Pre-steady-state phosphorylation and dephosphorylation of detergent-purified plasma-membrane Ca2+-ATPase

2002 ◽  
Vol 361 (2) ◽  
pp. 355-361 ◽  
Author(s):  
Luis M. BREDESTON ◽  
Alcides F. REGA
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Time Course ◽  
Slow Phase ◽  
Fast Phase ◽  
Lauryl Ether ◽  
Phosphorylation Reaction ◽  
Steady Level ◽  
Biexponential Kinetics ◽  
Acidic Phospholipids

Pre-steady-state phosphorylation and dephosphorylation of purified and phospholipid-depleted plasma-membrane Ca2+-ATPase (PMCA) solubilized in the detergent polyoxyethylene 10 lauryl ether were studied at 25°C. The time course of phosphorylation with ATP of the enzyme associated with Ca2+, probably the true phosphorylation reaction, showed a fast phase (kapp near 400s−1) followed by a slow phase (kapp = 23s−1). With asolectin or acidic phosphatidylinositol, the concentration of phosphoenzyme (EP) increased at as high a rate as before, passed through a maximum at 4ms and stabilized at a steady level that was approx. half that without lipids. Calmodulin (CaM) did not change the rate of the fast phase, accelerated the slow phase (kapp = 93s−1) and increased [EP] with small changes in the shape of the time course. Dephosphorylation was slow (kapp = 30s−1) and insensitive to CaM. Asolectin accelerated dephosphorylation, which followed biexponential kinetics with fast (kapp = 220s−1) and slow (kapp = 20s−1) components. CaM stimulated the fast component by nearly 50%. The results show that the behaviour of the PMCA is complex, and suggest that acidic phospholipids and CaM activate PMCA through different mechanisms. Acceleration of dephosphorylation seems relevant during activation of the PMCA by acidic phospholipids.

Fast-phase transvascular fluid flux and the Fahraeus effect

1987 ◽  
Vol 62 (4) ◽  
pp. 1513-1520 ◽  
Author(s):  
W. N. Richardson ◽  
D. Bilan ◽  
M. Hoppensack ◽  
L. Oppenheimer
Keyword(s):  
Mechanical Properties ◽  
Slow Rate ◽  
Time Course ◽  
Rate Of Change ◽  
Distributed Model ◽  
Slow Phase ◽  
Fluid Flux ◽  
Fast Phase ◽  
Constant Rate ◽  
Fahraeus Effect

Transvascular fluid flux was induced in six isolated blood-perfused canine lobes by increasing and decreasing hydrostatic inflow pressure (Pi). Fluid flux was followed against the change in concentration of an impermeable tracer (Blue Dextran) measured directly with a colorimetric device. The time course of fluid flux was biphasic with an initial fast transient followed by a slow phase. Hematocrit changes unrelated to fluid flux occurred due to the Fahraeus effect, and their contribution to the total color signal was subtracted to determine the rate of fast fluid flux (Qf). Qf was related to Pi to derive fast-phase conductance (Kf). Slow-phase Kf was calculated from the constant rate of change of lobe weight. For a mean change in Pi of 7 cmH2O, 40% of the color signal was due to fluid flux. Fast- and slow-phase Kf's were 0.86 +/- 0.15 and 0.27 +/- 0.05 ml X min-1. cmH2O–1 X 100 g dry wt-1. The fast-phase Kf is smaller than that reported for plasma-perfused lobes. Possible explanations discussed are the nature of the perfusate, the mechanical properties of the interstitium, and the slow rate of rise of the driving pressure at the filtration site on the basis of a distributed model of pulmonary vascular compliance.

scholarly journals Two Temporal Phases of Light Adaptation in Retinal Rods

2002 ◽  
Vol 119 (2) ◽  
pp. 129-146 ◽  
Author(s):  
Peter D. Calvert ◽  
Victor I. Govardovskii ◽  
Vadim Y. Arshavsky ◽  
Clint L. Makino
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Light Adaptation ◽  
Dynamic Range ◽  
Continuous Illumination ◽  
Slow Phase ◽  
Fast Phase ◽  
Slow Adaptation ◽  
Adaptation Phase

Vertebrate rod photoreceptors adjust their sensitivity as they adapt during exposure to steady light. Light adaptation prevents the rod from saturating and significantly extends its dynamic range. We examined the time course of the onset of light adaptation in bullfrog rods and compared it with the projected onset of feedback reactions thought to underlie light adaptation on the molecular level. We found that adaptation developed in two distinct temporal phases: (1) a fast phase that operated within seconds after the onset of illumination, which is consistent with most previous reports of a 1–2-s time constant for the onset of adaptation; and (2) a slow phase that engaged over tens of seconds of continuous illumination. The fast phase desensitized the rods as much as 80-fold, and was observed at every light intensity tested. The slow phase was observed only at light intensities that suppressed more than half of the dark current. It provided an additional sensitivity loss of up to 40-fold before the rod saturated. Thus, rods achieved a total degree of adaptation of ∼3,000-fold. Although the fast adaptation is likely to originate from the well characterized Ca2+-dependent feedback mechanisms regulating the activities of several phototransduction cascade components, the molecular mechanism underlying slow adaptation is unclear. We tested the hypothesis that the slow adaptation phase is mediated by cGMP dissociation from noncatalytic binding sites on the cGMP phosphodiesterase, which has been shown to reduce the lifetime of activated phosphodiesterase in vitro. Although cGMP dissociated from the noncatalytic binding sites in intact rods with kinetics approximating that for the slow adaptation phase, this hypothesis was ruled out because the intensity of light required for cGMP dissociation far exceeded that required to evoke the slow phase. Other possible mechanisms are discussed.

scholarly journals Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca2+-ATPase

1996 ◽  
Vol 315 (2) ◽  
pp. 673-677 ◽  
Author(s):  
Claudio J. HERSCHER ◽  
Alcides F. REGA ◽  
Hugo P. ADAMO
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Atp Hydrolysis ◽  
State Level ◽  
Red Cells ◽  
Catalytic Cycle ◽  
Steady State Kinetic ◽  
Phosphorylation Reaction ◽  
Apparent Concentration ◽  
Hydrolysis Of

The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis by the calcium ion-dependent ATPase from plasma membrane (PM-Ca2+-ATPase) were studied at 37 °C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 μM [γ-32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the kapp of formation by 140% to 84±11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4±0.17 pmol/mg of protein. If added together with [γ-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 → E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the kapp of the transition by 55% to 14±3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its kapp by 60% to 91±12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3 mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca2+-ATPase under physiological conditions.

Time course of end-expired carbon monoxide concentration is important in studies of cigarette smoking

1987 ◽  
Vol 73 (5) ◽  
pp. 553-555 ◽  
Author(s):  
G. Woodman ◽  
D. M. Wintoniuk ◽  
R. G. Taylor ◽  
S. W. Clarke
Keyword(s):  
Carbon Monoxide ◽  
Cigarette Smoking ◽  
Time Course ◽  
Slow Phase ◽  
Fast Phase ◽  
Carbon Monoxide Concentration ◽  
The Mean

1. Fifteen asymptomatic habitual smokers each smoked one of their usual cigarettes, not having smoked for 2 h. End-expired carbon monoxide concentration (EECO) was measured with an Ecolyzer 2000 series analyser before smoking (pre-S value), 1 min after finishing smoking (post-S value) and then at intervals up to 1 h. 2. The mean EECO boost (increase) over all subjects declined biphasically after smoking, with an initial fast phase from 1 to 5 min, and then a slow phase from 5 to 60 min. EECO fell by as much in the first 5 min as in the next hour. 3. Post-S EECO was related to pre-S EECO (r = 0.89, P < 0.001), but EECO boost was not related to pre-S (r = 0.00). EECO boost was unaffected by the sampling manoeuvre. 4. EECO measurements in epidemiological and smoking studies should not be made for at least 5 min after a cigarette is finished

Abstract 5385: Transport of Free Fatty Acids in Cardiac Myocytes is Mediated by a Plasma Membrane Pump as Revealed by Monitoring Cytoplasmic Unbound Free Fatty Acids

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Andrew N Carley ◽  
J P Kampf ◽  
Alan M Kleinfeld
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Steady State ◽  
Free Fatty Acids ◽  
Cardiac Myocytes ◽  
Time Course ◽  
Plasma Membranes ◽  
Initial Rate ◽  
Ffa Metabolism ◽  
Ffa Transport

The transport of FFA across the plasma membrane represents one of the earliest points at which FFA metabolism can be controlled by cardiac myocytes. Using novel methods to measure the intracellular unbound concentration of FFA ([FFA i ]), the first direct measurements of FFA transport across cardiac plasma membranes have been performed in freshly isolated cardiac myoctyes. Measurements of the unbound concentrations of FFA (FFA u ) in the aqueous phase were performed using the fluorescent ratio probe ADIFAB. Cardiac myocytes were microinjected with ADIFAB, and the transport of oleate and palmitate was determined by monitoring [FFA i ] using fluorescence ratio microscopy. FFA influx was initiated by rapidly increasing the extracellular concentration of FFA u ([FFA o ]) using FFA-BSA complexes, which clamped [FFA o ] at fixed values. The time course of influx was monitored from the change in [FFA i ], which rose exponentially to a steady state level (k influx ~ 0.01 s −1 ). Once steady state was achieved, efflux was initiated by changing the extracellular media back to zero [FFA o ]. Efflux was monitored by the decrease in [FFA i ] which, like influx, revealed exponential behavior (k efflux ~ 0.02 s −1 ). At steady state [FFA i ] was greater than [FFA o ] by a factor of ~3.5, indicating that during influx FFA are pumped up a concentration gradient. Both the initial rate of transport and the gradient ([FFA i ] > [FFA o ]) revealed saturation with increasing [FFA o ]. The initial rate of influx saturated at [FFA o ] > 200 nM, while the [FFA i ] > [FFA o ] gradient was relatively constant (~ 3.5) but began to decrease and approached 1 at [FFA o ] > 200 nM. The efflux rate constant decreased for [FFA o ] > zero, suggesting that efflux may be regulated by a mechanism that senses the level of circulating FFA u . Our results indicate that the mechanism of FFA transport across cardiac myocytes is regulated by the plasma membrane and allows for the efficient storage and release of FFA from cardiac myocytes. We suggest that this mechanism involves an as yet unknown membrane protein pump which enables the cells to accumulate surprisingly high concentrations of FFA. The ability to measure [FFA i ] and the demonstration of efflux are significant steps in understanding cardiac FFA metabolism. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).

scholarly journals A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a ‘binding-change’ mechanism

1999 ◽  
Vol 341 (2) ◽  
pp. 329-337 ◽  
Author(s):  
Jamie D. VENNING ◽  
J. B JACKSON
Keyword(s):  
Steady State ◽  
Equilibrium Constant ◽  
Protein Complexes ◽  
Hydride Transfer ◽  
Order Rate Constant ◽  
Slow Phase ◽  
Single Turnover ◽  
Fast Phase ◽  
Transfer Step ◽  
Steady State Kinetics

In mitochondria and bacteria, transhydrogenase uses the transmembrane proton gradient (δp) to drive reduction of NADP+ by NADH. We have investigated the pre-steady-state kinetics of NADP+ reduction by acetylpyridine adenine dinucleotide (AcPdADH, an analogue of NADH) in complexes formed from the two, separately prepared, recombinant, peripheral subunits of the enzyme: the dI component, which binds NAD+ and NADH, and the dIII component, which binds NADP+ and NADPH. In the stopped-flow spectrophotometer the reaction proceeds as a single-turnover burst of hydride transfer to NADP+ on dIII before product NADPH release becomes limiting in steady state. The burst is biphasic. The results indicate that the fast phase represents direct hydride transfer from AcPdADH to NADP+ in dI:dIII complexes, and that the slow phase, which predominates when [dI] < [dIII], corresponds to dissociation of the protein complexes during multiple turnovers of dI. Measurements on the amplitude of the burst, and on the apparent first-order rate constant of the fast phase, indicate that the equilibrium constant of the hydride-transfer step on the enzyme is shifted relative to that in solution. This has consequences for a model proposed earlier, in which δp is used, not at the hydride-transfer step, but to change the binding affinities of NADP+ and NADPH.

scholarly journals The reaction of fully reduced cytochrome c oxidase with oxygen studied by flow-flash spectrophotometry at room temperature. Evidence for new pathways of electron transfer

1984 ◽  
Vol 218 (3) ◽  
pp. 913-921 ◽  
Author(s):  
B C Hill ◽  
C Greenwood
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Time Course ◽  
Visible Region ◽  
Slow Phase ◽  
Second Phase ◽  
Difference Spectra ◽  
Fast Phase ◽  
O2 Concentration ◽  
Sensitive Phase

Absorption changes during the O2 reaction of reduced bovine cytochrome c oxidase were investigated by the rapid-reaction technique of flow-flash spectrophotometry in the Soret, visible and near-i.r. spectral regions. New features in the time courses of absorption change were observed relative to the earlier findings reported by Greenwood & Gibson [(1967) J. Biol. Chem. 242, 1782-1787]. These new features arise in the Soret and near-i.r. regions and allow the reaction to be described at all wavelengths as a composite of three exponential processes. There is a rapid O2-sensitive phase detectable in the Soret and visible region. The second phase has a rate that is somewhat less dependent on O2 concentration than is the fastest phase rate and is detectable in all three spectral regions. The rate of the third phase is almost independent of the O2 concentration and is also detectable in all spectral regions. Analysis of the three phases gives their rates and absorption amplitudes. The fast phase reaches a rate of 2.5 × 10(4) s-1 at the highest O2 concentration available at 20 degrees C, whereas the phase of intermediate rate is limited at a value of 7 × 10(3) s-1 and the slow phase rate is limited at 700 s-1. The ratios of the kinetic difference spectra for the fast phase and the slow phase do not correspond to the spectra of the individual haem centres. A branched mechanism is advanced that is able to reconcile the kinetic and static difference spectra. This mechanism suggests that some of the cytochrome a is oxidized along with cytochrome a3 in the initial O2-sensitive phase. In addition, the model requires that CuA is oxidized heterogeneously. This fits with the complex time course of oxidation observed at 830 nm while retaining CuA as virtually the sole contributor to absorbance at this wavelength.

Recovery from dynamic exercise

1995 ◽  
Vol 268 (6) ◽  
pp. H2311-H2320 ◽  
Author(s):  
R. E. Williams ◽  
S. M. Horvath
Keyword(s):  
Exercise Intensity ◽  
Time Course ◽  
Minute Ventilation ◽  
Dynamic Exercise ◽  
Slow Phase ◽  
Co2 Uptake ◽  
Exercise Tests ◽  
Fast Phase ◽  
Baseline Levels ◽  
Minimal Information

Minimal information is available on the basic interactions within the metabolic and cardiovascular systems during recovery from exercise. Nine men participated in three experiments: one control and two cost-equivalent (52 liters O2) exercise tests of 30 (EX30) and 45 (EX45) min. Exercise intensities were adjusted accordingly. During recovery, all parameters reestablished baseline levels within 10 min, except for heart rate (30 min). Correlations for each parameter for EX30 and EX45 were obtained by evaluating each subject's exercise cost and recovery "payback." A split, two-factor analysis of variance was run separately on the "fast" (minutes 1-7) and "slow" (minutes 10-60) phases of recovery to determine if the time course of recovery was related to exercise intensity. It was concluded that for a work cost of approximately 300 kcal, 1) the slow phase of recovery was unaffected by the exercise intensity, 2) the fast phase of cardiovascular recovery was unaffected by exercise intensity while minute ventilation and O2 and CO2 uptake were affected, and 3) cardiac output and the ventilatory equivalents for O2 and CO2 correlated well between work cost and recovery payback.

scholarly journals METABOLIC CONTROL OF LUMINESCENCE IN ISOLATED PHOTOPHORES OF PORICHTHYS: EFFECTS OF GLUCOSE ON OXYGEN CONSUMPTION AND LUMINESCENCE

1993 ◽  
Vol 181 (1) ◽  
pp. 279-293
Author(s):  
J. Mallefet ◽  
F. Baguet
Keyword(s):  
Oxygen Consumption ◽  
Time Course ◽  
Light Emission ◽  
Potassium Cyanide ◽  
Cellular Respiration ◽  
Slow Phase ◽  
Light Reaction ◽  
Fast Phase ◽  
Light Production ◽  
Rate Of Oxygen Consumption

1. Basal oxygen consumption of isolated photophores from Porichthys sp. at rest, i.e. without light emission, increased significantly from 0.101+/− 0.021 nmol min-1 to 0.173+/−0.016 nmol min-1 in response to the addition of 5.5 mmol l-1 glucose. 2. 5.5 mmol l-1 glucose pretreatment modified the time course of the two phases of adrenaline-induced luminescence; an increase in oxygen consumption was observed during the fast phase of light production but a decrease occurred during the slow phase of luminescence. 3. Pretreatment of isolated photophores with 5.5 mmol l-1 glucose totally inhibited the light emission induced by 1 mmol l-1 potassium cyanide. With this treatment, the respiration rate decreased progressively and after 40 min reached a value not significantly different from zero. 4. Even after blockage of cellular respiration by cyanide, an increase in the rate of oxygen consumption was observed during the fast adrenaline- induced luminescence. 5. Glucose utilisation by glycolysis or by oxidative metabolism may provide energy to an inhibitory mechanism that maintains the photophores in a non- luminescent state. 6. We suggest that the oxygen consumed during the fast phase of adrenaline luminescence could represent the activity of an extramitochondrial oxidative pathway involved in the light reaction.

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