Role of NAD+ in the stimulation of protein synthesis in rabbit reticulocyte lysates.

The stimulation of protein synthesis by NAD⁺ in rabbit reticulocyte lysates has been reported. [Lennon M. B., Wu, J., and Suhadolnik, R. J., (1976) Biochem. Biophys. Res. Commun. 72, 530–538]. NAD⁺ can replace the creatine phosphate-creatine phosphokinase ($\text{CP}\text{CPK}$) energy regenerating system normally used in in vitro protein synthesizing systems. The replacement of $\text{CP}\text{CPK}$ by NAD⁺ is optimal at 37 °C. A significant lag in the rate of protein synthesis with NAD⁺ is observed with decreasing temperatures. Analysis of the adenylate energy charge with NAD⁺ shows an initial rapid decrease. This decrease in the energy charge recovers with increasing NAD⁺ concentrations. The energy level correlates with the rates of incorporation of d,l-[4,5-³H(N)]leucine into protein. ATP production via NAD⁺ pyrophosphorylase or oxidative phosphorylation does not explain the stimulation by NAD⁺. Rather, the stimulation is correlated with the activation of glycolysis. Glycolysis is not active in lysate preparations because NAD⁺ is absent. Additional possible roles of NAD⁺ in protein synthesis are discussed.     

Activation of low and null activity isozymes of maize alcohol dehydrogenase by antibodies

Summary Antisera were raised against several purified, high specific acitivity isozymes of maize alcohol dehydrogenase (ADH1). The various antisera had different effects on the activity of immunoprecipitated ADH. One antiserum completely inactivated maize ADH. This inactivation could be blocked by preicubation of the enzyme with NAD⁺, its cofactor, or with NADP. The different antisera were used to analyze variant froms of ADH1. Isozymes having lowered specific activity were activated to wild-type levels by precipitation of the enzymes with noninactivating antisera. Isozymes having no detectable ADH activity (CRM⁺ nulls) were activated by immunoprecipition with noninactivating antisera when preincubated with NAD⁺ or NADP. All of the CRM⁺ nulls were shown to be unable to bind NAD⁺, a flaw which can account for their lack of activity. The results indicate that a conformational equilibrium between active and inactive forms of maize ADH in solution controls the specific activity of the various isozymes. Both controls the specific activity of the various isozymes. Both NAD⁺ and antibodies raised against high specific activity enzymes can interact with low activity isozymes to shift the balance of the equilibrium toward the active form, thus increasing their specific activity.     

Cyclic AMP inhibition of reactions of glyceraldehyde 3-phosphate dehydrogenase

Adenosine 3′,5′-monophosphate (cyclic AMP) is an inhibitor of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate and benzaldehyde. Inhibition appears to be competitive toward glyceraldehyde 3-phosphate and of a mixed type toward NAD⁺. In the absence of arsenate a plot of $\text{1}\text{V}$ vs (I) is sigmoidal at constant concentrations of glyceraldehyde 3-phosphate and NAD⁺ and linear at constant concentrations of benzaldehyde and NAD⁺. Thus, sigmoidal inhibition plots are dependent on the nature of the aldehyde substrate as was found previously to be the case with inhibition of these reactions by highly branched acyl phosphates. In the presence of 0.013 m arsenate the plots of $\text{1}\text{V}$ vs [I] are linear.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

Metabolic regulation of malic enzyme activity from Paracoccus denitrificans by glyoxylate and acetylCoA.

$\text{Paracoccus denitrificans}$ contains both NAD⁺- and NADP⁺-linked malic enzyme activities when grown on malate/nitrate. The enzyme is inactive in the absence of NH₄⁺. AcetylCoA inhibits both activities competitively with respect to L-malate. Glyoxylate (0.5 mM) causes 60% inhibition of the NADP⁺-linked activity but has little effect on the NAD⁺-linked activity. Citrate, aspartate, AMP, ADP, and ATP, at 0.5mM, have little effect on either of the two activities. The results are discussed with regards to the control of malic enzyme activity within the cell.     

The influence of NAD+-linked substrates on energy conservation at sites 2 and 3 in mitochondria treated with inorganic arsenate.

Exposure of rat liver mitochondria to inorganic arsenate followed by reisolation and washing to remove the added arsenate results in uncoupled respiration with succinate and ascorbate ($\text{ADP}\text{0}\text{=0}$), but $\text{ADP}\text{0}$ and $\text{ATP}\text{0}$ values of 1.3 to 1.6 with 3-hydroxybutyrate or glutamate. $\text{ADP}\text{0}$ and $\text{ATP}\text{0}$ values greater than 1.0 with NAD⁺-linked substrates arise as a result of partial reactivation of coupling at sites 2 and 3 by these substrates. In the presence of rotenone, NAD⁺-linked substrates can still reactivate coupling with succinate or ascorbate at these sites. The extent of reactivation in the presence of rotenone by 3-hydroxybutyrate is decreased by simultaneous addition of acetoacetate. The results suggest that the coupling at sites 2 and 3 is amenable to control through changes in the reduction state of some specific components of the respiratory chain located remotely from these sites.     

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase–ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E·NAD⁺) and three ternary (E·NAD⁺·acetone, E·NAD⁺·3-pentanone and E·NAD⁺·cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.     

Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.     

Metabolic effects of glucose deprivation and of various sugars in normal and transformed fibrobalst cell lines.

The sugar composition of the growth medium influenced the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, pyruvate and lactate production, and ATP levels in both normal and transformed fibroblast cell lines growing in tissue culture. Removal of glucose led to a rapid three- to fourfold rise in the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, followed by a slower decline in the content of ATP. However, there was no change in the adenylate energy charge [$\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP + ADP + AMP}\text{)}$] over a 2-h period. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio was restored to the original level within 10 s of glucose readdition. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ratios in cell lines growing on galactose were as high as for those incubated without sugars; growth on mannose or fructose produced intermediate ratios. There was an inverse relationship between the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio and pyruvate-lactate production for glucose, fructose and galactose. Thus, all cell lines had a high production of pyruvate and lactate but a low $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio when grown on glucose. In contrast, when galactose served as the sugar source, acid production was low, while the ratio was high. All cell lines had comparable hexokinase activity, and glucose was the best substrate, mannose intermediate and fructose poorest. Hexokinase activity did not correlate with the relative degree of utilization of the sugars. These results suggest that the sugar composition of the growth medium affects the metabolic pattern of a cell line, including the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, the ATP content and the production of pyruvate and lactate.     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

The interaction of phosphonate and homophosphonate analogues of 3-deoxy-D-arabino heptulosonate 7-phosphate with 3-dehydroquinate synthetase from Escherichia coli

Phosphonate and homophosphonate analogues of 3-deoxy-D-$\text{arabino}$ heptulosonate 7-phosphate and D-$\text{gluco}$ heptulosonate 7-phosphate behave as competitive inhibitors of 3-dehydroquinate synthetase. Phosphonates have better affinities than homophosphonates and protect efficiently the enzyme against thermal denaturation. No evidence has been obtained for 5-keto phosphonate intermediate formation in the interaction of such analogues with 3-dehydroquinate synthetase and NAD⁺.     

Inhibition of mouse brain synaptosomal gamma-aminobutyric acid transport by pyridoxal 5'-phosphate

Crude lysates from a strain of enterotoxigenic $\text{E. coli}$ have been shown to catalyse the incorporation of [³²P] from [adenylate-³²P] NAD⁺ into an 11,000 dalton protein in rat liver membranes. [³²P] incorporation paralleled adenylate cyclase activation and the results suggest that the mechanism of action of the heat-labile $\text{E. coli}$ enterotoxin may involve ADP-ribosylation of an intracellular acceptor protein.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

The inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The influence of fatty acid on the interconversion of the pyruvate dehydrogenase complex (PDH) between its active (dephospho-) and inactive (phospho-) forms and on the intramitochondrial $\text{ATP}\text{ADP}$, $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{acetyl-CoA}\text{CoASH}$ ratios was studied in isolated rat liver mitochondria. Conditions were found in which the PDH activity was inversely correlated only with the $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio. Under other conditions the PDH activity was inversely correlated solely with the $\text{acetyl-CoA}\text{CoASH}$ ratio. These experiments suggest that the activity of the regulatory enzymes involved in the inactivation and reactivation of the pyruvate dehydrogenase multienzyme complex may be controlled by both the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{acetyl-CoA}\text{CoASH}$ ratios.     

Interaction between the nitrate respiratory system of Escherichia coli K12 and the nitrogen fixation genes of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{chl}$^? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from $\text{K. pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$_Kp⁺ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by $\text{chl}$ genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.     

The use of 3-acetyl-pyridine adenine dinucleotide in the spectrophotometric assay of enzymatic activities coupled to dehydrogenases of unfavorable equilibrium

The high redox potential of the couple $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ has limited in the past the application of dehydrogenases in the coupled assay of enzymes. Here the way to obviate this difficulty is presented, by using the chemical analog 3-acetyl-NAD⁺ and an auxiliary enzyme after the dehydrogenase. A general kinetic scheme of the multienzyme systems is presented in the discussion that helps to explain why this approach succeeds in making these dehydrogenases satisfactory auxiliary enzymes even in the unfavorable direction.     

Regeneration of NAD(H) by alcohol dehydrogenase in gel-entrapped yeast cells

Anaerobically grown cells of Saccharomyces cerevisiae entrapped in polyacrylamide gel have been shown to provide a stable source of alcohol dehydrogenase [(ADH) alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1] for effective regeneration of NAD(H). This system was able to provide the coenzyme required for the operation of other dehydrogenases, such as lactate dehydrogenase [(LDH) l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27] and malate dehydrogenase [(MDH) l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37]. Yeast cells coimmobilized with a dehydrogenase are capable of the reversible regeneration of the reduced or oxidized coenzyme, depending on the additions made. A two-cell system can also be constituted using the same strain of yeast, adapted differently. Cells grown anaerobically and aerobically as sources of ADH and MDH, respectively, can operate efficiently on coimmobilization. The system can be used repeatedly without measurable loss of efficiency.     

Identification and quantitative determination of the flavin component of soluble hydrogenase from Alcaligeneseutrophus

The flavin component of soluble hydrogenase (hydrogen: NAD⁺ oxidoreductase, EC 1.12.1.2) from $\text{Alcaligenes}\text{eutrophus}$ was identified as FMN by thin layer chromatography in two solvent systems and by binding studies with apoflavodoxin from $\text{Megasphaera}\text{elsdenii}$. The flavin of hydrogenase reacted rapidly with apoflavodoxin with almost complete quenching of the fluorescence at 525 nm. Quantitative determination of FMN was performed by fluorimetric titration with a standardized solution of apoflavodoxin. From the determined FMN content of different enzyme preparations and from the percentage of stimulation of hydrogenase activity by exogenous FMN it is concluded that hydrogenase contains 2 FMN per molecule.     

The identification of MYO-inositol:NAD(P)+ oxidoreductase in mammalian brain.

$\text{myo}$-Inositol:NAD(P)⁺ oxidoreductase ($\text{myo}$-inositol oxidoreductase) has been identified in bovine brain. This enzyme elutes from DEAE cellulose with 0.3 M KCl in 50 mM Tris buffer, pH 7.5. Using NADH as cofactor $\text{myo}$-inosose-2 is reduced selectively to $\text{myo}$-inositol. With NADPH the enzyme forms both $\text{myo}$-inositol and $\text{scyllo}$-inositol, however, at a lower rate. The enzyme was chromatographed on G-100 Sephadex and found to have an apparent molecular weight of 74,000. This enzyme differs in DEAE binding, molecular weight and cofactor specificity from the previously described $\text{scyllo}$-inositol oxidoreductase which utilizes NADPH exclusively to produce 3 fold more $\text{scyllo}$-inositol than $\text{myo}$-inositol.