Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents.

1. O-Iodosobenzoate and 2,2'-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol. 2. The inhibition of the Ca2+-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2'-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide. 3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher Km for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Interactions of the chelating agent 2,3-dimercapto-propane-1-sulfonate with red blood cells in vitro. I. Evidence for carrier mediated transport

Uptake of the water soluble 1,2-dimercaptopropanol (BAL) derivative 2,3-dimercapto-1-sulfonate (DMPS) into human red blood cells was found in vitro and the mode of penetration studied in detail. The compound entered erythrocytes in a concentration dependent manner. In contrast to sealed ghosts where inside and outside concentrations reached the same value, DMPS accumulated in intact erythrocytes. Since no binding of DMPS could be detected, the reason for accumulation was assumed to be a conversion of DMPS into chelates or metabolites which penetrated the membrane in a slower rate. A facilitated transport of DMPS mediated by the anion carrier protein was concluded on the basis of the following similarities with the anion transport: inhibition of [¹⁴C]DMPS-uptake by N-ethylmaleimide (NEM), tetrathionate (90%), sulfate (50%), 5,5′-dithio bis(2-nitrobenzoic acid) (DTNB) (25%); inhibition of uptake and efflux by 4,4′-diisothiocyano-2,2′-stilbene disulfonate (DIDS) (80%), dipyridamole (55%); temperature dependency (activation energy 24 K_cal/mol); pH-dependency (pH optimum about 6.9); counter-transport; activation of uptake by preincubation with DMPS (transmembrane effect).     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF₁) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the ³²P_i-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

Evidence for solvent-induced conformational changes of the soluble Dunaliella chloroplast coupling factor 1 (CF1)

The ATPase activity of the chloroplast coupling factor 1 (CF₁) isolated from the green alga Dunaliella is completely latent. A brief heat treatment irreversibly induces a Ca²⁺ -dependent activity. The Ca²⁺ dependent ATPase activity can be reversibly inhibited by ethanol, which changes the divalent cation dependency from Ca²⁺ to Mg²⁺. Both the Ca²⁺ -dependent and Mg²⁺ -dependent ATPase activities of heat-treated Dunaliella CF₁ are inhibited by monospecific antisera directed against Chlamydomonas reinhardi CF₁. However, when assayed under identical conditions, the Ca²⁺ -dependent ATPase activity is significantly more sensitive to inhibition by the antisera than is the Mg²⁺ -dependent activity. These data are interpreted as indicating that soluble Dunaliella CF₁ can exist in a variety of conformations, at least one of which catalyzes a Ca²⁺ -dependent ATPase and two or more of which catalyze an Mg²⁺ -dependent ATPase.     

Effects of internal ph on the nonselective cation channel from the mouse collecting tubule

We investigated the effects of internal pH on Ca-activated, nucleotide-inhibited nonselective cation channels in the basolateral membranes of mouse collecting tubules, using the inside-out variant of the patch clamp technique. pH modulated the channel open probability (P _o ), giving a bell-shaped curve peaking at pH 6.8/7.0: P _o at pH 6.0 was 11±6% of P _o at pH 7.2 and 32 ±7% at pH 8.0. The open and closed time distributions, best fitted to the sum of two exponentials, were differently sensitive to acid and alkaline conditions. Low pH reduced the short and long open times to 38 and 24% of their pH 7.2 values, while high pH produced a 4-fold increase in the long closed time. As previously reported, 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) induced a quasi-permanent opening of the channel. The inhibition of the channel produced by high pH disappeared in the presence of SITS, while the inhibition produced by low pH was unaffected. These results suggest that the pH dependence of the channel is due to two separate mechanisms. pH was without effect on the ATP-evoked inhibition of the channel, while high pH profoundly reduced the steepness of the AMP inhibition curve, without altering the half-maximal inhibitory AMP concentration.     

Membrane-Bound ATPase Contributes to Hop Resistance of Lactobacillus brevis

The activity of the membrane-bound H⁺-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 μM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N′-dicyclohexylcarbodiimide, a known inhibitor of F_oF₁-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the α- and β-subunits of F_oF₁-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds.     

Limited proteol ysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solublized coupling factor with chymotrypsin rapidly produced an A′-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the ATPase enzyme. Secondary chymotryptic cleavage yielded an A″-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at ?20°C in the presence of 4 mM MgCl₂ with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4°C in the presence or absence of MgCl₂ both decreased ATPase activity and generated A′-type alpha subunit species. Since presence of phenylmethylsulfonyl fluoride prevented these changes, an unknown protease was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparently located within the same small segment of alpha subunit polypeptide chain.     

The epsilon Subunit of the Chloroplast Coupling Factor 1 from Euglena gracilis: A Possible Role in Controlling ATPase Activity

The coupling factor from chloroplasts (CF₁) of Euglena gracilis Z strain is an active ATPase in situ, and its activity cannot be increased by treatment with trypsin or heating as is the case with the CF₁ from other sources. The smallest subunit of CF₁, the ε subunit, is supposed to be involved in controlling the ATPase activity. We have devised a simple technique for rapid and large-scale isolation of this subunit. The ε subunit from Euglena CF₁, although having only a limited inhibitory effect on Euglena CF₁, drastically inhibited the ATPase activity of heat-activated spinach CF₁. The inhibition of spinach CF₁ could be reversed by passage through Sephadex G-50 or by a second heat activation. An antibody to the ε subunit of Euglena CF₁ cross-reacted only weakly with CF₁ from spinach, Sorghum, Kalanchoë, or Anacystis nidulans, but reacted well with whole Euglena CF₁ in addition to its ε subunit. The antibody increased the ATPase activity of Euglena and Anacystis CF₁ and of unactivated or partially activated spinach CF₁. The results suggest that the function of the ε subunit in Euglena CF₁ is similar to its function in CF₁ from other sources. The data also suggest that changes induced in spinach CF₁ by activation involves modifications in subunits other than the ε one.     

Effects of fluorescamine labeling on mitochondrial ATPase activity.

Fluorescamine labeling of rat liver mitochondria enhances the ATPase activity. It reached maximum stimulation when mitochondria were treated with 30–34 nmol fluorescamine per mg of mitochondrial protein. This stimulation is inhibited by N,N′-dicyclohexylcarbodiimide. The maximum stimulation caused by labeling is the same as that obtained from uncoupler with optimum concentration. The chemiosmotic potential (Δμ_H⁺) decreases as the labeling increased. However, Δμ_H⁺ is not abolished completely even when ATPase activity reaches a maximum. The results suggest that primary amino groups may be involved in controlling mitochondrial ATPase activity.     

Synthesis and vasodilative activity of tanshinone IIA derivatives

A series of 2,2′-(substituted methylene)bis-(1,6,6-trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan-10,11-dione) derivatives were synthesized by the reaction of tanshinone IIA (D₁) and aromatic aldehyde in the presence of p-TsOH. Bromination derivative of D₁ and hydrolysis product of cryptotanshinone (D₂) were also prepared in this work. Vasodilation activity in vitro of them was valuated on the contractile response of vascular thoracic aorta smooth muscle from Wistar rats for the first time. Most of them exhibited a concentration-dependent inhibition on the contractile response of norepinephrine.     

The stimulation of coupling factor 1 ATPase by tentoxin

Tentoxin at 10–1000 μM causes a marked species-selective stimulation of coupling factor 1 Ca²⁺-dependent ATPase activity (K_a 6.3 · 10³ M^?1). This effect decreases the K_m for ATP to about 0.3 mM and increases V 2.75-fold. Above 1.6 μM tentoxin the rate of coupled electron transport was reduced to basal without uncoupling.     

α and β subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ^?) and can survive complete loss of the organellar genome (ρ^o), the genetic factor(s) that permit(s) survival of ρ^? and ρ^o mutants remain(s) unknown. In this report we show that a function associated with the F₁-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ? subunit of F₁, renders cells petite-negative. The F₁ complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρ^o cells and those impaired in electron transport. The activity of F₁ that suppresses ρ^o lethality is independent of the membrane F_o complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F₁ subunits to suppress ρ^o lethality has been achieved by simultaneous expression of S. cerevisiae F₁α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ^? or ρ^o mutations in S. cerevisiae.     

Properties of a plasmalemma ATPase of the maize scutellum

Properties of a plasmalemma phosphatase of the maize scutellum, tentatively identified as an ATPase in a previous paper, were investigated. Fresh and frozen-thawed scutellum slices, that had been treated with 10 mM HCl to destroy acid phosphatases, were used as a source of enzyme. With the exceptions of the Na⁺, K⁺ and dinitrophenol experiments, the two kinds of slices gave similar results. ATP and CTP were the best substrates for the enzyme followed by TTP, UTP, CDP, ADP and GTP. UDP, nucleoside monophosphates, sugar phosphates, inorganic pyrophosphate and p-nitrophenyl phosphate were relatively ineffective as substrates. The K_m's for ATP and ADP were 0.65 and 5 mM, respectively, but the two substrates gave the same V_max (49.8 μmol P_i/hr/g slices). Previously, it was shown that the products of ATP hydrolysis are ADP, AMP and P_i. Using these previous results and from the time courses of ATP disappearance from the bathing solution and the appearance of P_i and ADP, it was concluded that ATP and ADP were hydrolysed by the same enzyme. The ATPase was not inhibited by oligomycin. N-N′-Dicyclohexylcarbodiimide (DCCD) was a poor inhibitor, and a water soluble analog of DCCD, 1-ethyl-3 (3 dimethyl-aminopropyl)-carbodiimide, gave only 33% inhibition. The relative effectiveness of divalent cations for stimulating ATPase activity was Mn²⁺ > Mg²⁺ ? Ca²⁺ > Co²⁺ · Na⁺ and K⁺ gave a small additional stimulation in the presence of Mg²⁺. However, Na⁺ and K⁺ gave a much greater stimulation when no divalent cation was added, and this occurred only when fresh slices were used. Dinitrophenol also increased ATPase activity only when fresh slices were used. Since it is likely that both the uptake of Na⁺ and K⁺ and the action of dinitrophenol would lower the electrochemical gradient of protons across the plasmalemma, the different results obtained with fresh slices indicate that the ATPase in these slices was under the constraint of a proton gradient.     

Photoaffinity labeling of soluble chloroplast adenosine 5′-triphosphatase with 3′-O-(4-benzoyl)benzoyl ADP

3′-O-(4-benzoyl)benzoyl ADP (BzADP) acts as a reversible inhibitor of the chloroplast coupling factor 1 ATPase (CF₁) when incubated with the enzyme in the dark. The V_max of ATP hydrolysis is decreased and the kinetics of the reaction are altered from noncooperative to cooperative with respect to ATP. Photoactivation of the benzophenone group in BzADP by irradiation with ultraviolet light (366 nm) results in the covalent binding of BzADP to the enzyme and inactivation of its enzymic activity. Polyacrylamide gel electrophoresis of CF₁-ATPase in the presence of sodium dodecyl sulfate shows that the analog is bound primarily to the enzyme's β subunit. Complete inactivation of the activated CF₁-ATPase occurs upon covalent binding of 2.45 mol BzADP/mol CF₁. Binding of BzADP and inactivation of the ATPase are prevented if ADP, but not ATP, is present during the photoactivation step. The presence of Ca²⁺ during irradiation enhances the rate of BzADP covalent binding as well as the rate of inactivation of the enzyme.     

Inhibition of the transthylakoid gradient of electrochemical proton potential by the local anesthetic dibucaine

The effects of the local anesthetic dibucaine on coupling between electron transport and ATP synthesis-hydrolysis by the coupling-factor complex (CF₀CF₁ ATPase) were investigated in thylakoid membranes from Spinacia oleracea L. cv. Monatol. Evidence is presented that inhibition of ATP synthesis was produced by a specific uncoupling mechanism which was based on dibucaine-membrane surface interactions rather than on the interaction of dibucaine with the ATPase complex. Dibucaine reduced the osmotic space of thylakoid vesicles. At low pH of the medium it stimulated ATP hydrolysis beyond the rates obtained with optimum concentrations of ‘classical’ uncouplers. After addition of dibucaine, there was displacement of membrane-bound Mg²⁺ and strong thylakoid stacking in the presence of only low Mg²⁺ concentrations. Inhibition of ATP synthesis and transmembrane pH gradient increased with medium pH. Hydrolysis of ATP by isolated CF₁ and the CF₀CF₁ complex was only slightly affected by dibucaine. The data are discussed assuming the involvement of localized proton channels on the membrane surface in protonic coupling of electron transport and ATP synthesis. A hypothesis for the mechanisms of action of local anesthetics at the thylakoid membrane is presented.     

The Role of Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH^o) and entropy (ΔS^o) upon channel gating. The magnitude, sign, and temperature dependence of ΔH^o and ΔS^o, the last given by an associated change in heat capacity (ΔC_p), can determine a channel’s temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔC_p makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH^o and ΔS^o, including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH^o/ΔS^o ratio associated with them, can also determine a channel’s temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔC_p-independent manner.     

Solubilization of a phospholipid-stimulated adenosine triphosphatase complex from membranes of Escherichia coli.

A phospholipid-stimulated adenosine triphosphatase (ATPase) complex was solubilized and partially purified from membrane particles of Escherichia coli ML308-225. The complex was of large molecular size and contained 16 polypeptides, five of which were subunits of the F₁-type ATPase of E. coli. Components of the respiratory chain were absent. Enzyme activity was stimulated by lysophosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, and cardiolipin but not by phosphatidylethanolamine. The ATPase activity of the complex was inhibited by N,N′-dicyclohexylcarbodiimide and by Dio-9 at lower inhibitor:protein ratios than required for inhibition of the F₁-type ATPase of E. coli. However, the ATPase complex was less sensitive than the membrane-bound enzyme to inhibition by these compounds.