Adenosine triphosphatase and adenosine diphosphate/adenosine triphosphate isotope-exchange activities of the chromaffin-granule membrane.
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ABSTRACT:
SUBMITTER: Apps DK
PROVIDER: S-EPMC1183652 | biostudies-other | 1977 Oct
REPOSITORIES: biostudies-other
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Project description:1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.
| S-EPMC1265009 | biostudies-other
Project description:1. Highly purified resealed chromaffin-granule ;ghosts' were assayed for ATPase and ATP-driven H(+)-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H(+)-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H(+)-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the ;ghosts' with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H(+)-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded ;ghosts', whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with (14)C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[(14)C]carbodi-imide are labelled predominantly in a protein of low molecular weight, which may be analogous to the mitochondrial H(+)-conducting proteolipid. 8. It is concluded that Nbf-Cl and silicotungstate inhibit the H(+)-translocating ATPase of the granule membrane; that dicyclohexylcarbodi-imide inhibits the ATPase, and inhibits 5-hydroxytryptamine accumulation by accelerating leakage of the amine; and that the effects of tributyltin chloride are due to inhibition of the ATPase, and collapse of the transmembrane H(+)-gradient through OH(-)-anion exchange.
| S-EPMC1162091 | biostudies-other
Project description:Catecholamines are accumulated by bovine chromaffin-granule "ghosts" in the presence of MgATP at 25 degrees C. With low concentrations of catecholamine, ratios of internal to external amine concentration of up to 20 000 were obtained. These values fit well with a transport model in which amine accumulation is both electrogenic and dependent on a pH gradient across the membrane.
| S-EPMC1162331 | biostudies-other
Project description:The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.
| S-EPMC1161965 | biostudies-other
Project description:The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.
| S-EPMC1168088 | biostudies-other
Project description:1. K(+) did not affect the Mg(2+)-dependent transphosphorylation but markedly increased the Na(+)-stimulated ADP-ATP exchange rate mediated by a microsomal fraction from guinea-pig kidney. 2. Rb(+), Cs(+), NH(4) (+) and Li(+) were equally effective in stimulating the Na(+)-dependent ADP-ATP exchange activity. 3. Treatment of the microsomal fraction with N-ethylmaleimide or increased concentrations of Mg(2+) prevented stimulation of the Na(+)-dependent exchange reaction by K(+). 4. Ouabain (2.5mum) inhibited ATP hydrolysis by 33% but did not decrease the K(+)-stimulated Na(+)-dependent ADP-ATP exchange rate. 5. A possible mechanism for stimulation of exchange activity by K(+) is discussed.
| S-EPMC1174179 | biostudies-other
Project description:The proton-translocating adenosine triphosphatase (ATPase) of bovine chromaffin granules contains up to five different polypeptides. Its activity is inhibited by N-ethylmaleimide, and ATP protects the enzyme from inhibition. After treatment of membranes with N-[2-3H]ethylmaleimide, only one polypeptide is strongly radiolabelled: this is the largest (70 kDa) subunit of the proton-translocating ATPase. This subunit therefore contains the ATP-hydrolysing site. Two-dimensional electrophoresis reveals heterogeneity in this polypeptide.
| S-EPMC1147241 | biostudies-other
Project description:1. The preparation of a fraction containing highly purified chromaffin granules from the bovine adrenal medulla is described. 2. The fraction contains an adenosine-triphosphatase activity that is stimulated by Mg(2+) and that cannot be explained by contamination with mitochondria or microsomes. 3. It is suggested that the adenosine-triphosphatase activity is related to the uptake of cate-cholamines by the chromaffin granules.
| S-EPMC1214348 | biostudies-other
Project description:The influence of isotopically enriched magnesium on the creatine kinase catalyzed phosphorylation of adenosine diphosphate is examined in two independent series of experiments where adenosine triphosphate (ATP) concentrations were determined by a luciferase-linked luminescence end-point assay or a real-time spectrophotometric assay. No increase was observed between the rates of ATP production with natural Mg, (24)Mg, and (25)Mg, nor was any significant magnetic field effect observed in magnetic fields from 3 to 1,000 mT. Our results are in conflict with those reported by Buchachenko et al. [J Am Chem Soc 130:12868-12869 (2008)], and they challenge these authors' general claims that a large (two- to threefold) magnetic isotope effect is "universally observable" for ATP-producing enzymes [Her Russ Acad Sci 80:22-28 (2010)] and that "enzymatic phosphorylation is an ion-radical, electron-spin-selective process" [Proc Natl Acad Sci USA 101:10793-10796 (2005)].
| S-EPMC3277194 | biostudies-literature
Project description:'Ghosts' of bovine chromaffin granules, in which the complex mixture of proteins and solutes normally found in the granule matrix is replaced by buffered sucrose are osmotically sensitive. They shrink when the osmotic pressure of the suspension medium is increased, and swell if solute entry is facilitated by the addition of ionophores. Swelling in the presence of ionophores has been used to investigate the passive ion permeability of these membranes. They have a very low permeability to K+ ions (of the order of 10(-10) cm/s); their permeability to protons, Na+ and choline ions is too low to be detected by these methods. Their passive permeability to anions decreases in the order: CNS- greater than I- greater than CCl3CO2- greater than Br- greater than Cl- greater than SO4(2)- greater than CH3CO2-, HCO3-, F-, PO4(3)- the permeability to hiocyanate being of the order of 10(-7) cm/s. Coupled proton and anion entry is extremely slow, except for weak acids. Fluoride, unexpectedly, also appears to enter rapidly when proton/K+ exchange is facilitated by nigericin. In the presence of K+ salts, nigericin, like valinomycin, induces lysis of intact granules, an effect that is not dependent on the presence of a permeant anion, but is dependent on the pH gradient across the membrane.
| S-EPMC1183763 | biostudies-other