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D C Phelps

Publications and source records attributed to D C Phelps.

18 recordsLinked to original sources
PubMed1989

Changes in Fructose 2,6-Bisphosphate Levels in Green Pepper (Capsicum annuum L.) Fruit in Response to Temperature.

The regulatory metabolite, fructose 2,6-bisphosphate (Fru 2,6-P(2)) was found in green pepper (Capsicum annuum L.). The Fru 2,6-P(2) level was found to: (a) rise rapidly in response to heat; (b) drop rapidly, followed by recovery, in response to cold storage of fruit and, (c) oscillate during cold storage of fruit. The possible existence of a relationship between chilling injury and Fru 2,6-P(2) is considered.

Journal Article↗
PubMed1985

Mitochondrial nicotinamide nucleotide transhydrogenase: nonidentical modification by N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline at the NAD(H) binding site.

The energy-linked nicotinamide nucleotide transhydrogenase (TH) purified from bovine heart mitochondria is inhibited by the carboxyl group modifiers, N,N'-dicyclohexylcarbodiimide (DCCD) and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ). With either reagent, complete activity inhibition corresponds to modification of one carboxyl group per 2 mol (monomers) of this dimeric enzyme, suggesting half-site reactivity toward DCCD and EEDQ [D. C. Phelps, and Y. Hatefi (1984) Biochemistry 23, 4475-4480; 6340-6344]. It has also been shown in the former reference that DCCD appears to modify TH at the NAD(H)-binding site. The present paper presents data suggesting that EEDQ also binds at or near the NAD(H)-binding domain of TH, but at a site not identical to that of DCCD: TH modified with and inhibited approximately 85% by EEDQ could be further labeled with [14C]DCCD to the extent of 70% of the maximum in the same time period that unmodified TH was modified by [14C]DCCD to near saturation (1 mol DCCD/TH dimer); DCCD-modified TH did not bind to NAD-agarose, while EEDQ-modified TH showed partial affinity for NAD-agarose; 5'-AMP completely protected TH against modification by DCCD, but showed only a weak protective effect against EEDQ; by contrast, NMNH, which is a TH substrate and binds to the NADH site, did not protect TH against DCCD, but completely protected the enzyme against attack by EEDQ. The results are consistent with the possibility that DCCD modifies TH where the 5'-AMP moiety of NAD(H) binds, while EEDQ modifies the enzyme where the NMN(H) moiety of NAD(H) resides.

Adenosine Monophosphate↗
PubMed1985

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.

Adenosine↗
PubMed1984

Effects of N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline on hydride ion transfer and proton translocation activities of mitochondrial nicotinamidenucleotide transhydrogenase.

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the mitochondrial energy-linked nicotinamidenucleotide transhydrogenase (TH). Our studies [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221; Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480] suggested that the inhibition site of DCCD is near the NAD(H) binding site, because NAD(H) and competitive inhibitors protected TH against inhibition by DCCD and, unlike the unmodified TH, the DCCD-modified TH did not bind to NAD-agarose. Others [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] could not demonstrate protection by NADH, obtained data indicating DCCD inhibits proton translocation by TH much more than hydride ion transfer from NADPH to 3-acetylpyridine adenine dinucleotide (AcPyAD), and concluded that DCCD modifies an essential residue in the proton channel of TH. The present studies show that N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inhibits TH. The inhibition is pseudo first order at several EEDQ concentrations, and the reaction order with respect to [EEDQ] is unity, suggesting that inhibition involves the interaction of one molecule of EEDQ with one active unit of TH. The EEDQ-modified TH reacts covalently with [3H]aniline, suggesting that the residue modified by EEDQ is a carboxyl group. More significantly, it has been shown that the absorbance change of oxonol VI at 630 minus 603 nm is a reliable reporter of TH-induced membrane potential formation in submitochondrial particles and that TH-catalyzed hydride ion transfer from NADPH to AcPyAD and the membrane potential induced by this reaction are inhibited in parallel by either DCCD or EEDQ.

Animals↗
PubMed1984

Interaction of purified nicotinamidenucleotide transhydrogenase with dicyclohexylcarbodiimide.

The inhibition of the energy-linked nicotinamidenucleotide transhydrogenase (TH; EC 1.6.1.1) by dicyclohexylcarbodiimide (DCCD) has been further studied because of its important mechanistic implications. We had shown earlier that TH bound to submitochondrial particles from bovine heart is inhibited by DCCD and that NAD(H) protects the enzyme against this inhibition [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221]. By contrast, Pennington and Fisher [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] working with purified TH concluded that NAD(H) does not protect against DCCD inhibition and that DCCD inhibition involves the TH proton channel rather than the nucleotide-binding active site. The present study shows that NAD(H) as well as AMP and ADP, which are known to bind to the NAD(H) binding site from competitive inhibition studies, protect the purified TH against inhibition by DCCD, whereas 2'-AMP and 3'-AMP, which bind to the NADP(H) site on TH, do not protect. In addition, it is shown that whereas the unmodified TH binds to NAD-agarose such that it is elutable by buffer containing NADH, the DCCD-modified enzyme does not bind to NAD-agarose. These results suggest strongly that DCCD binds at or near the NAD(H) binding site on TH. Another less likely possibility is that NAD(H) and DCCD bind to separate sites, but their bindings are mutually exclusive. With the use of [14C]DCCD, it has been shown that 100% activity inhibition corresponds to 0.5 mol of DCCD binding per mol of TH (Mr approximately 11 X 10(4].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1982

Substrate binding affinity changes in mitochondrial energy-linked reactions.

The effects of uncouplers and valinomycin plus nigericin (in the presence of K+) were studied on the apparent Km for substrates and apparent Vmax of the following energy-linked reactions catalyzed by submitochondrial particles: oxidative phosphorylation, NTP-33Pi exchange, ATP-driven electron transfer from succinate to NAD, and respiration-driven transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate. In all cases, partially uncoupling (up to 90%) concentrations of uncouplers of valinomycin plus nigericin were found to decrease apparent Vmax and to increase apparent Km. Results plotted as ln (Vmax/Km) versus the concentration of uncouplers or ionophores showed a linear decrease of the former as a function of increasing perturbant concentration (i.e., decreasing free energy). Because Vmax/Km may be considered as a measure of the apparent first-order rate constant for enzyme-substrate interaction and reflects the affinity between enzyme and substrate to form a complex, the results are consistent with the interpretation that membrane energization leads to a change in enzyme conformation with the resultant increase in enzyme-substrate affinity and facilitation of the reaction rate under consideration. The significance of these findings with respect to the mechanism of action of the energy-transducing systems studied is discussed.

Adenosine Triphosphate↗
PubMed1981

Inhibition of the mitochondrial nicotinamide nucleotide transhydrogenase by dicyclohexylcarbodiimide and diethylpyrocarbonate.

The mitochondrial nicotinamide nucleotide transhydrogenase enzyme (EC 1.6.1.1) is inhibited by treatment with dicyclohexylcarbodiimide or diethylpyrocarbonate. Both inhibitions are pseudo first order with respect to incubation time, and both reaction orders with respect to inhibitor concentration are close to unit, indicating that in each case inhibition results from the binding of one inhibitor molecule per active unit of the transhydrogenase enzyme. In the presence of either inhibitor, both the energy-linked and the nonenergy-linked transhydrogenation reactions are inhibited at about the same rate. The water-soluble carbodiimide, N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide, showed no inhibition, however, NAD(H) and reduced or oxidized 3-acetylpyridine adenine dinucleotide protected the enzyme against inhibition by dicyclohexylcarbodiimide, while NADP (but not NADPH) appeared to increase the rate of inhibition. Substrates did not protect the enzyme against inhibition by diethylpyrocarbonate. [14C]dicyclohexylcarbodiimide labeled the transhydrogenase enzyme in submitochondrial particles. Treatment of labeled particles with trypsin resulted in fragmentation of the transhydrogenase enzyme and loss of a labeled polypeptide of Mr = approximately 100,000 as determined by polyacrylamide gel electrophoresis.

Animals↗
PubMed1981

Inhibition of D(--)-beta-hydroxybutyrate dehydrogenase by modifiers of disulfides, thiols, and vicinal dithiols.

D(--)-beta-hydroxybutyrate dehydrogenase of beef heart mitochondria catalyzes the reversible oxidation of D-(--)-beta-hydroxybutyrate to acetoacetate in the presence of NAD. Both the membrane-bound and the soluble forms of the enzyme are inhibited by modifiers of thiols [N-ethylmaleimide (NEM) and p-(chloromercuri)phenylsulfonate (pCMS)], vicinal dithiols [phenylarsine oxide and diazenedicarboxylic acid bis(dimethylamide) (diamide)], and disulfides (sulfite, sulfide, and cyanide). NAD and NADH, but not beta-hydroxybutyrate and acetoacetate, protect the enzyme against inhibition by NEM, pCMS, phenylarsine oxide, and diamide. As tested with NEM and diamide, the inhibitions caused by mono- and dithiol modifiers were pseudo first order, and the reaction order with respect to the concentration of either inhibitor was unity, thus indicating the modification of a single essential thiol and/or dithiol. Sulfite and sulfide inhibitions appeared to be competitive with respect to beta-hydroxybutyrate, with Ki values of 10-15 and about 240 microM, respectively. Sulfite inhibition was uncompetitive with respect to NAD, NADH, and acetoacetate. The above results have suggested the presence in D(--)-beta-hydroxybutyrate dehydrogenase of an essential thiol and/or a vicinal dithiol associated with the binding site(s) of NAD and NADH. The inhibition by sulfite, sulfide, and cyanide might be indicative of the presence of an essential disulfide or due to a ternary complex formation involving the enzyme, NAD, and the above nucleophiles.

Acetoacetates↗
PubMed1981

Inhibition of D(--)-beta-hydroxybutyrate dehydrogenase by butanedione, phenylglyoxal, and diethyl pyrocarbonate.

D(--)-beta-Hydroxybutyrate dehydrogenase of beef heart mitochondria is inhibited by modifiers of disulfides, thiols, and vicinal dithiols [Phelps, D. C., & Hatefi, Y. (1981) Biochemistry 20 (preceding paper in this issue)]. The vicinal dithiol can be reversibly oxidized by diamide, resulting in activity inhibition, and rereduced by dithiothreitol, resulting in reactivation. The diamide-treated enzyme can no longer be irreversibly inhibited by N-ethylmaleimide, indicating the absence of an essential sulfhydryl group other than the vicinal dithiol. beta-Hydroxybutyrate dehydrogenase also appears to contain essential arginyl residues modifiable by phenylglyoxal or butanedione, and essential residue(s) modifiable at pH 6.0 by diethyl pyrocarbonate. Substrates protect against inhibitions by butanedione, phenylglyoxal, and diethyl pyrocarbonate, suggesting that the essential, modifiable residues are at or near the substrate binding sites. On the basis of these results and pH profiles, tentative mechanisms have been proposed for the oxidation of beta-hydroxybutyrate and the reduction of acetoacetate, involving the participation of the essential residues described above.

Acetoacetates↗
PubMed1980

Energy-linked transhydrogenation from NADPH to [14C]NADP.

Submitochondrial particles catalyze transhydrogenation from NADPH to [14C]NADP. This transhydrogenation is energy-linked, since its rate increases several-fold when the system is energized by succinate oxidation in the presence of rotenone (inhibitable by antimycin A or uncouplers), or by ATP hydrolysis (inhibitable by rutamycin or uncouplers). As in the case of transhydrogenation reactions from NAD(P)H to 3-ace-tylpyridine adenine dinucleotide phosphate and to thionicotinamide adenine dinucleotide phosphate, transhydrogenation from NADPH to [14C]NADP is also sensitive to treatment of the particles with trypsin or the arginyl residue modifier, butanedione. However, unlike the former reactions, transhydrogenation from NADPH to [14C]NADP cannot accumulate energy in the concentrations of the products, because, except for radioactivity, the nature and concentrations of the reactants and products remain unchanged throughout the course of the reaction. Therefore, the unrecoverable energy utilization by this region could be ascribed to an entropic component of the process, very likely an enzyme conformation change necessary for facilitation of hydride ion transfer from NADPH to [14C]NADP. This interpretation is in agreement with our previous kinetic evidence for enzyme conformation change associated with energy-linked transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate and thionicotinamide adenine dinucleotide phosphate, and with our conclusions regarding the mechanism of action of the transhydrogenase enzyme (Galante, Y.M., Lee, Y., and Hatefi, Y. (1980) J. Biol. Chem. 255, 9641-9646).

Animals↗
PubMed1980

Energy-linked mitochondrial transhydrogenation from NADPH to NADP analogs.

The mitochondrial energy-linked transhydrogenase enzyme catalyzes hydride ion transfer between NAD and HADP, of which the reaction NADH leads to NADP is slow in the absence of energy and is accelerated 10-fold or more when the mitochondrial membrane is energized by ATP hydrolysis or respiration. The enzyme is a proton pump and effects proton translocation coupled to hydride ion transfer from NADPH to NAD (Earle, S.R., and Fisher, R.R. (1980) J. Biol Chem. 255, 827-830). The present studies have shown that submitochondrial particles also catalyze transhydrogenation from NADPH to two NADP analogs, namely 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP) and thionicotinamide adenine dinucleotide phosphate (thioNADP). Both reaction rates are greatly accelerated when the system is energized by ATP hydrolysis (inhibitable by uncouplers or rutamycin) or succinate oxidation (inhibitable by uncouplers or antimycin A). As in the case of NAD(H) in equilibrium with NADP(H) reactions, the transhydrogenations from NADPH to AcPyADP and thioNADP are inhibited by treatment of submitochondrial particles with trypsin or the arginyl residue modifier, butanedione. The Km values of the above substrates and the Vmax values under energy-linked conditions have been determined. The finding that the mitochondrial energy-linked transhydrogenase enzyme catalyzes transhydrogenation from NADPH to NADP analogs has revealed features regarding substrate site specificities and the effect of substrates on the directionality of proton translocation by the enzyme.

Animals↗
PubMed1975

Inhibition of mitochondrial electron transport by hydroxy-substituted 1,4-quinones.

Mitochondrial electron transport from NADH, succinate, or duroquinol to oxygen is inhibited by 5-omega-cyclohexyl-n-pentyl-6-hydroxy-2,3-dimethoxy-1,4-benzoquinone and 2-omega-cyclohexyl-n-pentyl-3-hydroxy-1,4-napthoquinone. Assays of partial electron transport activities indicate a site of inhibition in the region between the site of duroquinol oxidation and cytochrome c reduction. Effects of the inhibitors on cytochrome spectra indicate the principle site of inhibition is between cytochromes of the b group and cytochrome c1. The quinones do not induce increased reduction of the b566 as does antimycin A. The benzoquinone can induce a partial antimycin insensitive bypass of the cytochromes b-c1 region. Uncoupling agents do not affect the extent of inhibition as has been reported for analogous naphthoquinone inhibitors.

Animals↗