7.5) transhydrogenase dissociates into subunits. These are highly unfolded as judged by hydrodynamic criteria. Retransfer of the enzyme to conditions stabilizing the native conformation (cGuHcl < 0.3 M) leads to reactivation provided that FAD is also present in the reactivation mixture. The yield of the reactivation is low, 3–8% of the theoretical maximum, and the process is slow. Delaying the time of addition of FAD to the reactivation mixture causes a decrease in reactivation yield. The results indicate that FAD is inserted in an intermediate stage in the assembly path leading to active transhydrogenase.Polyacrylamide gel electrophoresis in combination with the Western blotting technique shows the presence of a covalent dimer in purified transhydrogenase preparations, which is not dissociated by treatment with dodecylsulphate and 2‐mercaptoethanol. Since a dimer or other form of high relative molecular mass is not detected in cell extracts it follows that such forms do not play a role in the assembly of the complex quaternary structure of the enzyme in vivo.

Subunit specific antisera to the Escherichia coli ATP synthase: Effects on ATPase activity, energy transduction, and enzyme assembly

We obtained antisera to each of the five subunits (α, β, γ, δ, and ϵ) of the F 1 portion of the proton-translocating ATPase from Escherichia coli (ECF 1). No cross-reaction between the antiserum to a given subunit and any of the other four subunits was observed by Ouchterlony immunodiffusion. The α antiserum reacted only with the denatured α chain. Antibodies to either subunit β or subunit γ inhibited the ATPase activity of the enzyme. The ATPase activity of the holoenzyme in the everted membrane vesicles was just as sensitive as purified ECF 1 to inhibition by the anti-β or anti-γ serum. A prolonged digestion of ECF 1 with trypsin removed intact γ from ECF 1, but did not alter the sensitivity of the ATPase to inhibition by the anti-γ serum. Proteolytic fragments were isolated from the trypsinized enzyme. They gave an immunoprecipitation band with the anti-γ serum, but none of the other subunit antisera. The antiδ serum detached ECF 1 from everted membrane vesicles and completely blocked both the ATP- and respiration-dependent pyridine nucleotide transhydrogenase, an energylinked membrane function. The δ antiserum had no effect on the ATPase activity of the ECF 1. The e antiserum stimulated the ATPase activity of purified ECF 1 as shown previously (P. P. Laget and J. B. Smith, Arch. Biochem. Biophys. 197, 83, 1979), but strongly inhibited the holoenzyme in membrane vesicles. The α antiserum completely blocked the ATP-driven transhydrogenase. The same antiserum maximally inhibited the respiratory chain-driven reaction by only 35%. These observations indicate that the antiserum selectively affected energy transduction mediated by the ATPase. The protonmotive force generated by substrate oxidation was probably not dissipated by the ϵ antiserum. Adsorbing the δ or ϵ antiserum with everted membrane vesicles selectively removed those antibodies that reacted with membrane-bound ATPase. The adsorbed sera still reacted strongly with purified ECF 1, and prevented it from restoring ATP-dependent proton translocation in ECF 1-depleted vesicles. Therefore, it appears that more of the δ and the ϵ subunit is exposed in the purified ECF 1 molecule than in the membrane-bound enzyme.

Effect of thyroidectomy on the rat adrenal cortex enzyme activities involved in corticosterone and aldosterone biosynthesis

The effect of thyroidectomy on corticosterone and aldosterone biosynthesis, in rat adrenal cortex, were investigated in vitro. Thyroidectomy slowed down by about 30% the activity of microsomal 21 hydroxylase and mitochondrial 11β hydroxylase, the two stages leading to corticosterone formation from progesterone. The plasma corticosterone concentration also diminished in thyroidectomized rat. The mitochondrial enzyme system catalyzing the conversion of corticosterone into aldosterone included both 18 hydroxylase and 180H dehydrogenase. In the absence of thyroid hormone, values of both enzyme activity slowed down by about 30%. Thus thyroidectomy induced a general decrease in all mitochondrial hydroxylation processes requiring NADPH as energetic cofactor. Mitochondrial NADPH was mainly synthesized by the activity of NADP +malic enzyme and pyridine nucleotide transhydrogenase; the activity of both enzymes was determined in submitochondrial particles from adrenal cortex of normal and thyroidectomized rats. The activity of NADP + malic enzyme and non energy-dependent transhydrogenase in the direction of NADPH formation dropped significantly after thyroidectomy, thus reducing the amount of NADPH available for hydroxylating mechanisms. The results should be compared to the large rise in the cytosolic malic enzyme level caused by thyroidectomy. The latter also ensured that the transhydrogenase reaction did not vary in the direction of NADP + formation. Further, the drop in NADPH synthesis after thyroidectomy was coupled with a decrease in phosphorylation rate of added ADP by adrenal cortex mitochondria of thyroidectomized rat. These findings might account for the general decline in the energy-dependent processes involved in steroidogenesis, a decline mainly due to the lowering of the H + ions gradient driving ATP and NADPH synthesis through electron transfer.

Modification of the thiol residues of pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Activity modulation by the divalent thiol reagent p-aminophenylarsenoxide.

1. Purified pyridine nucleotide transhydrogenase from Azotobacter vinelandii contains three thiol residues as judged by titration with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions. 2. In the native conformation of the transhydrogenase only a single thiol residue is titrated. Modification of this exposed thiol does not influence transhydrogenase activity. 3. The two less exposed thiol residues can be reacted in part with either p-chloromercuribenzoate or N-ethyl-maleimide. Modification of one residue leads to loss of 40-60% of the enzyme activity in both the forward (NAD+ + NADPH leads to NADH + NADP+) and reverse reaction. The strong inhibitory action of phosphate ions on the reverse reaction [Voordouw et al. (1980) Eur. J. Biochem. 107, 337-344] is abolished after treatment with p-chloromercuribenzoate. Reaction with phenylmercurichloride or p-aminophenylmercuriacetate causes a similar activity loss without affecting the inhibitory action of phosphate. 4. The interaction of the divalent thiol inhibitor p-aminophenylarsenoxide with transhydrogenase was found to be reversible and is characterized by an association constant of 6.3 x 10(5) M-1 at 25 degrees C in 50 mM sodium phosphate pH 7.50. This reversibility indicates formation of a cyclic dithiolarsinite derivative with considerable ring strain. The activity of p-aminophenylarsenoxide-transhydrogenase is modulated by phosphate and magnesium ions. The activity of the transhydrogenase . p-aminophenylarsenoxide complex in the forward reaction is inhibited by phosphate and stimulated by magnesium ions. The reverse reaction is not catalyzed by the enzyme-inhibitor complex. 5. The presence of an activity modulating site in transhydrogenase which binds phosphate ions and has the two less exposed thiol residues in close proximity is indicated by the results.

Temperature-dependent changes in the activity and tryptic susceptibility of membrane-bound transhydrogenase.

Beef heart mitochondrial pyridine nucleotide transhydrogenase undergoes a 2-fold changes in apparent activation energy between 5 degrees and 40 degrees C, from 30 kcal/mol at the lower end to 15 kcal/mol at the upper end of this range. In order to determine whether changes in the structural domain of transhydrogenase accompany the large change in activation energy, the degree of proteolytic inactivation of membrane-bound transhydrogenase was monitored as a function of temperature. Changes in the susceptibility of transhydrogenase to proteolytic inactivation over the 5 degrees -40 degrees C interval correlate well with the observed alteration in activation energies. No temperature-dependent change in either apparent activation energy or susceptibility to proteolysis were observed in detergent-solubilized transhydrogenase. Interactions between transhydrogenase and its microenvironment in the mitochondrial membrane may be an important factor in determining its activity.

Pyridine nucleotide transhydrogenase from Azotobacter vinelandii. Improved purification, physical properties and subunit arrangement in purified polymers.

1. Pyridine nucleotide transhydrogenase from Azotobacter vinelandii was purified with a scaled-up procedure. In a typical purification 500 ml cell-free extract from 200 g cells is loaded on an Ado-2',5'-P2--Sepharose 4B affinity column (20 ml bed volume). After washing, the enzyme is desorbed with 2'AMP at neutral pH and further purified by Sephadex G-200 gel chromatography. The enzyme (10--12 mg) is obtained in 40--60% yield and is homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The homogeneity of the purified enzyme is also apparent from electron microscopy studies, where the enzyme appears as a polydisperse set of polymers without contaminating structures and from fluorescence lifetime studies by the method of single-photon counting. The flavin fluorescence appears to decay with a single lifetime tau = 2.5 ns. The polymeric nature of transhydrogenase can be aptly demonstrated by density gradient centrifugation in the presence of KBr. After centrifuging for 50 h at 160 000 X g and 10 degrees C the enzyme is concentrated in a narrow fluorescent band with buoyant density rho b = 1.305 g cm-3. 3. The arrangement of subunits in the transhydrogenase polymer has been derived from optical diffraction studies of electron micrographs. The polymers are built up from a linear assembly of tetramers. Four subunits are placed in a rhomb with sides of 13.5 mm and an angle of 45 degrees (135 degrees) between the sides. A second tetramer is located staggered on top of the first one. Since a variety of other studies have indicated that the polymers dissociate into octamers under alkaline conditions [Voordouw, G. et al. (1979 Eur. J. Biochem. 98,447--454] we conclude that this smallest functional unit is build up from two tetramers.

Structural aspects of the membrane-bound Escherichia coli pyridine nucleotide transhydrogenase (EC 1.6.1.1)

Escherichia coli, when grown on complex media containing high levels of amino acids, produce low levels of pyridine nucleotide transhydrogenase (EC 1.6.1 .l) both energy-linked and independent [ 1,2]. Washing and replacement of such cells into a glucose minimal medium resulted in the ‘de nova’ synthesis of both transhydrogenase activities after 3 h [2]. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in activity. We have here used this induction system, in which a 5-lo-fold increase in transhydrogenase is produced, to label newly synthesized polypeptides. By incorporating [ 3H] casamino acids in the initial growth phase and non-repressive levels of [ “C]leucine in the induction phase, then partially purifying the transhydrogenase by slight modifications of a described procedure, we have been able to define the synthesized polypeptides on the basis of their high 14(J3H ratios after analysis on polyacrylamide gels in the presence of SDS,

Effects of an insertion mutation in a locus affecting pyridine nucleotide transhydrogenase (pnt::Tn5) on the growth of Escherichia coli.

The effects of a pnt::Tn5 insertion mutation on the growth of strains lacking phosphoglucoisomerase or glucose 6-phosphate dehydrogenase were examined. The results support the idea that the energy-linked transhydrogenase is an important source of reduced nicotinamide adenine dinucleotide phosphate for Escherichia coli under some conditions.

The relationship between energy generation and cholesterol side-chain cleavage reaction in the mitochondria from human term placenta

1. 1. The interrelationship between progesterone (from cholesterol) biosynthesis and oxidative phosphorylation in human placental mitochondria was examined. 2. 2. ADP and ATP stimulated the malate, succinate and α-ketoglutarate-supported progesterone biosynthesis probably via the energy-dependent pyridine nucleotide transhydrogenase activation. The effect of ADP was abolished by rotenone and antimycin in the presence of malate or α-ketoglutarate. 3. 3. In the non-energized state of mitochondria malate may supported progesterone biosynthesis by the malic enzyme-dependent pathway. 4. 4. The inhibitory effects of antimycin or cyanide, and the stimulatory effect of rotenone on the succinate-supported progesterone biosynthesis indicate that the succinate to malate conversion is a necessary condition for the stimulation of progesterone biosynthesis from cholesterol. 5. 5. α-Ketoglutarate plus malonate did support progesterone biosynthesis also in the presence of ADP or ATP and to a lesser degree in the presence of DNP and rotenone. Arsenate in the presence of α-ketoglutarate, malonate, dinitrophenol and rotenone did not affect significantly progesterone biosynthesis. These results indicate that NADPH may be generated also by a non-energy-dependent transhydrogenation in placental mitochondria.

Structure of pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy. 2. At high pH (8.5--9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris-Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient s20, w = 15 S and its Stokes' radius Rs = 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 +/- 2) X 10(3) it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit ('unimer') from which the polymeric form of transhydrogenase is constructed. 3. Gel filtration and sucrose gradient centrifugation studies of cell-free extracts from A. vinelandii show the unimer to be the predominant active species.

Genetic mapping of a mutation affecting pyridine nucleotide transhydrogenase in Escherichia coli

A mutation, pnt-1, causing loss of pyridine nucleotide transhydrogenase activity in Escherichia coli, was mapped by assaying for the enzyme in extracts of recombinant strains produced by conjugation, F-duction, and P1 transduction. The site of this mutation was near min 35, counterclockwise from man, and it co-transduced 59% with man. The mutation was associated with loss from the cell membrane fraction of energy-independent and adenosine 5'-triphosphate-dependent transhydrogenase activities, but reduced nicotinamide adenine dinucleotide dehydrogenase activity was not affected. Strains were constructed which lack phosphoglucoisomerase (pgi-2) and which carry either pnt+ or pnt-1. Although such strains, when grown on glucose, are expected to produce a large excess of reduced nicotinamide adenine dinucleotide phosphate, the growth rate was not affected by the pnt-1 allele.

The kinetic mechanism of pyridine nucleotide transhydrogenase from Escherichia coli.

The kinetic mechanism of pyridine nucleotide transhydrogenase from Escherichia coli.

Isolation and partial characterization of a mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase

A mutant of Escherichia coli lacking pyridine nucleotide transhydrogenase (EC 1.6.1.1) was isolated by assaying activity in clones of cells mutagenized with N-methyl- N′-nitro- N-nitrosoguanidine. The mutant is missing both energy-independent and energy-dependent transhydrogenase, but has normal NADH dehydrogenase and ATPase activities. Compared to the parental strain, the mutant has normal growth rates with glucose, glycerol, or succinate aerobically and with glucose or glycerol plus fumarate anaerobically. The aerobic growth yield with limiting glucose concentrations is also normal. These growth properties indicate that the enzyme is not an essential source of NADPH or ATP in vivo.

Kinetics of pyridine nucleotide transhydrogenase from Chlorella

A pyridine nucleotide transhydrogenase which could facilitate the transfer of hydrogen between chloroplastic and non-chloroplastic pools of pyridine nucleotides was isolated from the unicellular green alga Chlorella. The kinetics of this enzyme with respect to the substrate NADPH demonstrated sigmoid characteristics and substrate inhibition.

Repression of Escherichia coli pyridine nucleotide transhydrogenase by leucine

Addition of 0.1% casein hydrolysate to a minimal growth medium decreased membrane-bound transhydrogenase activity in Escherichia coli by about 80%. Of the amino acids added individually to the growth medium, only leucine and, to a lesser extent, methionine and alanine were effective, alpha-Ketoisocaproate- and leucine-containing peptides repressed the activity, and leucine also repressed activity in adenyl cyclase-deficient and relaxed strains. Derepression of transhydrogenase followed the removal of leucine from the growth medium and was sensitive to rifampin and chloramphenicol. A phosphoglucoisomerase-deficient strain that was forced to use the hexose monophosphate shunt exclusively had normal levels of transhydrogenase, which was repressed by leucine. Transhydrogenase activity doubled in mutants lacking either of the shunt dehydrogenases but was still repressed by leucine. In strains constitutive for the leucine biosynthetic operon, transhydrogenase was repressed by leucine but in strains livR and lst R, with leucine transport resistant to leucine repression, transhydrogenase was not repressed by leucine. These data suggest that transhydrogenase may have a function in the transport of branched-chain amino acids. In a hisT strain (which has altered leucyl-tRNA), transhydrogeanse was at a repressed level without the addition of leucine, suggesting that leucyl-tRNA may be involved in the regulation.