Interactions of beef heart mitochondrial adenosine triphosphatase and aurovertin.

Aurovertin forms a complex with soluble beef heart mitochondrial ATPase (F₁) while exhibiting a biphasic fluorence enhancement. The effect of substrate, activators and inhibitors of F₁¹ of the fluorescence of the aurovertin-F₁ complex is reported. The aurovertin-F₁ complex can exist in two different states, one showing low fluorescence and the other with high fluorescence. Transition into the low fluorescence state is induced by various nucleoside triphosphates (ATP ± Mg²⁺, ITP ± Mg²⁺, GIP + Gg²⁺, and AMP-P(NH)P ± Mg²⁺). The rate and extent of fluorescence decrease caused by nucleotide addition (except that caused by ATP) is dependent on the presence of added Mg²⁺. The inhibitors of ATPase activity (AMP-P(NH)P, GMP-P(NH)P and EDTA) at concentrations that inhibit hydrolysis of ATP did not prevent the ATP induced decrease of aurovertin fluorescence. EDTA at high concentration (>0.4 mM) enhanced the effect of ADP.The complex of aurovertin with F₁ that had previously been treated with butanedione loses sensitivity to ATP. Addition of ADP to the system containing butanedione-treated enzyme caused a 2-fold greater enhancement of fluorescence than the addition of ADP to the control system. In contrast to the butanedione-treated enzyme, the complex of aurovertin with F₁ previously treated at pH 5.6 loses sensitivity to ADP. Addition of ATP to this system lowered the fluorescence as in the system containing native enzyme.On the basis of the analyses of the aurovertin fluorescence changes and hydrolytic activity of F₁, the existence of several types of ligand binding sties with varying degrees of specificity are proposed. It is further proposed that these sites are important in control of the conformation and the catalytic properties of the ATPase molecule.     

Thermal perturbation difference spectra and D2O vs H2O isothermal difference spectra of protein-model aromatic chromophores.

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D₂O and H₂O spectra of these chromophores. For phenols approximately equal Δ? values are obtained in both types of spectra, but for their methyl ethers Δ? values of D₂O vs H₂O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25–4% D₂O or H₂O. For phenols in D₂O vs H₂O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D₂O and H₂O. The refractive index difference between D₂O and H₂O was shown to be a minor contribution by means of experiments in which D₂O was at 5 dgC and H₂O at 47 dgC, conditions of equal refractive index (Na_D). D₂O vs H₂O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D₂O vs H₂O spectra of proteins are discussed.     

Determination of Fatty Acid in Surfactin and Elucidation of the Total Structure of Surfactin

Several properties of ATPase bound to the inner membrane of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 were examined. The membrane-bound ATPase had two optimal peaks of the activity at pH 5.8 and 7.3. The ATPase activity was strongly inhibited by N,N’- dicyclohexylcarbodiimide (DCCD) and NaN₃ at pH 5.8 and 8.0, and stimulated by MgCl₂ and CaCl₂ at pH 8.0. At pH 8.0, the enzyme hydrolyzed GTP and ITP as well as ATP but not AMP or p-nitrophenylphosphate. CTP, UTP, and ADP were poor substrates. These characteristics indicate that there is a F₀F₁-type ATPase in the inner membrane of this bacterium. In addition, the ATPase activity was also significantly inhibited by Na₃ Vo₄, suggesting the coexistence of a P-type ATPase as a minor constituent. The membrane-bound ATPase activity was maximum at 50°C, but the strong DCCD-sensitivity observed at 20°C was greatly reduced at this temperature.     

Mg2+-induced ADP-dependent inhibition of the ATPase activity of beef heart mitochondrial coupling factor F1.

Incubation of F₁ in the presence of Mg²⁺ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg²⁺ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F₁ treated to remove tightly bound nucleotides was preincubated in the presence of Mg²⁺. Mg²⁺-induced inhibition of ATPase activity of nucleotide-depleted F₁ can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg²⁺-induced inhibition of the ATPase activity of F₁ is due to the formation of an inactive F₁. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, $\text{86}$, 434/ is directed to F₁ component of the complete mitochondrial ATPase system.     

Complete Assignment of the 500 MHz 1H-NMR Spectra of Bleomycin A2 in H2O and D2O Solution by means of Two-dimensional NMR Spectroscopy

Abstract

¹H-NMR spectra of bleomycin A2 recorded at 500 MHz in D₂O and H₂O at 24°C and 3°C were investigated. Resonances of the individual spin systems were identified by using two-dimensional correlated spectroscopy (COSY), two-dimensional spin echo correlated spectroscopy (SECSY) and by the application of two-dimensional Nuclear Overhauser Effect spectroscopy (NOESY). Employment of these techniques allowed the assignment of 13 exchangeable and 59 non-exchangeable protons in the ¹H NMR spectrum of bleomycin A2. By means of 2D NOE spectroscopy also interresidual connectivities could be observed. Comparison of the NOESY spectra at 3°C and 24°C suggest that at low temperatures the central part of the bleomycin A2 molecule tends to adopt an extended conformation.     

Differential Stability of the Triple Helix of (Pro-Pro-Gly)10 in H2O and D2O: Thermodynamic and Structural Explanations

Abstract

(Pro-Pro-Gly)₁₀ [(PPG₁₀)], a collagen-like polypeptide, forms a triple-helical, polyproline-II structure in aqueous solution at temperatures somewhat lower than physiological, with a melting temperature of 24.5°C. In this article, we present circular dichroism spectra that demonstrate an increase of the melting temperature with the addition of increasing amounts of D₂O to an H₂O solution of (PPG)₁₀, with the melting temperature reaching 40°C in pure D₂O. A thermodynamic analysis of the data demonstrates that this result is due to an increasing enthalphy of unfolding in D₂O vs. H₂O. To provide a theoretical explanation for this result, we have used a model for hydration of (PPG)₁₀ that we developed previously, in which inter-chain water bridges are formed between sterically crowded waters and peptide bond carbonyls. Energy minimizations were performed upon this model using hydrogen bond parameters for water, and altered hydrogen bond parameters that reproduced the differences in carbonyl oxygen-water oxygen distances found in small-molecule crystal structures containing oxygen-oxygen hydrogen bonds between organic molecules and H₂O or D₂O. It was found that using hydrogen bond parameters that reproduced the distance typical of hydrogen bonds to D₂O resulted in a significant lowering of the potential energy of hydrated (PPG)₁₀. This lowering of the energy involved energetic terms that were only indirectly related to the altered hydrogen bond parameters, and were therefore not artifactual; the intra-(PPG₁₀) energy, plus the water-(PPG₁₀) van der Waals energy (not including hydrogen bond interactions), were lowered enough to qualitatively account for the lower enthalpy of the triple-helical conformation, relative to the unfolded state, in D₂O vs. H₂O. This result indicates that the geometry of the carbonyl-D₂O hydrogen bonds allows formation of good hydrogen bonds without making as much of an energetic sacrifice from other factors as in the case of hydration by H₂O.     

Interaction of Mg+2 with beef heart mitochondrial ATPase (F1).

The soluble mitochondrial ATPase, F₁, can be slowly inactivated by incubation with Mg⁺² in a manner consistent with the observations of Moyle and Mitchell $\text{(}\text{FEBS}\text{Lett}\text{.}\text{56}$, 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F₁ which has been incubated with Mg⁺². This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F₁ concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F₁ is unstable if incubated with EDTA in the absence of free nucleotides or Mg⁺². The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg⁺² in excess of the EDTA or 50 μM ADP stabilize the F₁ against the inactivation but cannot reverse prior denaturation.     

Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO₄, 1.4% K₂HPO₄, 0.02% CaCl₂·2H₂O, 0.002% FeSO₄·7H₂O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F₂ fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70°C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90°C, the t_1/2 values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90°C, the enzyme retained its activity up to 8 h in the presence of 1mM MnCl₂. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K _{m app} values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the V_max values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F₂ chitosanase at 24 h incubation (70°C) were pentasaccharide (GlcN)₅ and hexasaccharide (GlcN)₆. The prelimiaary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium. Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.     

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.

The three major subunits (α, β and γ) of the coupling factor, F₁ ATPase, of $\text{Escherichia coli}$ were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgCl₂. A mixture containing α, β and γ regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with α plus β. The reconstituted ATPase had a K_m of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000.     

The interaction between the mitochondrial ATPase (F1) and the ATPase inhibitor

1. The naturally occurring mitochondrial ATPase inhibitor inhibits the mitochondrial ATPase (F₁) non-competitively.2. The interaction between inhibitor and inhibitor-depleted F₁ or submitochondrial particles is diminished when the ratio of ATP/ADP is low or when energy is generated by substrate oxidation.3. The dissociation of the inhibitor from coupled Mg-ATP particles is promoted when substrates are being oxidized. This results in the appearance of a large uncoupler-stimulated ATPase activity. Activation of the uncoupler-stimulated ATPase activity is also achieved by incubation of the particles with ADP.4. The ATPase activity of Mg-ATP particles is determined by the turnover capacity of F₁. When endogenous inhibitor is removed, energy dissipation becomes the rate-limiting step. This energy dissipation can be activated by an uncoupler.5. Evidence is presented for the existence of a non-inhibited intermediate F₁-inhibitor complex.     

The use of aurovertin to determine the F1 content of submitochondrial particles and the ATPase complex

(1) The concentration of aurovertin-binding sites calculated from fluorimetric titrations of submitochondrial particles is equal to the F₁ concentration, calculated from the concentration of F₁-binding sites in stripped particles.(2) Direct binding experiments show that the fluorescence enhancement of aurovertin bound to submitochondrial particles and the isolated ATPase complex is less (or absent) at higher concentrations than at lower concentrations. The binding data can be described by ‘specific’ and ‘non-specific’ binding. The concentration of the ‘specific’ sites is twice that derived from fluorimetric titrations.(3) After dissociation of the bound F₁ with LiCl, fluorimetric titrations with aurovertin yield linear Scatchard plots. The fluorescence enhancement and K_D are equal to those of the β-subunit-aurovertin complex. The concentration of β-subunits is double the concentration of F₁.(4) It is concluded that both for submitochondrial particles and the isolated ATPase complex the most reliable and simple way to determine the F₁ content is to dissociate the F₁ with LiCl, spin down the insoluble material and titrate the supernatant (containing free β-subunit) with aurovertin.     

Flow cytometric analysis of mitochondrial populations in HL-CMS systems of rice under H2O2 stress

Cytoplasmic male sterility (CMS) has often been associated with mitochondrial dysfunction. In this report, the heterogeneity of mitochondria was analyzed in both Honglian (HL) CMS (YtA) rice seedlings and those of its corresponding maintainers (YtB) by flow cytometry and staining with rhodamine-123 (Rh-123). Both lines revealed two distinct fluorescence populations: high fluorescence populations (HFP) and light fluorescence populations (LFP), and a somewhat lower LFP/HFP ratio was detected in conjunction with the higher reactive oxygen species (ROS) content in YtA. In addition, use of the specific effector hydrogen peroxide (H₂O₂) demonstrated a correlation between the LFP/HFP ratio and ROS levels in both lines. Higher ROS content caused a more swift decrease of F₀F₁-ATPase activity and ATP contents in YtA than those in YtB, which accompanied with an obvious decline of the LFP/HFP ratio in YtA. Furthermore, a mitochondrial genomic DNA smear was detected by pulsed field gel electrophoresis. Taken together, these results implied that HL-CMS line rice seedlings and those of its corresponding maintainer have different proportion of Rh-123 staining mitochondria populations, which may be accounted for by ROS contents on the basis of ATPase activity and ATP contents.     

The equilibrium between the mitochondrial ATPase (F1) and its natural inhibitor in submitochondrial particles

1. The reversible equilibrium between the mitochondrial ATPase (F₁) and its naturally occurring inhibitor in Mg-ATP submitochondrial particles has been studied under different conditions.2. High ionic strength favours dissociation of the ATPase inhibitor as tested by ATPase and ATP-driven transhydrogenase activities.3. Dissociation of the ATPase inhibitor results in an increased maximal velocity of the ATPase activity measured in the presence of uncoupler and an increased affinity for adenine nucleotides, in particular for ATP.4. Association of the ATPase inhibitor with inhibitor-depleted Mg-ATP particles causes a slowing of the initial rate of succinate oxidation.5. The antibiotic aurovertin stimulates the ATPase activity of Mg-ATP particles preinculbated in the presence of a supply of oxidative energy. Bound aurovertin impedes the association of inhibitor-deficient particles with ATPase inhibitor.6. The fluorescence of aurovertin bound to inhibitor-containing particles is much less than that of aurovertin bound to inhibitor-depleted particles.7. The oligomycin-sensitivity-conferring protein, added either alone or in the presence or absence of membranous components of the ATPase complex, has little or no effect on the fluorescence of the F₁-aurovertin complex.8. It is suggested that the ATPase inhibitor brings F₁ in a conformation denoted 1F₁ that binds aurovertin with a low quantum yield, a decreased affinity and an increased binding capacity.     

NADP-linked 15-hydroxyprostaglandin dehydrogenase for prostaglandin D2 in human blood platelets

Two forms of NADP-linked 15-hydroxyprostaglandin dehydrogenase for prostaglandin D₂ were found in the cytosol fraction of human blood platelets. These enzymes were purified by ammonium sulfate fractionation, Blue Sepharose, and Sephadex G-100 column chromatography. The two enzymes differed in molecular weights (65,000 for peak I enzyme and 31,000 for peak II as estimated by gel filtration) and their substrate specificities. The relative rates for reaction with peak I enzyme were: prostaglandin D₂, 100(%); E₂, 14; F_2α, 2; I₂, 29; and B₂, 0; whereas for peak II enzyme, D₂, 100; E₂, 23; F_2α, 61; I₂, 29; and B₂, 131. Prostaglandin D₂ was converted to 15-ketoprostaglandin D₂ and then 13,14-dihydro-15-ketoprostaglandin D₂, which were identified by spectrophotometry and gas chromatography/mass spectrometry, respectively. These metabolites were three orders of magnitude less potent in inhibiting human platelet aggregation than prostaglandin D₂. The results indicated that NADP-linked dehydrogenases participated in the metabolic inactivation of prostaglandin D₂ in the platelets. Furthermore, the dehydrogenase activity for prostaglandin D₂ was high in monkey (0.128 nmol/min · mg at 24 °C) and human platelets (0.066), but was not detectable (less than 0.007) in the rabbit, rat, and chicken. Because prostaglandin D₂, which was demonstrated by several authors to be synthesized in platelet-rich plasma during platelet aggregation, exhibited significant antiaggregatory activity only in human and monkey platelets, these prostaglandin dehydrogenases appear to play a physiological role in the circulatory system.     

Warming-induced enhancement of soil N2O efflux linked to distinct response times of genes driving N2O production and consumption

Temperature responses of denitrifying microbes likely play a governing role in the production and consumption of N₂O. We investigated temperature effects on denitrifier communities and their potential to produce N₂O and N₂ by incubating grassland soils collected in multiple seasons at four temperatures with ¹⁵N-enriched NO₃ ^? for ~24 h. We quantified [N₂O] concentration across time, estimated its production and reduction to N₂, and quantified relative abundance of genes responsible for N₂O production (cnorB) and reduction (nosZ). In all seasons, net N₂O production was positively linked to incubation temperature, with highest estimates of net and gross N₂O production in late spring soils. N₂O dynamics were tightly coupled to changes in denitrifier community structure, which occurred on both seasonal and incubation time scales. We observed increases in nosZ abundance with increasing incubation temperature after 24 h, and relatively larger increases in cnorB abundance from winter to late June. The difference between incubation and in situ temperature was a robust predictor of cnorB:nosZ. These data provide convincing evidence that short-term increases in temperature can induce remarkably rapid changes in community structure that increase the potential for reduction of N₂O to N₂, and that seasonal adaptation of denitrifying communities is linked to seasonal changes in potential N₂O production, with warmer seasons linked to large increases in N₂O production potential. This work helps explain observations of high spatial and temporal variation in N₂O effluxes, and highlights the importance of temperature as an influence on denitrification enzyme kinetics, denitrifier physiology and community adaptations, and associated N₂O efflux and reduction.     

Temperature-dependence of spectroscopic and catalytic properties of the eel (Anguilla anguilla) liver mitochondrial F1-ATPase

• 1.1. F₁-ATPase from eel liver mitochondria at low concentrations preserves unaltered the enzymatic activity for more than 20 min over a temperature range of 6–36°C.
• 2.2. The Arrhenius plot of ATP hydrolysis at saturating substrate concentration appears biphasic with a break-point at 16°C and activation energies of 14.4 and 56.1 kJ/mol.
• 3.3. The ultraviolet, fluorescence and circular dichroism spectra of the enzyme, below and above 16°C, have been recorded; the fluorescence emission spectra of F₁-ATPase excited at 275 nm, and the circular dichroism spectra, are different at the two temperatures examined.
• 4.4. It is concluded that temperature induces two different conformational states of F₁-ATPase with different catalytic properties.
• 5.5. Ultraviolet spectroscopic features and temperature-dependence of eel liver mitochondrial F₁-ATPase are discussed in relation to mammalian F₁-ATPases.

     

Rapid relaxation processes in pig heart lipoamide dehydrogenase revealed by subnanosecond resolved fluorometry

The decay kinetics of the FAD-fluorescence in lipoamide dehydrogenase from pig heart have been reinvestigated using phase fluorometric methods and sophisticated laser pulse techniques. Both pulse and modulation methods lead to distinct heterogeneity in lifetimes. The two different techniques lead to good correspondence in the longer lifetime component of a biexponential decay model, whereas the more rapidly decaying component is distinctly shorter and has a larger amplitude using the phase technique with two available modulation frequencies (15 and 60 MHz). Lifetime measurements as a function of temperature and in the presence of D₂O instead of H₂O illustrate that the quenching of the FAD fluorescence in lipoamide. dehydrogenase is mainly dynamic in nature and that solvent comes into contact with the fluorophor. Mobility of the flavin itself, free and bound to the enzyme, has been measured by both differential polarized phase fluorometry and experimental fluorescence anisotropy decay after ps laser pulse excitation. By employing flavin models it has been shown that both techniques have ps time resolution. Measurements with the latter more direct method indicate a rapid subnanosecond motion of the FAD bound within the enzyme, only visible at temperatures lower than about 15°C, where the protein rotational diffusion is slowed down. The significance of rapid transient conformational fluctuations for catalysis is discussed with reference to recently developed insights reported in the literature.     

Regulation of mitochondrial FoF1ATPase activity by Sirt3-catalyzed deacetylation and its deficiency in human cells harboring 4977 bp deletion of mitochondrial DNA

Sirt3, a mitochondrial NAD⁺-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F_oF₁ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F_oF₁ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F_oF₁ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F_oF₁ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F_oF₁ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ? 85% of mtDNA with 4977 bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F_oF₁ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5–10 μM of menadione impaired the Sirt3-mediated deacetylation and activation on F_oF₁ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F_oF₁ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation.