NAD Analogues Research Articles

Preparation of an affinity chromatographic system for the separation of ADP binding proteins.

[4-(3-Bromoacetylpyridinio)-butyl]adenosine pyrophosphate as a structural analog of NAD+ reacts covalently with the sulfhydryl groups of thiopropyl agarose. 10-20 mumol can be bound to 1 ml gel. Stabilization of the insoluble coenzyme is attained by treatment with sodium boro hydride (NaBH4). This complex when applied to column chromatography, allows the separation of various dehydrogenases as a result of their different complex stability coefficients. Alcohol dehydrogenase from liver, lactate dehydrogenase, and adenylate kinase, which all bind to the ADP-analog residues of the gel matrix, can thus be separated by different salt gradients. Alcohol dehydrogenase from yeast, however, does not form a complex and can easily be eluted from the column with phosphate buffer. Glyceraldehyde-3 phosphate and aldehyde dehydrogenases can be eluted by the addition of NAD+ or NADH to the buffer. The uncharged 1,4-dihydropyridine ring of the reduced coenzyme produces a more stable complex with the dehydrogenases than the oxidized form.

Preparation of analogues of NAD+ as substrates for a sensitive fluorimetric assay of nucleotide pyrophosphatase.

1,N6-Etheno derivatives of pyridine analogues of NAD+ were synthesized, characterized and tested as substrates for a fluorimetric assay of nucleotide pyrophosphatase (EC 3.6.1.9). Upon cleavage of their pyrophosphate bond, the fluorescence of pyridine-1,N6-ethenoadenine dinucleotide (epsilon PdAD+) and of 4-hydrazinocarbonyl-pyridine-1,N6-ethenoadenine dinucleotide (epsilon hy4PdAD+) increased respectively 15-and 73-fold, at pH 7.4. This property allows a convenient steady-state assay of nucleotide pyrophosphatase by continuous monitoring of reaction progress. Both compounds were good substrates of this class of enzyme. The relative insensitivity of the fluorescence of epsilon PdAD+ and epsilon hy4PdAD+ to pH changes allowed assays under conditions preserving cellular integrity. epsilon PdAD+ is useful as a substrate for measuring nucleotide pyrophosphatase activity on the outside of mammalian cells because it is not a substrate for the external NAD+ glycohydrolase. epsilon Hy4PdAD+ proved useful when high sensitivity was needed.

The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD + and their use in EPR studies of dehydrogenases

Two spin-labeled analogues of NAD + were synthesized with an 15N and perdeuterated nitroxide radical, 4-amino-2,2,6,6-[ 2H 17, 15N]tetramethylpiperidone-1-oxyl, which was attached to either the C-6 or C-8 position of the purine ring. The EPR spectra of these derivatives exhibit an approx. 6-fold increase in sensitivity compared with the corresponding 14N, protonated analogues due to a decrease in both the number of nuclear manifolds (from three to two) and the linewidth. The enhanced spectral resolution obtained with ( 2H 17, 15N)spin-labeled-NAD + analogues has facilitated simulation of the EPR lineshape of the nucleotide bound to lactate dehydrogenase ( l-lactate:NAD + oxidoreductase, EC 1.1.1.27). The spin-label moiety exhibits highly constrained motion indicative of a single environment. The motion of the spin label does not reflect the overall motion of the enzyme; rather, it is characteristic of some limited mobility relative to the lactate dehydrogenase. By contrast, the spin label on the membrane-bound enzyme, d-β-hydroxybutyrate dehydrogenase ( d-β-hydroxybutyrate:NAD + oxidoreductase, EC 1.1.1.30), is completely immobilized and exhibits two distinct spectral components for spin-labeled NAD +, which appear to differ in the polarity of the environment of the nitroxide.

Kinetic mechanism in the direction of oxidative decarboxylation for NAD-malic enzyme from Ascaris suum.

Measurement of the initial rate of the malic enzyme reaction varying the concentration of NAD at several different fixed levels of Mg2+ (0.25-1.0 mM) and a single malate concentration gave a pattern which intersects to the left of the ordinate. Repetition of this initial velocity pattern at several additional malate concentrations and treatment in terms of a terreactant mechanism suggests an ordered mechanism in which NAD adds prior to Mg2+ which must add prior to malate. On the other hand, when a broader concentration range of Mg2+ (0.25-50 mM) is used, data are consistent with a random mechanism in which Mg2+ must add prior to malate. By use of product inhibition studies, pyruvate is competitive vs. malate and noncompetitive vs. NAD, while NADH is competitive vs. NAD and noncompetitive vs. malate. These results are consistent with the random addition of substrates and further suggest rapid equilibrium random release of products. Tartronate, a dead-end analogue of malate, is competitive vs. malate and noncompetitive vs. NAD. Thio-NAD is a slow substrate which is used at 2.4% the maximum rate of NAD. When used as a dead-end analogue of NAD, thio-NAD is competitive vs. NAD and gives a complex inhibition pattern vs. malate in which competitive inhibition is apparent at low concentrations of malate (less than 12.5 mM), and this changes to uncompetitive inhibition at high concentrations of malate (greater than 12.5 mM). These data are consistent with a steady-state random mechanism in the direction of oxidative decarboxylation in which Mg2+ adds in rapid equilibrium prior to malate.(ABSTRACT TRUNCATED AT 250 WORDS)

NAD[S], an NAD analogue with reduced susceptibility to phosphodiesterase. Chemical synthesis and enzymic properties.

The chemical synthesis of adenosine(5') [alpha-thio]diphospho(5')ribofuranosyl-nicotinamide (NAD[S]) is described. The product occurs as a pair of diastereomers with different configuration at the sulfur-bearing phosphorus atom. The diastereomers were separated by high-performance liquid chromatography and their absolute configuration was determined after chemical degradation to the ADP[alpha S] diastereomers and chromatographic comparison with enzymically synthesized ADP[alpha S] diastereomers of known absolute configuration. Additional support for this assignment is based on different rates in the phosphodiesterase-catalyzed hydrolysis. Furthermore the synthesis of [14C]NAD[S] is described. The coenzyme activity of NAD[S] in the reaction with alcohol dehydrogenase from baker's yeast and lactate dehydrogenase from pig heart is very similar to that of beta-NAD. Also, NAD and NAD[S] serve equally well as substrates for NAD glycohydrolase from calf spleen. In contrast, no reaction was detected with NAD pyrophosphorylase, and hydrolysis of the separated NAD[S] diastereomers with snake venom phosphodiesterase showed a 26-fold and a 33-fold slower reaction rate than that of NAD. Nucleotide pyrophosphatase was less sensitive to the S substitution, hydrolyzing NAD[S] 14-times slower than NAD. Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cell nuclei accepted NAD[S] as a substrate but the reaction was significantly slower and approached saturation at much lower values than with NAD. Alkaline hydrolysis of the products insoluble in trichloroacetic acid yielded AMP[S] as the main derivative. It is concluded that with NAD[S] as a substrate the nuclear acceptors were nearly exclusively mono(ADP-ribosyl) ated .

Identification of the NADH-NAD+ transhydrogenase peptide of the mitochondrial NADH-CoQ reductase (Complex I). A photodependent labeling study utilizing arylazido-beta-alanyl NAD+.

Incubation of Complex I (NADH-CoQ reductase) of ox heart mitochondria at 4 degrees C in the presence of 0.5 M NaClO4 followed by ammonium sulfate fractionation of the solubilized proteins results in the isolation of a resolved preparation still capable of catalyzing NADH-NAD+ transhydrogenation but having only low levels of NADH dehydrogenase activity. A number of NAD(H) analogues, including the photoaffinity probes, arylazido-beta-alanyl NAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]NAD+ and arylazido-beta-alanyl AcPyAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]AcPyAD+ can be utilized as substrates for transhydrogenation in this preparation. A further incubation (10 min) of the resolved NADH-NAD+ transhydrogenase in the presence of 0.5 M NaClO4, but now at 30 degrees C, results in the complete loss of this transhydrogenase activity. Photoaffinity labeling experiments utilizing arylazido-[3-3H]beta-alanyl NAD+ and arylazido-[3-3H]beta-alanyl AcPyAD+ with the resolved NADH-NAD+ transhydrogenase preparation prior to and following NaClO4 (30 degrees C) treatment indicates that the 42,000 molecular weight component of Complex I is the pyridine nucleotide binding site responsible for the major NADH-NAD+ (DD) transhydrogenase activity of Complex I.

Steady-state kinetics of horse-liver alcohol dehydrogenase with a covalently bound coenzyme analogue.

The steady-state kinetics of the enzyme modified by affinity labelling with NAD analogue, nicotinamide-N6-[N-(6-aminohexyl)carbamoylmethyl]-adenine dinucleotide, has been investigated using a recycling reaction with p-nitrosodimethylaniline and n-butanol as substrates and compared to the kinetics of native alcohol dehydrogenase. The modified enzyme obeys a ping-pong mechanism involving two inactive enzyme forms (enzyme-NAD and enzyme-NADH complexes in the 'open' conformations, the nicotinamide moieties of the coenzymes being out of the active center). The rate of p-nitrosodimethylaniline reduction in the reaction catalyzed by the modified enzyme is comparable to that observed in the presence of the native enzyme. On the other hand, the oxidation of butanol by the modified enzyme is essentially slower under our experimental conditions (pH 8.5). The measurements in the presence of specific alcohol dehydrogenase inhibitors competing with substrates and coenzymes (isobutyramide, pyrazole and AMP) revealed that the relative portion of the inactive 'open' form of the enzyme-NADH complex is negligible, whereas the 'open' form of the enzyme-NAD complex seems to represent a more significant portion (about 30%) under the conditions used.

Reaction of the Z isomer of 4-trans-(N,N-dimethylamino)cinnamaldoxime with the liver alcohol dehydrogenase-oxidized nicotinamide adenine dinucleotide complex.

The Z isomer of 4-trans-(N,N-dimethylamino)-cinnamaldoxime, (Z)-DMOX (lambda maxH2O 354 nm), forms a ternary complex with NAD+ and equine liver alcohol dehydrogenase. The 3-acetyl (3-acetyl-PdAD+), 3-thiocarboxamide (3-thio-NAD+), 3-iodo (io3PdAD+) and nicotinamide mononucleotide (NMN+) analogues of NAD+ also form ternary complexes with enzyme and (Z)-DMOX. These complexes are characterized by large red-shifts in the UV-visible spectrum of bound (Z)-DMOX (lambda max 428 nm for the NAD+ complex) and new spectral bands in the 280-340-nm region associated with the pyridine moieties of NAD+ and the NAD+ analogues. The ternary enzyme-NAD+-(Z)-DMOX complex is weakly fluorescent (lambda ex 430 nm; lambda em max 505 nm) and strongly quenches the residual tryptophan fluorescence of the enzyme-NAD+ binary complex. (Z)-DMOX binds with high affinity to the enzyme-NAD+ complex (Kd less than or equal to 4 X 10(-9) M at pH 8.75 and 25 degrees C), and similarly high affinities were found for the 3-acetyl-PdAD+, 3-thio-NAD+, and io3PdAD+ complexes. Binding is much weaker to the enzyme-NMN+ complex. The active site specifically substituted Co(II), Ni(II), Cu(II), and Cd(II) enzyme derivatives and the enzyme species lacking any metal ion at the active site (apoenzyme) also form ternary complexes with (Z)-DMOX in which the DMOX UV-visible spectrum is red-shifted (ranging from 43 to 83.5 nm). The complexes formed with the Zn(II) and Co(II) enzymes are characterized by relatively high affinities for (Z)-DMOX and by spectra that are independent of pH over the range 6-10. The affinity of the apoenzyme-NAD+ complex for (Z)-DMOX is much lower, and the spectrum of the complex is pH dependent with lambda max = 430 nm at pH 7 and lambda max = 397 nm at pH 10. The rate of (Z)-DMOX dissociation from the apoenzyme complex was found to be approximately 10(3)-fold greater than the rates observed for the metal ion substituted enzymes. The 280-340-nm spectral bands appear to result from the dihydropyridine moieties of covalent adducts formed between (Z)-DMOX and NAD+ and the NAD+ analogues. The large red-shifts of the (Z)-DMOX spectrum result from the bonding of the oxime nitrogen to a strong electrophilic center (either the active site zinc ion or the nicotinamide ring of NAD+.)(ABSTRACT TRUNCATED AT 400 WORDS)

Novel nucleoside inhibitors of guanosine metabolism as antitumor agents

A detailed study of the inhibition of DR And TR in the SkLu-1 line of human lung adenocarcinoma has shown that TR significantly inhibits this tumor line, probably via inhibition of IMP dehydrogenase by the corresponding TAD analog of NAD. DR exhibiteda similar degree of inhibition in this cell line. In a system devised to detect the inhibition of cloning efficiency of the SkLu cells, DR showed a 50% inhibition at 4 × 10 −3 m and TR at 1 × 10 −4 m . When DR and TR were used in combination, the ID 50 was decreased to 3 × 10 −5 m . The study of DR in a number of human carcinoma cell lines revealed that de novo purine biosynthesis was significantly inhibited; however, in the SkLu-1 lung carcinoma cells this inhibition was not observed. The synergism observed in this cell line is presently viewed as potentially due to both agents acting on IMP dehydrogenase at different sites.

Reconstituted mitochondrial transhydrogenase is a transmembrane protein

Bovine heart mitochondrial transhydrogenase, a redox-linked proton pump, can be functionally and asymmetrically inserted into liposomes by a cholate-dialysis procedure such that the active site faces the external medium. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), a membrane-impermeant photoprobe, when encapsulated in the vesicles, covalently modified the enzyme and inhibited transhydrogenation between NADPH and the 3-acetylpyridine analog of NAD + (AcPyAD +) in a light-dependent manner. External AcPyAD + increased the rate of inactivation several fold, whereas NADPH, NADP +, and NADH were without effect. Labelling of the enzyme by intravesicular [ 35S]NAP-taurine was enhanced by AcPyAD + and NADP +, decreased by NADH, and not significantly affected by NADPH. These results indicate that transhydrogenase spans the membrane and that substrate binding alters the conformation of that domain of the enzyme protruding from the inner surface of the membrane.

The role of nucleotide pyrophosphatase in Mullerian duct regression

Mullerian inhibiting substance (MIS), a glycoprotein from the fetal testis causing regression of the embryonic Mullerian duct, can be inhibited in vitro in the presence of Mn 2+ by a wide range of nucleotides including GTP, NAD, ATP, AMP, and several nonhydrolyzable synthetic ATP analogs. Extracellular nucleotide pyrophosphatase (NPPase), an enzyme able to hydrolyze the wide variety of the nucleotides and analogs found to inhibit Mullerian duct regression, was studied by histochemical staining (H. Sierakowska and D. Shugar (1963). Biochem. Biophys. Res. Commun. 11, 70–74) to determine if NPPase localized in or around the Mullerian duct during regression. Frozen sections of urogenital ridges from 14 1 2 - to 17 1 2 - day rat fetuses ( n = 77) were incubated with a-naphthyl thymidine-5′-phosphate (naphthyl TMP) and Fast Red TR. Nucleotide pyrophosphatase hydrolyzes naphthyl TMP, releasing naphthol, which then reacts with Fast Red to produce color at the enzyme site. Nucleotide hydrolysis was detected around regressing male ( n = 16) Mullerian duct cells at 16 1 2 days of gestation, but no hydrolysis was detected around female ( n = 17) Mullerian duct cells at any stage. Controls ( n = 24) incubated without substrate did not stain. Addition of exogenous ATP ( n = 20) to the histochemical incubation medium inhibited nucleotide hydrolysis on male Mullerian ducts, suggesting that this staining is specific for pyrophosphatase activity. Results in vivo were confirmed in vitro by incubating 14 1 2 day female rat urogenital ridges with MIS for 72 hr prior to histochemical staining. The addition of testosterone to MIS was obligatory to detect staining in vitro ( n = 10). The localized NPPase activity around the regressing Mullerian duct suggests that NPPase may appear as a consequence of duct regression and may act to control the degree of membrane phosphorylation by degrading excess trinucleotides.

Properties of horse liver alcohol dehydrogenase modified by the affinity label 3-chloroacetylpyridine-adenine dinucleotide

The reactive analogue of NAD +, CPAD +, was incorporated in the horse liver alcohol dehydrogenase (EC 1.1.1.1) linearly with its inactivation, to stoichiometry, with no apparent subunit interaction. No hydride transfer could take place in the modified enzyme, nor the interaction of trans-4- N, N-dimethylaminocin-namaldehyde with its reduced form, indicating an impairment of the accessibility to the catalytic zinc atom. The labeling in the enzyme, alkylated by [ carbonyl- 14C]CPAD + was not stable, with a half-life of 32 h.

Conversion of 2-β-D-ribofuranosylselenazole-4-carboxamide to an analogue of nad with potent imp dehydrogenase-inhibitory properties

Conversion of 2-β-D-ribofuranosylselenazole-4-carboxamide to an analogue of nad with potent imp dehydrogenase-inhibitory properties

Characterization of pyridine nucleotide binding site of UDP-glucose 4-epimerase from Saccharomyces fragilis.

UDP-glucose 4-epimerase from Saccharomyces fragilis has 1 mol of NAD firmly bound per mol of the dimeric apoenzyme. This prevents a direct study of the coenzyme binding site of the protein. Dissociation of the dimer with p-chloromercuribenzoate and its reconstitution with exogenous NAD or one of its analogues and 2-mercaptoethanol provides an indirect method of study of the site. Depending on the reconstitution properties, the analogues can be classified in the following groups: (i) analogues that have no affinity for the site; (ii) analogues that have affinity but are not incorporated into the apoenzyme; (iii) analogues that produce catalytically inactive holoenzymes; and (iv) analogues that produce catalytically active holoenzymes. Minimum structural requirements that lead to affinity for the coenzyme site and to binding to the apoenzyme can also be discerned from these studies. Reconstitution with etheno-NAD, a fluorescent analogue of NAD, indicates the presence of a hydrophobic pocket for the adenosine subsite.

Adenosine diphosphoribose transfer reactions catalyzed by Bungarus fasciatus venom NAD glycohydrolase.

Reactions catalyzed by purified Bungarus fasciatus venom NAD glycohydrolase were demonstrated to include ADP-ribose transfer from NAD to alcohols and to imidazole derivatives to produce a variety of ADP-ribosides. The formation of products was monitored by high performance liquid chromatography. In the enzyme-catalyzed alcoholysis of NAD, the ratio of n-alkyl-ADP-riboside formed to the hydrolytic product, ADP-ribose, increased linearly with alcohol concentration. The effectiveness of alcohols as acceptors of the ADP-ribose moiety in these reactions increased with increasing chainlength of the alcohol used. Linear positive chainlength effects extended from methanol to pentanol suggesting facilitation of these reactions by nonpolar interactions. In the methanolysis reaction, NADP, thionicotinamide adenine dinucleotide, nicotinamide-1, N6-ethenoadenine dinucleotide, and 3-acetylpyridine adenine dinucleotide were shown to be as effective as NAD as donor substrates. The NAD glycohydrolase-catalyzed ADP-ribose transfer to pyridine bases to form NAD analogs was studied at pyridine base concentrations above those determined to be saturating for the base exchange reaction. Under these conditions, the ratio of base exchange to hydrolysis of NAD was directly related to the pKa of the ring nitrogen of the pyridine base employed. In addition to alcoholysis and pyridine-base exchange reactions, the snake venom enzyme was demonstrated to catalyze an ADP-ribose transfer reaction to imidazole derivatives. Arginine methyl ester was ineffective as an ADP-ribose acceptor molecule in these reactions.

Studies on the mechanism of action of tiazofurin metabolism to an analog of NAD with potent IMP dehydrogenase-inhibitory activity

Following the parenteral administration of tiazofurin, 2-β- d-ribofuranosyl-thiazole-4-carboxamide (thiazole ncleoside, TR), a potent but reversible inhibitor of IMP dehydrogenase is generated in subcutaneous nodules of the P388 leukemia. The compound responsible for this effect has been isolated from homogenates of the tumor by ion-exchange HPLC, and its presence monitored by enzyme-inhibition assay. The inhibitor has also been prepared by incubation of tiazofurin with P388 cells in culture. Chromatographically, the inhibitory principle exhibits a moderately strong set negative charge at pH 3, and elutes in the general vicinity of the nucleoside-5′-diphosphates; its absorption maximum in aqueous solution (pH 7) lies at 252 nm. Exposure of the molecule to snake-venom phosphodiesterase or to nucleotide pyrophosphatase destroys its inhibitory potency, whereas other phosphodeisterases are either less effective or inert. Since these results suggested that the anabolite might be a dinucleotide with a phosphodiester linkage of the kind found in NAD, attempts were made to synthesize such an analogue from the 5′-monophosphate of thiazole nucleoside and ATP-Mg 2+, using a purified preparation of NAD pyrophosphorylase; modest yields were obtained of a compound with chromatographic, spectral and enzyme-inhibitory properties identical to those of the material isolated from P388 tumor nodules. This enzyme-synthesized material was radioactive when [ 3H]ATP was used as co-substrate, and yielded both AMP and thiazole nucleoside-5′-monophsophate on treatment with phosphodiesterase. It resisted attack by NAD glycohydrolase. An apparently identical dinucleotide was also synthesized chemically by means of the Khorana condensation. Mass spectral analysis and nuclear magnetic resonance studies with homogeneous preparations of both the enzymically and chemically synthesized compound were compatible with its being a dinucleotide in which the nicotinamide of NAD has been replaced by thiazole-4-carboxamide. Versus IMP dehydrogenase, the dinucleotide exhibited a K 1 of 2 × 10 −7 M and was non-competitive with NAD as the variable substrate. Other NAD utilizing enzymes, including representative dehydrogenases and poly ADP ribose polyemrase, were, by comparison to mammalian IMPD, resistant to inhibition by TAD. The properties of this novel dinucleotide are compared and contrasted with those of analogs of NAD containign modifications in the pyridine, adenine or ribofuranose rings, as well as in the pyrophosphate bridge.

A word of caution: 1,4,5,6-tetrahydronicotinamide adenine dinucleotide (phosphate) should be used with care in acidic and neutral media

The decomposition of 1,4,5,6-tetrahydronicotinamide adenine dinucleotide (phosphate) (5,6-THN)AD(P) under neutral and acidic conditions is catalyzed by general acid. Adenosine 5′-diphosphoribose (ADPR) has been isolated in high yield by high-performance liquid chromatography from decomposition of (5,6-THN)AD in phosphate buffer, pH 6.8. The limitation of conditions under which (5,6-THN)AD(P) should be used as an analog of NAD(P)H are emphasized.

The conversion of 2-β-D-ribofuranosylthiazole-4-carboxamide to an analogue of NAD with potent IMP dehydrogenase-inhibitory properties

The conversion of 2-β-D-ribofuranosylthiazole-4-carboxamide to an analogue of NAD with potent IMP dehydrogenase-inhibitory properties

An enzymatically inactive variant of human lactate dehydrogenase-LDH BGUA-1 Study of subunit interaction

The LDH BGUA-1 variant is an electrophoretic variant which is enzymatically inactive. It is only detectable because of its ability to form heterotetramers with A and/or active B subunits and alter the electrophoretic pattern, although all evidence suggests the B-GUA-1 subunits are always enzymatically inactive. All tetrameric combinatious of active plus inactive subunits including either an A or active B plus three inactive B subunits possess enzymatic activity. The heterotetramers composed of A and B-GUA-1 subunits are more thermostable than A 4 homotetramers but less thermostable than normal AB heterotetramers. The AB-GUA-1 heterotetramers composed of active A and inactive B subunits have a K m for pyruvate, for lactate and for NADH which is similar to that observed for normal AB heterotetramers. The substrate specificity of the A plus normal B heterotetramer and the A plus variant B heterotetramer with α-hydroxybutyrate or the acetylpyridine analog of NAD as substrate are similar, while differences between the normal and variant erythrocyte isozymes are observed when glyoxalate is the substrate. Interaction with the B-GUA-I subunit reduces the sensitivity of the A subunit to urea inhibition (independent of dissociation), as does the active B subunit, but the inactive B subunit does not modulate the inhibition by oxalate. Although a single active subunit in a tetrameric conformation is sufficient for enzymatic activity, many of the kinetic properties of the lactate dehydrogenase molecule reflect the tetrameric structure rather than the sum of independent subunits. Thus communication among the subunits must exist and conformational changes which affect the catalytic properties of the enzyme ( l-lactate:NAD + oxidoreductase, EC 1.1.1.27) must occur during tetramer formation.

The synthesis of deuterium-substituted, spin-labeled analogues of AMP and NAD + and their use in ESR studies of lactate dehydrogenase

Two spin-labeled analogues of AMP and NAD + were synthesized, in which a perdeuterated nitroxide radical (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, TEMPAMINE) was attached to C-6 or C-8 position of the adenine ring. The ESR spectra of these derivatives exhibit a 4-fold increase in sensitivity and a concomitant decrease in line-width as compared to the corresponding protonated analogues. The improved resolution of composite spectra consisting of freely tumbling and immobilized components is demonstrated in ternary complexes of the spin-labeled NAD + derivatives with lactate dehydrogenase ( l-lactate:NAD + oxidoreductase, EC 1.1.1.27) and oxalate.