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PubMed1995

Inhibition of neutrophil superoxide generation by hypericin, an antiretroviral agent.

We previously reported that phorbol 12-myristate 13-acetate (PMA)-induced superoxide (O2.-) generation of neutrophils was inhibited by hypericin, a photosensitizing pigment found in St. Johnswort (herb Hypericin triquetrifolium Turra), via a mechanism involving protein kinase C (PKC). To obtain further insights into the mechanism of inhibition, the effects of hypericin on stimulation-dependent O2.- generation and related enzymes of neutrophils were investigated. Hypericin inhibited O2.- generation of neutrophils induced by PKC-dependent and -independent stimuli in a light- and concentration-dependent manner. Oxygen was required for the light-dependent inhibition by hypericin. NADPH oxidase activity in a cell-free system and TNF-alpha-induced tyrosyl phosphorylation of neutrophil proteins were also inhibited by hypericin in a concentration- and light-dependent manner. However, tyrosine kinase of p60src, an enzyme not bound to a membrane, was not inhibited either in the light or in the dark. Oxygen uptake of neutrophils by photosensitization with hypericin resulted in the formation of singlet oxygen (1O2), O2.-, and hydroxyl radical (.OH) and enhanced lipid peroxidation. The formation of 1O2 was inhibited by azide, a quencher of 1O2, but not by desferrioxamine (DSF), a ferric ion chelator. By contrast, both generation of .OH and lipid peroxidation were inhibited by DSF but not by azide. Furthermore, PMA-induced O2.- generation inhibited by hypericin partially recovered in the presence of azide but not DSF. These results suggested that the light-dependent inhibition of O2.- generation by hypericin might be due to inhibition of tyrosine kinase, PKC, and NADPH oxidase via an oxygen-dependent mechanism, possibly through both Type I and II photosensitization mechanisms.

Anthracenes↗
PubMed1999

Metmyoglobin/azide: the effect of heme-linked ionizations on the rate of complex formation.

The kinetics of formation and dissociation of the horse metmyoglobin/azide complex has been investigated between pH 3.5 and 11.5. The ionic strength dependence of the reaction has been determined at integral pH values between 5 and 10. Hydrazoic acid, HN3, binds to metmyoglobin with a rate constant of (3.8 +/- 1.0) x 10(5) M-1 s-1. Protonation of a group with an apparent pKa of 4.0 +/- 0.3 increases the rate of HN3 binding 6.5-fold to (2.5 +/- 0.8) x 10(6) M-1 s-1. The ionizable group is attributed to the distal histidine, His-64. The azide anion, N-3, binds to metmyoglobin with a rate constant of (4.7 +/- 0.3) x 10(3) M-1 s-1, about two orders of magnitude slower than HN3. Conversion of aquometmyoglobin to hydroxymetmyoglobin slows azide binding significantly. Binding of HN3 to hydroxymetmyoglobin cannot be detected, while N-3 binds to hydroxymetmyoglobin with a rate of 5.7 +/- 3.2 M-1 s-1, almost three orders of magnitude slower than N-3 binding to aquometmyoglobin. Protonation of the distal histidine facilitates HN3 dissociation from the complex. HN3 dissociates from the metmyoglobin/azide complex with a rate constant of 18 +/- 6 s-1, while the azide anion dissociates with a rate constant of 0.16 +/- 0.02 s-1, about 100 times slower. The apparent pKa for His-64 is essentially the same in metmyoglobin and the metmyoglobin/azide complex, 4.0 +/- 0.3 and 4.4 +/- 0.2, respectively. The ionic strength dependence of the observed association rate constant is influenced by both primary and secondary kinetic salt effects. The primary kinetic salt effect is anomalous, with the rate of N-3 binding decreasing with increasing ionic strength above the isoelectric point of metmyoglobin where the protein has a net negative charge. The ionic strength dependence of the dissociation rate constant can be described solely in terms of the ionic strength dependence of the acid dissociation constant for His-64 in the metmyoglobin/azide complex, a secondary kinetic salt effect.

Animals↗
PubMed1979

The Pasteur effect and catabolite repression in an oxidative yeast, Kluyveromyces lactis.

The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation. Extracts from these cells contained ATP-sensitive phosphofructokinase activity. Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation. Phosphofructokinase in these cells was ATP-insensitive. The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation. These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K. lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase. Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity. The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts.

Adenosine Triphosphate↗
PubMed1980

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans.

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide Mr = 150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrance protein Mr = 150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are co-regulated and that the active enzyme has a role in regulating its own synthesis.

Azides↗
PubMed1991

Luminol chemiluminescence and active oxygen generation by activated neutrophils.

Upon stimulation by various ligands and membrane perturbers, neutrophils produce various active oxygen species. Since luminol chemiluminescence (LCL) in neutrophils can be blocked by azide, an inhibitor of myeloperoxidase, LCL has been believed to reflect mainly the myeloperoxidase-catalyzed reaction. When cells were stimulated by formyl-methionyl-leucyl-phenylalanine, LCL was strongly inhibited by superoxide dismutase (SOD) and uric acid, a scavenger for hydroxy radical (.OH) and singlet oxygen, whereas it was stimulated by azide. LCL was also inhibited by .OH scavengers, such as mannitol, ethanol, and dimethylsulfoxide. However, when stimulated by phorbol myristate acetate or opsonized zymosan, LCL was strongly inhibited by azide but not by uric acid, and the inhibitory action of SOD was low. Thus, the qualitative and quantitative aspects of reactive oxygen generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the metabolic fate of active oxygens in neutrophils and, hence, their effect on microorganisms and the surrounding tissues might differ depending on the stimulus.

Animals↗
PubMed1975

Biochemical and biophysical studies on cytochrome c oxidase. XX. Reaction with sulphide.

1. Upon addition of sulphide to oxidized cytochrome c oxidase, a low-spin heme sulphide compound is formed with an EPR signal at gx = 2.54, gy = 2.23 and gz = 1.87. Concomitantly with the formation of this signal the EPR-detectable low-spin heme signal at g = 3 and the copper signal near g = 2 decrease in intensity, pointing to a partial reduction of the enzyme by sulphide. 2. The addition of sulphide to cytochrome c oxidase, previously reduced in the presence of azide or cyanide, brings about a disappearance of the azido-cytochrome c oxidase signal at gx = 2.9, gy = 2.2, and gz = 1.67 and a decrease of the signal at g = 3.6 of cyano-cytochrome c oxidase. Concomitantly the sulphide-induced EPR signal is formed. 3. These observations demonstrate that azide, cyanide and sulphide are competitive for an oxidized binding site on cytochrome c oxidase. Moreover, it is shown that the affinity of cyanide and sulphide for this site is greater than that of azide.

Animals↗
PubMed1990

Investigation by amperometric methods of intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes in the presence of different inhibitors of cellular metabolism.

The reduction of 2,6-dichlorophenolindophenol (DCIP) was measured by amperometric methods in Morris hepatoma 3924A cells, normal isolated rat hepatocytes and in mitochondria isolated from normal rat liver. The influence of aerobic and anaerobic atmospheres and of various inhibitors of cellular metabolism, especially of the respiratory chain (KCN, NaN3, oligomycin), on DCIP-reduction were studied using glucose, succinate, beta-hydroxybutyrate, alpha-keto-glutarate and oxalacetate as substrates. Under the influence of KCN and oligomycin the velocity of DCIP-reduction was increased in both cell types. Azide showed a similar effect on tumour cells and to a lower extent on hepatocytes. Using isolated mitochondria total DCIPred was increased by KCN and azide using various mitochondrial metabolites as substrates and with ADP/Pi present. The effects of KCN, azide and oligomycin could be explained by taking DCIP as an artificial coupling site in mitochondria which is only used when oxygen is absent or when the respiratory chain is blocked by inhibitors of cytochrome oxidase. Evaluation of the reaction kinetics revealed differences between normal and transformed cells in terms of the pseudo-first-order rate constants and the activity of overall oxidoreductases. The results apparently reflect quantitative differences in enzymatic equipment and the metabolic pathways predominating in normal and neoplastic cells.

2,6-Dichloroindophenol↗
PubMed1982

4-Azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes.

A new phlorizin derivative (2'-O-(beta-D-glucopyranosyl)-4-azidophloretin, 4-azidophlorizin) has been synthesized and its affinity for the D-glucose, Na+ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) Ki', the constant for fully-competitive inhibition of (Na+, delta psi)-dependent D-glucose uptake, (2) Kd', the dissociation constant of 4-azido[3H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) Ki", the constant for fully-competitive inhibition of the specific ((Na+, delta psi)-dependent, D-glucose protectable) high-affinity [3H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (Ki', Kd' and Ki") were essentially equal and ranged between 3.2 and 5.2 microM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its Ki' values are some 15-20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244-248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.

Affinity Labels↗
PubMed1986

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers.

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.

Affinity Labels↗
PubMed1995

Effects of light and inhibitors of ATP-synthesis on the chloride carrier of the alga Valonia utricularis: is the carrier a chloride pump?

The effect of metabolic inhibitors, such as cyanide, antimycin A and azide was studied on the chloride transport system of the giant marine alga Valonia utricularis by using the charge pulse relaxation method. Two clearly defined voltage relaxations were resolved. The addition of 10-30 microM cyanide to the artificial sea water (ASW) bathing the algal cells increased the time constants of the slow voltage relaxation, tau 2, significantly when the algal cells were kept in the dark. The cyanide-effect reached a plateau value at 100-300 microM and was fully reversible when cyanide was removed from the ASW. Analysis of the charge pulse data in terms of the Läuger-model demonstrated that the translocation rates of the free, kS, and the charged carrier, kAS, decreased. The decrease of kS was more pronounced than that of kAS. 10 microM antimycin A and 3 mM azide had similar effects on the rate constants when the light was switched off. Upon illumination the cyanide- and antimycin A-, but not the azide-mediated effects disappeared. At concentrations higher than 1 mM cyanide caused a further, dramatic decrease of kS and kAS, while the surface concentration of the carrier molecules, N0, was not affected. This cyanide-effect was also reversible, but not light-dependent. Measurements of the ATP level showed that 3 mM cyanide reduced the ATP level by about 70% both under light and dark conditions. In the presence of 30 microM cyanide (or 10 microM antimycin A) the ATP level decreased by about 50%, but only in the dark. These results suggest two different effects of cyanide on the Cl(-)-carrier system: in the micromolar concentration range cyanide (and antimycin A) reduced predominantly the translocation of the free carrier by inhibition of ATP synthesis by oxidative phosphorylation, whereas in the millimolar concentration range cyanide apparently inhibits the translocation rates of both the free and charged carriers by its binding to the carrier. The results provide some evidence that the chloride transport of V. utricularis could be coupled to metabolic energy but it is an open question whether it is a pump or not.

Adenosine Triphosphate↗
PubMed1994

Morphology and volume alterations of human erythrocytes caused by the anion transporter inhibitors, DIDS and p-azidobenzylphlorizin.

p-Azidobenzylphlorizin (p-AzBPhz) is a potential photoaffinity labeling agent for the anion and glucose transporters in human RBCs. In the absence of light and at the same low concentrations which block these transport processes (only 1-2 million molecules bound/cell), this impermeable membrane probe produces rapid morphological and volume alterations. This high-affinity activity, called phase 1, can be rapidly and completely reversed by simply diluting the azide-treated cell suspension. Phase 2 effects, including formation of cells with multiple, long spicules (stage 3/4 echinocytes), followed by an influx of salt and water with eventual lysis, occur at two log units higher concentration by a different mechanism, probably by intercalating into and selectively expanding the outer lipid monolayer. Light scattering, electronic cell sizing, microhematocrit measurements and scanning electron microscopy have been employed to compare the effects of the azide and the anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), on red cells. DIDS produced only those changes analogous to the azide's low dose phase 1 action; cells swell, lose the ability to scatter 800 nm light and undergo a limited shape change (comparable to stage 1 echinocytosis). The mechanism by which the two ligands perturb the membrane is additive, suggesting that a Band 3-mediated transmembrane signaling is involved which leads to altered cytoskeleton dynamics.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1979

Transient intermediates in the reduction of Fe(III) myoglobin-ligand complexes by electrons at low temperature.

1. The reductions of a number of sperm-whale Fe(III) myoglobin-ligand complexes by electrons generated by gamma-irradiation in ethylene glycol/water glass, have been investigated by using low-temperature spectrophotometry. The ligands are azide, fluoride, imidazole and water. 2. The reduction of the Fe(III) myoglobin-ligand complexes at 77 K leads to the formation of low-spin liganded Fe(II) myoglobin, in the case of the azide, imidazole and water derivatives, while the reduction of the fluoride derivative proceeds both by a pathway involving prior dissociation of the ligand and with the ligand in position. 3. Investigation of the effect of temperature on the stability of the Fe(II) myoglobin-ligand complexes indicates that more than one bound states exists in dissociation of the ligand molecule from the ferrous heme iron of the reduced azide and imidazole derivatives. 4. The results are discussed in terms of the possible structure of the Fe(II) myoglobin complexes and it is suggested that the low-spin state is created by a strained configuration of the heme center with the iron atom in an intermediate position relative to the heme plane.

Animals↗
PubMed1986

Synthesis and enantioselective mutagenicity of azidoalanine in Salmonella typhimurium.

Azide mutagenicity involves the requisite formation of the putative novel aminoacid metabolite, beta-azidoalanine. The role of this metabolite, however, is unclear. In order to confirm the identity of this metabolite and provide additional information on possible stereochemical requirements for mutagenicity, authentic racemic and L-azidoalanine were synthesized by an unambiguous route and tested for mutagenicity in Salmonella typhimurium TA100, TA1535, hisG46 and Escherichia coli WP2-. A marked antipodal potency ratio was observed in strains TA100 and TA1535 when racemic and L-azidoalanine were compared. The mutagenic activity resided primarily in the L-isomer. The molar potency of L-azidoalanine in TA100 and TA1535 was nearly identical to that of azide. The lack of mutagenic response for racemic or L-azidoalanine in hisG46 and E. coli WP2- was like that reported for azide and is consistent with similar modes of action for these agents.

Alanine↗
PubMed1979

Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs.

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.

Azides↗
PubMed1984

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase.

The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (Rönnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.

Azides↗
PubMed1989

Prosthetic group content and ligand binding properties of a spinach catalase.

A recently discovered form of spinach catalase that contains both a novel heme and protoheme as prosthetic groups has been characterized using immunological and spectroscopic techniques. The enzyme appears to be a dimer of identical Mr 60,000 monomers. Extraction of the non-covalently bound prosthetic groups, followed by thin-layer chromatography of the extract, suggested that the novel heme contains four carboxylic acid side-chain groups. The resonance Raman spectrum of the resting enzyme indicates that the protoheme prosthetic group is five-coordinate and high-spin. The enzyme was shown to bind formate, azide and cyanide. Cyanide and azide binding to catalase are biphasic, suggesting the existence of two different binding sites for cyanide and azide in the enzyme. Results obtained from EPR and resonance Raman spectroscopies also support the hypothesis that two different ligand-binding sites are present in the enzyme. Western blots suggest that the Mr 60,000 peptide of the novel heme-containing catalase is similar or identical to that of a previously characterized, exclusively protoheme-containing, tetrameric catalase.

Azides↗
PubMed1982

The kinetics of ligand binding for diverse mammalian myoglobins and the effects of substitutions outside the heme cavity.

1. We report rate constants for oxygen dissociation and for oxygen, carbon monoxide, azide, and cyanide binding to whale, horse, dog, beef and human myoglobins. 2. For azide binding, rate constants can vary by at least a factor of two for substitutions outside the heme cavity. Azide binding may be affected by a substitution at residue 66 in the E helix, a site suggested by Case & Karplus (1979) J. molec. Biol. 132, 343-368, to be on a reactive path to the heme. 3. The oxygen and CO data show that substitutions outside the heme cavity can affect rate constants by at least a factor of 1.5. 4. The oxygen equilibrium constant was correlated with the metabolite rate of the corresponding species, in accord with the Wyman (1966) J. biol. Chem. 241, 115-121, model for facilitated diffusion of oxygen.

Animals↗
PubMed2006

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase.

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.

Adenosine Triphosphate↗