Transition state structure for ADP-ribosylation of eukaryotic elongation factor 2 catalyzed by diphtheria toxin

Bacterial protein toxins are the most powerful human poisons known, exhibiting an LD(50) of 0.1-1 ng kg(-)(1). A major subset of such toxins is the NAD(+)-dependent ADP-ribosylating exotoxins, which include pertussis, cholera, and diphtheria toxin. Diphtheria toxin catalyzes the ADP ribosylation of the diphthamide residue of eukaryotic elongation factor 2 (eEF-2). The transition state of ADP ribosylation catalyzed by diphtheria toxin has been characterized by measuring a family of kinetic isotope effects using (3)H-, (14)C-, and (15)N-labeled NAD(+) with purified yeast eEF-2. Isotope trapping experiments yield a commitment to catalysis of 0.24 at saturating eEF-2 concentrations, resulting in suppression of the intrinsic isotope effects. Following correction for the commitment factor, intrinsic primary kinetic isotope effects of 1.055 +/- 0.003 and 1.022 +/- 0.004 were observed for [1(N)'-(14)C]- and [1(N)-(15)N]NAD(+), respectively; the double primary isotope effect was 1.066 +/- 0.004 for [1(N)'-(14)C, 1(N)-(15)N]NAD(+). Secondary kinetic isotope effects of 1.194 +/- 0.002, 1.101 +/- 0.003, 1.013 +/- 0.005, and 0.988 +/- 0.002 were determined for [1(N)'-(3)H]-, [2(N)'-(3)H]-, [4(N)'-(3)H]-, and [5(N)'-(3)H]NAD(+), respectively. The transition state structure was modeled using density functional theory (B1LYP/6-31+G) as implemented in Gaussian 98, and theoretical kinetic isotope effects were subsequently calculated using Isoeff 98. Constraints were varied in a systematic manner until the calculated kinetic isotope effects matched the intrinsic isotope effects. The transition state model most consistent with the intrinsic isotope effects is characterized by the substantial loss in bond order of the nicotinamide leaving group (bond order = 0.18, 1.99 A) and weak participation of the attacking imidazole nucleophile (bond order = 0.03, 2.58 A). The transition state structure imparts strong oxacarbenium ion character to the ribose ring even though significant bond order remains to the nicotinamide leaving group. The transition state model presented here is asymmetric and consistent with a dissociative S(N)1 type mechanism in which attack of the diphthamide nucleophile lags behind departure of the nicotinamide.     

Remote nitrogen-15 isotope effects on addition of cyanide to NAD

The reversible reaction NAD + CN(-)----NAD-CN was examined for remote secondary 15N isotope effects caused by isotopic substitution at the ring nitrogen of the nicotinamide group. These were compared with analogous effects for dehydrogenase-catalyzed reactions, since both cyanide and the hydride ion add at the N-4 position of the nicotinamide ring. The 15N effects on the rate constants for the forward and reverse processes were examined directly by conducting both the normal and isotopic reactions simultaneously under carefully controlled conditions in the sample and reference cells of a dual-beam spectrophotometer. In both cases, the 15N kinetic isotope effect differed from 1.00 by considerably less than 0.01. The 15N equilibrium isotope effect, 15K, was obtained as the ratio of equilibrium constants measured separately with natural-abundance and labeled NAD by using a concentration jump procedure [1.004 +/- 0.002 (cyanide addition)]. A similar value for 15K of 1.010 +/- 0.008 was obtained in an analogous manner for the reaction catalyzed by lactate dehydrogenase: NAD + lactate----pyruvate + NADH + H+. The latter value is significantly smaller than a previously reported value obtained from kinetic studies [1.044 +/- 0.012; Cook, P. F., Oppenheimer, N. J., & Cleland, W. W. (1981) Biochemistry 20, 1817]. The present value also is smaller than might be expected for a change in bond order from 4 to 3 [Cleland, W. W. (1980) Methods Enzymol. 64, 104-125] on the basis of the canonical resonance structures for NAD and NADH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of biopterin. Studies on the mechanism of 6-pyruvoyltetrahydropteridine synthase

[1'-3H]- and [2'-3H]dihydroneopterin triphosphate (NH2TP) were prepared enzymatically from [4-3H]- and [5-3H]glucose and converted to tetrahydrobiopterin (BH4) by an extract from bovine adrenal medulla. The formation of BH4 from both [1'-3H]- and [2'-3H]-NH2TP proceeds with virtually complete loss of the respective tritium label. The breaking of the CH-bond at C-1' is characterized by a kinetic isotope effect of 2.6 +/- 0.5. A smaller kinetic isotope effect of 1.5 +/- 0.2 was found for the breaking of the CH-bond at C-2'.     

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.     

Dehydrogenase-dependent ethanol metabolism in deer mice (Peromyscus maniculatus) lacking cytosolic alcohol dehydrogenase. Reversibility and isotope effects in vivo and in subcellular fractions

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH-) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 +/- 0.1 and 3.2 +/- 0.8 in the two strains, respectively. To an equal extent in both strains, ethanol elimination was accompanied by an ethanol-acetaldehyde exchange with an intermolecular transfer of hydrogen atoms, indicating the occurrence of dehydrogenase activity. This exchange was also observed in perfused deer mouse livers. Based on calculations it was estimated that at least 50% of ethanol elimination in ADH- deer mice was caused by the action of dehydrogenase systems. NADPH-supported cytochrome P-450-dependent ethanol oxidation in liver microsomes from ADH+ and ADH- deer mice was not stereoselective and occurred with a D(V/K) of 3.6. The D(V/K) value of catalase-dependent oxidation was 1.8, whereas a kinetic isotope effect of cytosolic ADH in the ADH+ strain was 3.2. Mitochondria from both ADH+ and ADH- deer mice catalyzed NAD+-dependent ethanol oxidation and NADH-dependent acetaldehyde reduction. The kinetic isotope effects of NAD+-dependent ethanol oxidation in the mitochondrial fraction from ADH+ and ADH- deer mice were 2.0 +/- 0.1 and 2.3 +/- 0.3, respectively. The results indicate only a minor contribution by cytochrome P-450 to ethanol elimination, whereas the isotope effects are consistent with ethanol oxidation by the catalase-H2O2 system in ADH- deer mice in addition to the dehydrogenase systems.     

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of recombinant Aspergillus fumigatus mannitol-1-phosphate 5-dehydrogenase and its application for the stereoselective synthesis of protio and deuterio forms of D-mannitol 1-phosphate

A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a approximately 40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 degrees C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing (4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol 1-phosphate at pH 7.1 and 25 degrees C revealed a primary kinetic isotope effect of 2.9+/-0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes in culture

ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP-ribose production converted from [3H]NAD+ by ADP-ribosyl cyclase (2.015+/-0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained approximately 50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP-ribose from cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP-ribose by incubating cultures of intact astrocytes, the presence of Triton X-100 in the medium for permeabilizing cells increased cyclic ADP-ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP-ribose formation in crude membrane-associated cyclase activity. This isoproterenol-induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP-ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of beta-adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.     

Uridine diphosphate galactose 4-epimerase. pH dependence of the reduction of NAD+ by a substrate analog

Neutron activation analysis of UDP-galactose 4-epimerase from Escherichia coli for 53 metals shows that the enzyme does not contain any of these metals at significant levels. The substrate analog P1-5'-uridine-P2-glucose-6-yl pyrophosphate (UGP), a structural isomer of UDP-glucose with the sugar linked to UDP through the C-6 hydroxyl group, is an inactivator that irreversibly reduces epimerase.NAD+ to epimerase.NADH. The pH dependence of kobs reveals the essential involvement of an acidic group, kinetically measured pKa = 5.48 +/- 0.08, in unprotonated form and two weakly acidic or basic groups, apparent pKa values of 10.03 +/- 0.43, in protonated forms. Measurements of kobs as a function of [UGP] show that it is given by kobs = k[UGP]/(K + [UGP]) at a given pH, where K = 0.19 +/- 0.04 mM throughout the pH range 4.8-10.4. The pH-dependent first order rate constants range from 0.28 to 1.94 s-1, with the maximum value at pH 7.6 and decreasing at acidic and basic pH values. Reaction of [glucose-1-2H]UGP proceeds with kinetic isotope effects of 5.0, 2.1, 2.0, 1.9, and 3.5 at pH values 5.0, 6.2, 7.6, 9.0, and 10.0, respectively. Therefore, hydride transfer becomes rate-limiting at pH extremes but is not limiting at neutral pH, although deuteride transfer is significantly limiting at all pH values. The isotope effects facilitated correction of the kinetic pK values to the thermodynamic values 6.1-6.2 on the acid side and 9.0-9.6 on the alkaline side. We postulate that the group with pK1 = 5.5 (6.1-6.2 corrected) functions as an enzymic general base that abstracts the glucosyl C-1 hydroxyl proton in concert with transfer of the C-1 hydrogen and two electrons to NAD+. The pH dependence on the alkaline side may be related to the uridine nucleotide-dependent conformational transition that is an essential step in the reduction of epimerase.NAD+ to epimerase.NADH by sugars.     

Isotope effects in 3-dehydroquinate synthase and dehydratase. Mechanistic implications.

The mechanism of 3-dehydroquinate synthase was explored by incubating partially purified enzyme with mixtures of [1-14C]3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) and one of the specifically tritiated substrates [4-3H]DAHP, [5-3H]DAHP, [6-3H]DAHP, (7RS)-[7-3H]DAHP, (7R)-[7-3H]DAHP, or (7S)-[7-3H]DAHP. Kinetic and secondary 3H isotope effects were calculated from 3H:14C ratios obtained in unreacted DAHP, 3-dehydroquinate, and 3-dehydroshikimate. 3H was not incorporated from the medium into 3-dehydroquinate, indicating that a carbanion (or methyl group) at C-7 is not formed. A kinetic isotope effect kH/k3H of 1.7 was observed at C-5, and afforded support for a mechanism involving oxidation of C-5 with NAD. A similar kinetic isotope effect was found at C-6 owing to removal of a proton in elimination of phosphate, which is reasonably assumed to be the next step in 3-dehydroquinate synthase. Hydrogen at C-7 of DAHP was not lost in the cyclization step of the reaction, indicating that the enol formed in phosphate elimination participated directly in an aldolase-type reaction with the carbonyl at C-2. In the dehydration of 3-dehydroquinate to 3-dehydroshikimate the (7R) proton from (7RS)- or (7R)-[7-3H]DAHP is lost, indicating that the 7R proton occupies the 2R position in dehydroquinate. Hence the cyclization step occurs with inversion of configuration at C-7. A kinetic isotope effect kH/k3H = 2.3 was observed in the conversion of (2R)-[2-3H]dehydroquinate to dehydroshikimate. Hence loss of a proton from the enzyme-dehydroquinate imine contributed to rate limitation in the reaction.     

Inhibition of hepatic proteolysis by insulin. Role of hormone-induced alterations of the cellular K+ balance

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

The [4B-3H] NADH-H2O exchange reaction of the mitochondrial NADH dehydrogenase

The purified mitochondrial NADH dehydrogenase enzyme has been shown to catalyze a rapid [4B-3H] NADH-H2O exchange reaction. When the enzyme is subjected to a single freeze-thaw cycle there is a complete loss of NADH dehydrogenation without a measurable decrease in the [4B-3H] NADH-H2O exchange. Complete loss of the [4B-3H] NADH-H2O exchange follows brief exposure to ultraviolet photoirradiation. The differential sensitivity of the water exchange reaction and the dehydrogenase activity suggests a direct involvement of the enzymes flavin cofactor in the catalysis of the [4B-3H] NADH-H2O exchange. Arylazido-beta-alanyl NAD+ (A3'-0-[3-[N-4-azido-2-nitrophenyl)amino] propionyl]NAD+) is shown to be a potent photodependent inhibitor of the [4B-3H] NADH-H2O exchange activity following photoirradiation with visible light. This is consistent with the observed photodependent inhibition of the dehydrogenase activity by this photoprobe (Chen, S. and Guillory, R.J. (1981) J. Biol. Chem. 256, 8318-8323).     

Norepinephrine Metabolism in Man Using Deuterium Labelling: Origin of 4-Hydroxy-3-Methoxymandelic Acid

A double isotope labelling technique was used to simultaneously determine the in vivo turnover rates of 4-hydroxy-3-methoxyphenylglycol (HMPG) and 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) and the rate of HMPG oxidation to HMMA. Six healthy men were given intravenous injections of [2H3]HMPG and [2H6]HMMA and their plasma and urine samples analysed by gas chromatography--mass spectrometry (GC/MS) for the protium and deuterium species. HMPG and HMMA production rates were calculated by isotope dilution. The rate of HMPG oxidation to HMMA was obtained from the fraction of [2H3]HMPG recovered as [2H3]HMMA. The results showed that the entire production of HMMA, 1.11 +/- 0.21 mumol/h (mean +/- SE), could be accounted for by oxidation of HMPG, 1.49 +/- 0.31 mumol/h. In another experiment designed to avoid expansion of the HMPG body pool, a tracer dose of [14C]HMPG was given to the same subjects. The levels of [14C]HMPG and [14C]HMMA were measured in urine after extraction and separation by thin layer chromatography. Urinary excretion of endogenous HMPG and HMMA was determined by GC/MS. The results showed that the endogenous HMMA fraction of the total HMPG and HMMA urinary excretion rate, 0.57 +/- 0.04, was the same as the fraction of [14C]HMPG oxidized to [14C]HMMA, 0.62 +/- 0.01. Thus, HMPG is the main intermediate in the metabolic conversion of norepinephrine and epinephrine to HMMA in man.     

Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism

A neutral flavin semiquinone species was formed upon photoreduction of Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase whereas no flavin radical was detected by anaerobic reduction with NADH in the presence of m-hydroxybenzoate. In the latter case, the formation of flavin semiquinone is apparently thermodynamically unfavorable. A stereospecificity for the abstraction of the 4R-position hydrogen of NADH has been demonstrated for this hydroxylase. Deuterium and tritium isotope effects were observed with (4R)-[4-2H]NADH and (4R)-[4-3H]NADH as substrates. The DV effect indicates the existence of at least one slow step after the isotope-sensitive enzyme reduction by dihydropyridine nucleotide. A minimal kinetic mechanism has been deduced on the basis of initial velocity measurements and studies on deuterium and tritium isotope effects. Following this scheme, m-hydroxybenzoate and NADH bind to the hydroxylase in a random sequence. The flavohydroxylase is reduced by NADH, and NAD+ is released. Oxygen subsequently binds to and reacts with the reduced flavohydroxylase-m-hydroxybenzoate complex. Following the formation and release of water and gentisate, the oxidized holoenzyme is regenerated. The enzyme has a small (approximately 2-fold) preference for the release of NADH over m-hydroxybenzoate from the enzyme-substrates ternary complex.     

Permeability of Rickettsia prowazekii to NAD.

Rickettsia prowazekii accumulated radioactivity from [adenine-2,8-3H]NAD but not from [nicotinamide-4-3H]NAD, which demonstrated that NAD was not taken up intact. Extracellular NAD was hydrolyzed by rickettsiae with the products of hydrolysis, nicotinamide mononucleotide and AMP, appearing in the incubation medium in a time- and temperature-dependent manner. The particulate (membrane) fraction contained 90% of this NAD pyrophosphatase activity. Rickettsiae which had accumulated radiolabel after incubation with [adenine-2,8-3H]NAD were extracted, and the intracellular composition was analyzed by chromatography. The cells contained labeled AMP, ADP, ATP, and NAD. The NAD-derived intracellular AMP was transported via a pathway distinct from and in addition to the previously described AMP translocase. Exogenous AMP (1 mM) inhibited uptake of radioactivity from [adenine-2,8-3H]NAD and hydrolysis of extracellular NAD. AMP increased the percentage of intracellular radiolabel present as NAD. Nicotinamide mononucleotide was not taken up by the rickettsiae, did not inhibit hydrolysis of extracellular NAD, and was not a good inhibitor of the uptake of radiolabel from [adenine-2,8-3H]NAD. Neither AMP nor ATP (both of which are transported) could support the synthesis of intracellular NAD. The presence of intracellular [adenine-2,8-3H]NAD within an organism in which intact NAD could not be transported suggested the resynthesis from AMP of [adenine-2,8-3H]NAD at the locus of NAD hydrolysis and translocation.     

Pharmacokinetic studies of methionine in rats using deuterium-labeled methionine quantitative assessment of remethylation

The pharmacokinetics of methionine has been studied in rats by means of stable isotope methodology. After the i.v. bolus injection of [2H7]methionine (5 mg/kg body wt.), the plasma concentrations of [2H7]methionine, demethylated [2H4]homocysteine and remethylated [2H4]methionine were determined simultaneously with endogenous methionine and homocysteine by gas chromatography-mass spectrometry. The half-life for [2H7]methionine were 35.0 +/- 6.9 min. The appearance of the metabolites, [2H4]homocysteine and [2H4]methionine, in the plasma was very rapid. The fraction of [2H7]methionine that remethylated to [2H4]methionine through [2H4]homocysteine were 0.185 +/- 0.028. The administered [2H7]methionine did not influence the plasma levels of endogenous methionine and homocysteine. The present stable isotope methodology has made it possible to evaluate the pharmacokinetics of methionine, including the estimation of remethylation.     

Liberation of [3H]arachidonic acid and changes in cytosolic free calcium in fura-2-loaded human platelets stimulated by ionomycin and collagen.

Cytosolic Ca2+ levels and arachidonate liberation were investigated in platelets loaded with the fluorescent Ca2+ indicator dye fura-2, and labelled with [3H]arachidonate. Fura-2 was used in preference to quin2 because the latter interfered with [3H]arachidonate labelling of phospholipids. From a resting free Ca2+ level of around 100 nM, ionomycin (10-200 nM) evoked an instantaneous, concentration-dependent increase in cytosolic Ca2+ that only resulted in [3H]arachidonate liberation (up to 4-fold over control) at Ca2+ levels greater than 1 microM. Addition of collagen (10 micrograms/ml) evoked an elevation in Ca2+ up to 461 +/- 133 nM. These changes in Ca2+ were accompanied by a 2-4-fold elevation in [3H]arachidonate with depletion of [3H]phosphatidylcholine by 17 +/- 4% and [3H]phosphatidylinositol by 41 +/- 7%. Indomethacin (10 microM) reduced the elevation in Ca2+ by collagen to 115 +/- 18 nM but did not significantly inhibit the 2-4-fold increase in [3H]arachidonate. [3H]Phosphatidylcholine and [3H]phosphatidylinositol were decreased by 9 +/- 7% and 10 +/- 6%, respectively, with collagen in the presence of indomethacin. Stimulation of phosphoinositide turnover by collagen in the presence and absence of indomethacin was indicated by [32P]phosphatidate formation in cells prelabelled with [32P]Pi. This phosphatidate formation was decreased (75%) by the presence of indomethacin. In the presence of indomethacin, phorbol myristate acetate (20 nM) alone or in combination with ionomycin (30 nM) failed to stimulate arachidonate liberation despite a marked stimulation of aggregation. These results indicate that, whereas ionomycin requires Ca2+ in the microM range for arachidonate liberation, collagen, notably in the presence of indomethacin, does so at basal Ca2+ levels. The mechanisms underlying the regulation of arachidonate release by collagen are not clear, but do not appear to involve activation of protein kinase C, or an elevation of cytosolic free Ca2+.     

(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle

(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites.