Concentrations Of DPNH Research Articles

Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate.

Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)

DPNH peroxidase: Effector activities of DPN +

At suboptimal H 2O 2 concentrations, DPNH inhibits the peroxidase activity of the flavoprotein, DPNH peroxidase, by converting the enzyme to an unstable intermediate that decays slowly to inactive enzyme. It is postulated that at concentrations of DPNH that are saturating for peroxidase activity, this unstable intermediate is responsible for most of the DPNH oxidation that is supported by alternate electron acceptors, such as O 2 and menadione. DPN + behaves as an activator by reversing the equilibria that lead to the unstable intermediate, thus converting the enzyme to the kinetically active complex that reduces H 2O 2. The data show that DPN + binding will stimulate the peroxidase activity (by lowering the K m for H 2O 2) and simultaneously lead to strong inhibition of both the rate of enzyme inactivation and the rate at which DPNH is oxidized by alternate electron acceptors.

Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus

The rates of oxidation of ent-kaur-16-ene to ent-kaur-16-en-19-ol, ent-kaur-16-en-19-al, ent-kaur-16-en-19-oic acid, and ent-kaur-16-en-7alpha-ol-19-oic acid are maximal in microsomes prepared from the endosperm of immature Marah macrocarpus seeds in which the cotyledons are approximately one-half the overall length of the seed. The supernatant fraction remaining from the preparation of the microsomes contains factors which stimulate the rates of oxidation catalyzed by the microsomes. Added TPNH is more effective than added DPNH in meeting the requirement for reduced pyridine nucleotide. A mixture of DPNH, ATP, and TPN(+) is much more effective than DPNH alone. Experiments with 2,4-dinitrophenol as a selective inhibitor indicate that the ATP-stimulated synthesis of TPNH which occurs in these microsomes in the presence of this mixture of coenzymes provide TPNH for use in the mixed function oxidations. Relatively low concentrations of DPNH and TPNH together are much more effective than either alone at equivalent concentration. This is consistent with the involvement of two pathways of electron transfer associated with the mixed function oxidations, one of which preferentially utilizes TPNH and the other favoring DPNH. FAD added to microsomes at an optimal concentration of about 10 mum in the presence of TPNH stimulates the rate of the oxidations; higher concentrations are inhibitory. FMN by itself does not produce this stimulation. However, FMN and FAD added together at low concentrations (0.5 mum each) have approximately the same effectiveness as FAD alone at 10 mum. This suggests a role for both flavin nucleotides in the normal electron transfer pathways associated with these oxidations. Some of the stimulatory properties of the supernatant fraction may be accounted for by its content of reduced pyridine nucleotides, FAD, and FMN; the concentrations of FAD and FMN were determined to be 1.1 mum and 0.4 mum, respectively. However, the effects of the supernatant fraction are not completely explained by its content of these coenzymes since other experiments indicate the presence of a heat-labile, nondialyzable stimulatory factor(s) in the supernatant fraction in addition to heat-stable, dialyzable fractors.

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.

Affinity labeling of a regulatory site of bovine liver glutamate dehydrogenase.

A new adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), has been synthesized which reacts covalently with bovine liver glutamate dehydrogenase. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of DPNH. Both of these effects are irreversibly decreased upon incubation of the enzyme with the adenosine analog, 3'-p-fluorosulfonyl-benzoyladenosine (3'-FSBA), while the intrinsic enzymatic activity as measured in the absence of regulatory compounds remains unaltered. A plot of the rate constant for modification as a function of the 3'-FSBA concentration is not linear, suggesting that the adenosine derivative binds to the enzyme (Ki equals 1.0 times 10-4 M) prior to the irreversible modification. Protection against modification by 3'-FSBA is provided by ADP and by high concentrations of DPNH, but not by the inhibitor GTP, the substrate alpha-keto glutarate, the coenzyme TPNH, or low concentrations of the coenzyme DPNH. The isolated altered enzyme contains approximately 1 mol of sulfonylbenzoyladenosine per peptide chain, indicating that a single specific regulatory site has reacted with 3'-tfsba. the modified enzyme exhibits normal Michaelis constants for its substrates and is still inhibited by GTP, albeit at a higher concentration, but it is not inhibited by high concentrations of DPNH. Although ADP does not appreciably activate the modified enzyme, it does (as in the case of the native enzyme) overcome the inhibition of the modified enzyme by GTP. These results suggest that ADP can bind to the modified enzyme, but that its ability to activate is affected indirectly by the modification of the adjacent tdpnh inhibitory site. It is proposed that the regulatory sites for ADP and DPNH are partially overlapping and that 3'FSBA functions as a specific affinity label for the DPNH inhibitory site of glutamate dehydrogenase. It is anticipated that 3'-p-fluorosulfonylbenzolyadenosine may act as an affinity label of other dehydrogenases as well as of other classes of enzymes which use adenine nucleotides as substrates or regulators.

Microsomal electron transport reactions: II. The use of reduced triphosphopyridine nucleotide and/or reduced diphosphopyridine nucleotide for the oxidative N-demethylation of aminopyrine and other drug substrates

The ratio of formaldehyde formed to TPNH oxidized during aminopyrine oxidative demethylation as catalyzed by rabbit liver microsomes was found to be about 0.5. This is less than the expected 1:1 ratio for a mixed function oxidase reaction and may reflect the oxidation of TPNH by other reactions. Similar results were obtained when measuring the oxidative demethylation of codeine and ethylmorphine. In all cases the addition of DPNH significantly increased the yield of formaldehyde formed in the presence of TPNH. The stimulatory effect of DPNH was a linear function of the DPNH concentration added until the initial concentrations of DPNH and TPNH were equal. Increasing the DPNH concentration above a DPNH:TPNH ratio of 1:1 had no further effect upon the final concentration of formaldehyde formed. This observation, as well as the inhibition of DPNH-supported aminopyrine metabolism by TPN +, argue against the role of a transhydrogenase mechanism for the DPNH effect. The rate of DPNH oxidation catalyzed by liver microsome was also observed to increase markedly in the presence of TPNH.

Regulatory Mechanisms Involving Nicotinamide Adenine Nucleotides as Allosteric Effectors: I. Control characteristics of malate dehydrogenase

Abstract The malate dehydrogenase of Escherichia coli is controlled by DPNH in an allosteric manner. This is concluded from kinetic studies in which oxalacetate (in the forward direction) and malate (in the reverse direction) are, individually, the variable substrates and DPNH is the inhibitor. With oxalacetate as the variable substrate the initial velocity plots at very low (0.013 mm) and very high (0.65 mm) concentrations of DPNH are hyperbolic but become sigmoidal at intermediate ranges. Similarly, when malate is the variable substrate, the hyperbolic rate concentration plots in the absence of DPNH become sigmoidal in its presence. It has shown that ATP, ADP, and AMP also inhibit the enzyme in an allosteric manner, possibly by binding at the DPNH site. From a consideration of the kinetic data it is proposed that there is an active and an allosteric site for DPNH on the enzyme surface with only the active site releasing a product. The cooperativity of the rate concentration plots is explained on the basis of the development of alternate pathways for substrate binding and product release in the presence of DPNH. The physiological importance of inhibition of malate dehydrogenase (and other enzymes) by DPNH is considered and the conclusion is reached that this inhibition is necessitated in bacteria because of the absence of rigid, compartmentation controls and the desirability of preventing gluconeogenetic channels from functioning when growth is occurring on glucose.

Regulatory Mechanisms Involving Nicotinamide Adenine Nucleotides as Allosteric Effectors: II. Control of phosphoenolpyruvate carboxykinase

Abstract Phosphoenolpyruvate carboxykinase of Escherichia coli is inhibited in an allosteric manner by DPNH. This inhibition is relatively quite specific. Other coenzymes such as TPN+ and DPN+ are completely ineffective and TPNH is only 15% as effective as DPNH in the inhibition of the enzyme. In cells grown on glucose DPNH concentration is 1.5 to 2 times higher than in those grown on succinate. From this it is concluded that DPNH levels serve as indicators of the state of glycolysis, and DPNH inhibition of Penolpyruvate carboxykinase is seen as a mechanism whereby gluconeogenetic channels can be prevented from functioning when active glycolysis is progressing. A kinetic study of the enzyme in the presence and absence of DPNH is presented. Initial velocity and product inhibition patterns show that the free enzyme form possibly binds ADP (or ATP) first. The inhibitor and the remaining substrates possibly bind to the E-ADP or E-ATP complexes only. The sigmoidality of oxalacetate, P-enolpyruvate, and bicarbonate plots is postulated to arise as a result of an isomerization of the enzyme in the presence of DPNH.

A fluorescent spot test for creatine kinase

A fluorescent spot test for creatine kinase in serum is described. Sensitivity varied with DPNH concentration, incubation period, size, and type of sample. Under optimum conditions samples with varying degrees of excess enzyme were distinguished from those with normal activity (<12 Sigma units). The method is useful as both a screening and diagnostic procedure in the laboratory.

Binding by Glutamate Dehydrogenase of Reduced Diphosphopyridine Nucleotide: EFFECTS OF REGULATORY (“ALLOSTERIC”) REAGENTS AND IONIC STRENGTH

Glutamic dehydrogenase from bovine liver, studied by equilibrium dialysis, has a single reduced diphosphopyridine nucleotide-binding site per protein chain of molecular weight 52,000. Guanosine triphosphate increases DPNH binding, while adenosine diphosphate and high ionic strength decrease binding. DPNH binding is independent of the concentration-dependent aggregation of the enzyme, and shows a simple dependence on DPNH concentration. These studies, therefore, fail to show a separate regulator site for DPNH on the enzyme, and do not show evidence for cooperative binding in this allosteric protein.

Fluorescence assay of high concentrations of DPNH and TPNH in a spectrophotofluorometer

Under conditions usually employed to measure DPNH and TPNH fluorescence in spectrophotofluorometers, the curve of fluorescence vs. concentration (response curve) becomes nonlinear above a concentration of 10 −5 M. The nonlinearity is due to absorption of the incident light by the solution rather than reabsorption of the emitted light, because the corrected excitation and emission spectra of DPNH show little overlap. The response curve can be improved by use of a cell holder modified to allow eccentric placement of the cuvette, or by the use of a microcell with a short path length. Furthermore, since the absorbancy of DPNH and TPNH is low at 395 mμ, excitation with the monochromator set at this wavelength produces response curves which are linear up to 5 × 10 −4 M, if used in conjunction with a microcell.

Vitamin E deficiency in the rat: I. Effect of substrate concentration on respiratory decline in liver homogenates from rats fed a vitamin E-deficient diet

Male Holtzman rats were maintained for approximately 3 weeks on either a vitamin E-deficient or vitamin E-supplemented diet. The capacity of liver homogenates from these animals to oxidize succinate, α-ketoglutarate, β-hydroxybutyrate, pyruvate, malate, and DPNH was measured over extended periods of time, and in the presence of different substrate concentrations. The decline in rate of oxidation, with time of incubation, differed, depending on the substrate employed, the concentration of substrate, and whether vitamin E was present or absent in the diet of the donor animal. The concentration of α-ketoglutarate, β-hydroxybutyrate, pyruvate, and malate influenced the respiratory decline of vitamin E-deficient homogenates differently from the manner in which it affected the decline of supplemented homogenates. There was no apparent differences in respiratory decline between deficient and supplemented homogenates due to differences in succinate or DPNH concentration at any given substrate concentration studied.

RADIOLYSIS OF REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE IN AQUEOUS SOLUTION.

The radiolysis of DPNH solutions by 250-kvp x rays has been followed utilizing the absorption at 340 m mu , the corresponding fluorescence at 470 m mu , and the coenzymatic activity with alcohol dehydrogenase. All three parameters are decreased simultaneously and this reduction in DPNH concentration is neither linear nor logarithmic with dose. The coenzyme is protected in the presence of protein; the protection afforded by glutannic dehydrogenase, a DPNHbinding enzyme, is equal to that of serum albumin at the same weight concentration. The radiolysis of DPNH is independent of pH over the range 4.8--7.5 and is accompanied by an initial increase in pH, 260-m mu sbsorption, and cyanide reactivity. These observations suggest that DPNH is the first product formed which is, in turn, degraded logarithmically. A kinetic model for the radiolysis was formulated which gives a G(-DPNH) of 2.3 in air and 1.2 in oxygen-free nitrogen. (auth)

Reduced pyridine nucleotides as activators of certain reactions catalyzed by peroxidase

Conditions are described in which the aerobic oxidation of epinephrine and ferrocytochrome c by peroxidase is dependent on the presence of DPNH or TPNH in the reaction mixture and is stimulated by thyroxine, certain thyroxine analogues and estradiol. In the case of epinephrine oxidation, DPNH and thyroxine abolished a lag period which is associated with the aerobic oxidation of epinephrine in the absence of these substances. The oxidation of epinephrine and ferrocytochrome c by peroxidase in the presence of Mn ++, DPNH, thyroxine and oxygen is inhibited by catalase and by certain thyroxine analogues. The latter inhibition is overcome by an increase in DPNH concentration. Thyroxine is inactivated by the H 2O 2-peroxidase system and this inactivation is inhibited by the presence of a suitable hydrogen donor ( e.g. DPNH, epinephrine) in the reaction mixture. A DPNH dependent inactivation of thyroxine by the Mn ++-peroxidase-oxygen system also is described. It is suggested that the Mn ++ dependent aerobic oxidation of reduced pyridine nucleotides by peroxidase facilitates certain secondary oxidations as a result of the generation of hydrogen peroxide in the course of the reaction.