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PubMed1968

The effects of inhibitor mixtures and the specific effects of different anions on the oxidase activity of caeruloplasmin.

1. The interpretation of the effects of mixtures of inhibitors on enzymes is considered. 2. The effects of inhibitor mixtures on caeruloplasmin were determined. 3. Fluoride, chloride and cyanate inhibit at one type of site (alpha), whereas bromide and iodide inhibit at another type (beta) present in the same enzyme intermediate. 4. Effects of inhibitor mixtures containing azide or cyanide are consistent with previous indications (Speyer & Curzon, 1968) that these ligands form inhibited complexes with different enzyme intermediates. 5. Isobols of halides or of cyanate with azide indicate that azide inhibits caeruloplasmin by bridging two alpha sites, these being reduced copper atoms. 6. Iodide and cyanate give hyperbolic plots of 1/v against [I]. 7. It is suggested that in the cyanate-inhibited complex the inhibitor binds to a reduced copper atom (alpha site) but that binding of cyanate at another copper atom is sterically prevented. It is suggested that the less bulky alpha-site inhibitors, fluoride and chloride, cause complete inhibition by binding to both of these copper atoms, which can also be bridged by a single azide group. 8. Each halide shows a pattern of effects on caeruloplasmin that is qualitatively distinct from that of other halides.

Azides↗

PubMed1969

The nature of the sex-linked differences in glutathione peroxidase activity and aerobic oxidation of glutathione in male and female rat liver.

1. Glutathione peroxidase activity in the livers of sham-operated female rats was about 60% higher than in similarly treated male rats. The value in the ovariectomized female was about the same as that in the castrated or sham-operated male. 2. Glutathione peroxidase activity changed during the oestrous cycle. The highest value was in oestrus, and was about 50% higher than the lowest activity, which was found in dioestrus. The activity in proestrus and in metoestrus was respectively about 20 and 30% higher than in dioestrus. 3. In the pregnant female 1 or 2 days before term, glutathione peroxidase activity was about 20% higher than that in the female in oestrus. 4. Subcutaneous implants of both oestra-diol and progesterone in the gonadectomized rats increased the glutathione peroxidase activity approximately to the values found in the female at oestrus. 5. The rate of aerobic oxidation of GSH in the female rat liver was about 80% higher than in the male and about 110% higher than in the gonadectomized rats. Treatment of gonadectomized rats with subcutaneous implants of oestradiol and of progesterone increased the rate of oxidation of GSH by about 100%. 6. In the presence of azide the rate of GSH oxidation in the male and in the female was respectively about 3.5- and 2.1-fold that in the absence of azide. In castrated or ovariectomized rats the increase due to the presence of azide was about 2.4-fold. In the gonadectomized rats treated with oestradiol or progesterone the rate of GSH oxidation in the presence of azide was about 2.2-fold that in its absence. 7. The rate of lipid peroxidation in female was 15-30-fold that in male or in gonadectomized rats. Treatment of the gonadectomized rats with oestradiol or with progesterone increased the rate of lipid peroxidation up to values that were even higher than in the female. In the presence of GSH the formation of malonaldehyde from peroxides was virtually eliminated. 8. The results suggest that the sex-linked differences in glutathione peroxidase activity, in the rate of GSH oxidation and in the rate of lipid peroxidation are due to the female sex hormones. 9. It is suggested that both the catalase activity and the rate of hydrogen peroxide formation are higher in the male than in the female. 10. Sex-linked changes in glutathione peroxidase, in the rate of GSH oxidation and in the rate of lipid peroxide formation are discussed in relation to the metabolism of oestrogens in the liver and also to the possible nature of those sex-linked changes.

Aldehydes↗

PubMed1979

The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii.

1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture.

Amoeba↗

PubMed1989

Neutrophil degranulation inhibits potential hydroxyl-radical formation. Relative impact of myeloperoxidase and lactoferrin release on hydroxyl-radical production by iron-supplemented neutrophils assessed by spin-trapping techniques.

Hydroxyl radical (.OH) formation by neutrophils in vitro requires exogenous iron. Two recent studies [Britigan, Rosen, Thompson, Chai & Cohen (1986) J. Biol. Chem. 261, 17026-17032; Winterbourn (1987) J. Clin. Invest. 78, 545-550] both reported that neutrophil degranulation could potentially inhibit the formation of .OH, but differed in their conclusions as to the responsible factor, myeloperoxidase (MPO) or lactoferrin (LF). By using a previously developed spin-trapping system which allows specific on-line detection of superoxide anion (O2-) and .OH production, the impact of MPO and LF release on neutrophil .OH production was compared. When iron-diethylenetriaminepenta-acetic acid-supplemented neutrophils were stimulated with phorbol myristate acetate or opsonized zymosan, .OH formation occurred, but terminated prematurely in spite of continued O2- generation. Inhibition of MPO by azide increased the magnitude, but not the duration, of .OH formation. No azide effect was noted when MPO-deficient neutrophils were used. Anti-LF antibody increased both the magnitude and duration of .OH generation. Pretreatment of neutrophils with cytochalasin B to prevent phagosome formation did not alter the relative impact of azide or anti-LF on neutrophil .OH production. An effect of azide or anti-LF on spin-trapped-adduct stability was eliminated as a confounding factor. These data indicate that neutrophils possess two mechanisms for limiting .OH production. Implications for neutrophil-derived oxidant damage are discussed.

Antibodies↗

PubMed1999

Metabolic impairment induces oxidative stress, compromises inflammatory responses, and inactivates a key mitochondrial enzyme in microglia.

Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO.) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide-treated microglia were also treated with LPS. The combination of the azide-stimulated ROS and LPS-induced NO. would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO. and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.

Animals↗

PubMed2003

How does cyanide inhibit superoxide reductase? Insight from synthetic FeIIIN4S model complexes.

Superoxide reductases (SORs) are nonheme iron-containing enzymes that reduce HO(2) to H(2)O(2). Exogenous substrates such as N(3)(-) and CN(-) have been shown to bind to the catalytic iron site of SOR, and cyanide acts as an inhibitor. To understand how these exogenous ligands alter the physical and reactivity properties of the SOR iron site, acetate-, azide-, and cyanide-ligated synthetic models of SOR have been prepared. The x-ray crystal structures of azide-ligated [Fe(III)(S(Me2)N(4)(tren))(N(3))](+) (3), dimeric cyanide-bridged ([Fe(III)(S(Me2)N(4)(tren))](2)-mu-CN)(3+) (5), and acetate-ligated [Fe(III)(S(Me2)N(4)(tren))(OAc)](+) (6) are described, in addition to x-ray absorption spectrum-derived and preliminary crystallographic structures of cyanide-ligated [Fe(III)(S(Me2)N(4)(tren))(CN)](+) (4). Cyanide coordination to our model (4) causes the redox potential to shift anodically by 470 mV relative to acetate-ligated 6 and 395 mV relative to azide-ligated 3. If cyanide coordination were to cause a similar shift in redox potential with SOR, then the reduction potential of the catalytically active Fe(3+) center would fall well below that of its biological reductants. These results suggest therefore that cyanide inhibits SOR activity by making the Fe(2+) state inaccessible and thus preventing the enzyme from turning over. Cyanide inhibits activity in the metalloenzyme superoxide dismutase via a similar mechanism. The reduced five-coordinate precursor to 3, 4, and 6 [Fe(II)(S(Me2)N(4)(tren))](+) (1) was previously shown by us to react with superoxide to afford H(2)O(2) via an [Fe(III)(S(Me2)N(4)(tren))(OOH)](+) intermediate. Cyanide and azide do not bind to 1 and do not prevent 1 from reducing superoxide.

Azides↗

PubMed1999

Electron spin resonance investigation of the cyanyl and azidyl radical formation by cytochrome c oxidase.

Cyanide (CN(-)) is a frequently used inhibitor of mitochondrial respiration due to its binding to the ferric heme a(3) of cytochrome c oxidase (CcO). As-isolated CcO oxidized cyanide to the cyanyl radical ((.)CN) that was detected, using the ESR spin-trapping technique, as the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(.)CN radical adduct. The enzymatic conversion of cyanide to the cyanyl radical by CcO was time-dependent but not affected by azide (N(3)(-)). The small but variable amounts of compound P present in the as-isolated CcO accounted for this one-electron oxidation of cyanide to the cyanyl radical. In contrast, as-isolated CcO exhibited little ability to catalyze the oxidation of azide, presumably because of azide's lower affinity for the CcO. However, the DMPO/(.)N(3) radical adduct was readily detected when H(2)O(2) was included in the system. The results presented here indicate the need to re-evaluate oxidative stress in mitochondria "chemical hypoxia" induced by cyanide or azide to account for the presence of highly reactive free radicals.

Animals↗

PubMed2000

Competitive substrate and inhibitor interactions at the physiologically relevant active site of nitrogenase.

Nitrogenase catalyzes the MgATP-dependent reduction of dinitrogen gas to ammonia. In addition to the physiological substrate, nitrogenase catalyzes reduction of a variety of other multiply bonded substrates, such as acetylene, nitrous oxide, and azide. Although carbon monoxide (CO) is not reduced by nitrogenase, it is a potent inhibitor of all nitrogenase catalyzed substrate reductions except proton reduction. Here, we present kinetic parameters for an altered Azotobacter vinelandii MoFe protein for which the alphaGly(69) residue was substituted by serine (Christiansen, J., Cash, V. L., Seefeldt, L. C., and Dean, D. R. (2000) J. Biol. Chem. 275, 11459-11464). For the wild type enzyme, CO and acetylene are both noncompetitive inhibitors of dinitrogen reduction. However, for the alphaSer(69) MoFe protein both CO and acetylene have become competitive inhibitors of dinitrogen reduction. CO is also converted from a noncompetitive inhibitor to a competitive inhibitor of acetylene, nitrous oxide, and azide reduction. These results are interpreted in terms of a two-site model. Site 1 is a high affinity acetylene-binding site to which CO also binds, but dinitrogen, azide, and nitrous oxide do not bind. This site is the one primarily accessed during typical acetylene reduction assays. Site 2 is a low affinity acetylene-binding site to which CO, dinitrogen, azide, and nitrous oxide also bind. Site 1 and site 2 are proposed to be located in close proximity within a specific 4Fe-4S face of FeMo cofactor.

Acetylene↗

PubMed2003

Identification of the catalytic residues in family 52 glycoside hydrolase, a beta-xylosidase from Geobacillus stearothermophilus T-6.

beta-d-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a beta-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased approximately 106-fold, and this activity was enhanced 103-fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pKa). The mutant exhibited 103-fold reduction in activity, and the Brønsted plot of log(kcat) versus pKa revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 103-fold. The rates of the glycosylation step, as reflected by the specificity constant (kcat/Km), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pKa) but decreased 102-fold for those that require strong acid catalysis (high pKa). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp495 acting as the acid-base in XynB2.

Amino Acid Substitution↗

PubMed2006

Protective effect of hepatocyte growth factor against degeneration of the retinal pigment epithelium and photoreceptor in sodium iodate-injected rats.

PURPOSE: To investigate the possible protective effect of hepatocyte growth factor (HGF) against degeneration of photoreceptors and retinal pigment epithelium (RPE) in vivo. METHODS: Sprague-Dawley (SD) rats received an intravitreal injection of HGF in the right eye. The left eye was injected with vehicle as a control. Two days after the intravitreal injections, rats were administered 40 mg/kg of sodium iodate (NaIO3) intravenously. Scotopic ERGs were elicited by different stimulus intensities with a maximum luminance of 0.84 log cds/m2. To evaluate RPE function, the azide response was evoked by intravenous injection of 0.1 mg sodium azide. These electrophysiological measurements were conducted on days 4, 7, 14, and 28 after the NaIO3 injections. After recording ERGs or azide response, animals were sacrificed for quantification of the histological change and immunohistochemical analysis using antibodies against RPE 65. RESULTS: The threshold for the scotopic b-wave was significantly lower in HGF-treated eyes than in untreated control eyes (p < 0.005), and maximum b-wave amplitudes (Vbmax) were significantly larger in HGF-treated eyes (p < 0.05) across all experimental time points after NaIO3 injection. Azide response amplitudes were significantly larger in the HGF-treated eyes than in the untreated eyes (p < 0.05). The structure of the outer retina was preserved to a greater degree in the HGF-treated eyes than in the untreated eyes (p < 0.05). Immunohistochemical analysis demonstrated that irregular alignment of the outer nuclear layer was confined to the retinal area that was not stained with RPE 65. CONCLUSIONS: Our results indicated that an intravitreal injection of HGF provided significant protection against degeneration of the photoreceptor and RPE induced by systemic administration of NaIO3. This suggests that HGF could be used as a therapeutic agent for degeneration of photoreceptors as well as RPE.

Animals↗

PubMed1979

Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism.

The effects of energy metabolism inhibitors on the distribution of acetylcholine receptors (AChRs) in the surface membranes of non-innervated, cultured rat myotubes were studied by visualizing the AChRs with monotetramethylrhodamine-alpha-bungarotoxin. Incubation of myotubes with inhibitors of energy metabolism causes a large decrease in the fraction of myotubes displaying clusters of AChR. This decrease is reversible, and is dependent on temperature, the concentration of inhibitor, and the duration of treatment. Cluster dispersal is probably not the result of secondary effects on Ca++ or cyclic nucleotide metabolism, membrane potential, cytoskeletal elements, or protein synthesis. Sequential observations of identified cells treated with sodium azide showed that clusters appear to disperse by movements of receptors within the sarcolemma without accompanying changes in cell shape. AChR clusters dispersed by pretreating cells with sodium azide rapidly reform upon removal of the inhibitor. Reclustering involves the formation of small aggregates of AChR, which act as foci for further aggregation and which appear to be precursors of large AChR clusters. Small AChR aggregates also appear to be precursors of clusters which form on myotubes never exposed to azide. Reclustering after azide treatment does not necessarily occur at the same sites occupied by clusters before dispersal, nor does it employ only receptors which had previously been in clusters. Cluster reformation can be blocked by cycloheximide, colchicine, and drugs which alter the intracellular cation composition.

Acetylcholine↗

PubMed1978

Ethylene formation by polymorphonuclear leukocytes. Role of myeloperoxidase.

Ethylene formation from the thioethers, beta-methylthiopropionaldehyde (methional) and 2-keto-4-thiomethylbutyric acid by phagocytosing polymorphonuclear leukocytes (PMNs) was found to be largely dependent on myeloperoxidase (MPO). Conversion was less than 10% of normal when MPO-deficient PMNs were employed; formation by normal PMNs was inhibited by the peroxidase inhibitors, azide, and cyanide, and a model system consisting of MPO, H2O2, chloride (or bromide) and EDTA was found which shared many of the properties of the predominant PMN system. MPO-independent mechanisms of ethylene formation were also identified. Ethylene formation from methional by phagocytosing eosinophils and by H2O2 in the presence or absence of catalase was stimulated by azide. The presence of MPO-independent, azide-stimulable systems in the PMN preparations was suggested by the azide stimulation of ethylene formation from methional when MPO-deficient leukocytes were employed. Ethylene formation by dye-sensitized photooxidation was also demonstrated and evidence obtained for the involvement of singlet oxygen (1O2). These findings are discussed in relation to the participation of H2O2, hydroxyl radicals, the superoxide anion and 1O2 in the formation of ethylene by PMNs and by the MPO model system.

Azides↗

PubMed1965

Electrical properties of Neurospora crassa. Respiration and the intracellular potential.

The internal potential of Neurospora appears to have two components, one (a) which is reduced by anoxia or abolished by respiratory inhibitors such as azide and 2,4-dinitrophenol, and (b) a fraction that remains in the presence of respiratory inhibitors and is sensitive to the external potassium concentration. Under standard conditions 1 mM azide or dinitrophenol diminishes internal potentials from near -200 mv to about -30 mv within 1 minute and at a maximal rate of 20 mv/second. The internal potential usually recovers within 10 minutes after the inhibitor has been removed. The effect of carbon monoxide on the internal potential is similar to that of azide or dinitrophenol, but can be reversed by visible light, specifically of the wavelengths (430 mmicro and 590 mmicro) known to decompose cytochrome-CO complexes in yeast. Respiration and internal potentials vary proportionally with azide concentration, but dinitrophenol at low (3 x 10(-6)M) concentrations enhances oxygen consumption without affecting the internal potential. In the presence of 0.1 mM calcium, the fraction of the internal potential which persists during respiratory inhibition increases (becomes more negative) about 30 mv for each tenfold decrease of external potassium over the range 10 to 0.1 mM. The surface resistivity of Neurospora, normally about 5000 ohm.cm(2), is unchanged by respiratory inhibitors during the period of rapid potential shift.

Antimetabolites↗

PubMed1998

Survey of four different photoreactive moieties for DNA photoaffinity labeling of yeast RNA polymerase III transcription complexes.

In order to optimize the detection of protein-DNA contacts by DNA photoaffinity labeling, we attached four different photoreactive groups to DNA and examined their ability to crosslink yeast RNA polymerase III (Pol III) transcription complexes. Photoreactive nucleotides containing an aryl azide (AB-dUMP), benzophenone (BP-dUMP), perfluorinated aryl azide (FAB-dUMP) or diazirine (DB-dUMP) coupled to 5-aminoallyl deoxyuridine were incorporated into the SUP4 tRNATyr gene at bp -3/-2 or +11. Photo-crosslinking with diazirine revealed contacts of Pol III with DNA that are not detected by DNA photoaffinity labeling using an aryl azide, fluorinated aryl azide or benzophenone group attached to DNA. These novel contacts were of the 82 kDa subunit of Pol III with DNA at bp -3/-2 in the initiation complex and of the 82, 40(37) and 31 kDa subunits of Pol III with DNA at bp +11 in elongation complexes stalled at bp +17. These results provide evidence for the subcomplex of the 82, 34 and 31 kDa subunits of Pol III being positioned near the transcription bubble of actively transcribing Pol III, as all three proteins were crosslinked at bp +11 of the stalled transcription complex.

Affinity Labels↗

PubMed1985

Change of inhibitor sensitivities of Escherichia coli F1-ATPase due to a mutational substitution of Phe for Ser at residue 174 of the beta subunit.

The F1-ATPase from the uncD11 mutant of E. coli (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., & Futai, M. (1980) J. Biochem. 88, 695-703), showed different enzymological properties from the wild-type enzyme. The mutant F1-ATPase had biphasic kinetics and essentially the same Km values as the wild-type enzyme, although its Vmax values were lower. The mutant enzyme showed altered sensitivities to dicyclohexylcarbodiimide (DCCD), azide and quercetin; it was less sensitive than the wild-type to quercetin and DCCD, and its Mg2+-dependent ATPase activity was slightly more resistant to azide than that of the wild-type, whereas its Ca2+-dependent activity was more sensitive. On the other hand, the mutant and wild-type F1 were inhibited equally by 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). The fact that the Mg2+- and Ca2+-dependent F1-ATPase activities of the wild-type and mutant responded differently to quercetin and azide suggested that their mechanisms of action were different. Previous studies (Noumi, T., Mosher, M.E., Natori, S., Futai, M., & Kanazawa, H. (1984) J. Biol. Chem. 259, 10071-10075) indicated that Ser is replaced by Phe at residue 174 of the beta subunit of the mutant. Thus the Ser residue or its neighboring area(s) may constitute the binding site of DCCD, quercetin and azide.

Amino Acid Sequence↗

PubMed1999

Synergism of energy starvation and dextran-conjugated doxorubicin in the killing of multidrug-resistant KB carcinoma cells.

Here we report that 2-deoxyglucose/Na azide treatment and free/conjugated doxorubicin are synergistic in cell killing. As demonstrated by fluorescence confocal microscopy, KB-V1 cells retained more conjugated doxorubicin than free doxorubicin. Verapamil or 2-deoxyglucose/Na azide enhanced only the retention of the free drug and the small (<70 kDa) conjugates, indicating that P-glycoprotein (P-gp) is not effective against large conjugates. Conjugated doxorubicin was excluded from nuclei. Initially both free and conjugated doxorubicin accumulated in cytoplasmic organelles. Upon 2-deoxyglucose/Na azide treatment, fluorescence labeling of organelles dissipated. Prolonged (24 h) incubation of conjugate-preloaded cells resulted in redistribution of some of the organelle-associated fluorescence to nuclei, suggesting decoupling. The appearance of free doxorubicin was confirmed by capillary electrophoresis. 2-Deoxyglucose/Na azide treatment also retarded decoupling. Our results suggest that energy starvation, in addition to increasing cellular retention of P-gp substrates, may affect cellular fate of conjugated drugs with a possible enhancing effect in cancer cell killing.

Antineoplastic Agents↗

PubMed1987

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: intracellular pH and the effect of membrane depolarizing compounds.

Addition of plasma membrane depolarizing agents, such as dinitrophenol (DNP) and azide, to cells of Saccharomyces cerevisiae under aerobic conditions, is known to cause an increase in the cAMP level within 15 s. We found that both compounds lowered the intracellular pH (measured by in vivo 32P-NMR) drastically within the same time period. Plasma membrane depolarization, however, was much slower: DNP and azide had no effect on the membrane potential during, respectively, the first 2 min and the first 10 min after addition. Apparently, the intracellular pH of yeast is much more sensitive to perturbation than the membrane potential. The effect of both compounds on the cAMP level was highly dependent on the extracellular pH: when the latter was raised, the effect disappeared completely between pH 6 and 7. A similar dependence on the extracellular pH was observed for the lowering of intracellular pH. Addition of organic acids, such as acetate and butyrate, at low pH and under aerobic conditions, also caused an immediate increase in the cAMP level and an immediate drop in the intracellular pH. These results suggest that agents such as DNP and azide do not raise the cAMP level in yeast cells because of their membrane depolarizing properties but because they lower the intracellular pH. Under anaerobic conditions, DNP, azide and organic acids were much less effective in increasing the cAMP level. Addition of a small amount of glucose, however, restored their capacity to enhance the cAMP level. This suggests that under anaerobic conditions and in the absence of glucose the ATP level is a limiting factor for cAMP synthesis.

Azides↗

PubMed1981

Ligand binding to catalase and metmyoglobin. Direct measurements of proton involvement.

1. Spectroscopic measurements of the binding of the weak-acid ligands formate, azide and cyanide to catalase indicate interaction of a primary haem-binding site with the undissociated forms of the ligands between pH 5 and 8. A similar conclusion can be drawn from observations on the pH dependence of catalase activity in the presence of cyanide and formate. 2. Direct measurements of pH changes during catalase interaction with these ligands also indicate stoichiometric uptake of one proton per haem group upon addition of formate or azide at pH 6, but at greater pH values the proton uptake declines, suggesting the involvement of a secondary site in the liganding reaction. This secondary site can be occupied by formate and certain other anions inducing proton dissociation from the protein. The results also suggest a direct displacement of formate or azide from the primary haem site by cyanide. 3. Similar stoichiometric pH measurements on metmyoglobin indicate binding of the formate, azide and cyanide anions to a single (primary) site on the molecule.

Animals↗