scholarly journals Chromous ion reduction of mammalian cytochrome oxidase and some of its derivatives

1977 ◽  
Vol 165 (2) ◽  
pp. 413-416 ◽  
Author(s):  
C Greenwood ◽  
T Brittain ◽  
M Brunori ◽  
M T Wilson
Keyword(s):  
Mixed Valence ◽  
Slow Phase ◽  
High Concentration ◽  
Difference Spectra ◽  
Rapid Phase ◽  
Fast Phase ◽  
Two Phases ◽  
Rate Limit ◽  
Kinetics Of ◽  
Inhibited Enzyme

The reduction of cytochrome c oxidase by Cr2+, followed by means of stopped-flow spectrophotometry, exhibits two phases: the faster Cr2+-concentration-dependent reaction has an initial rate constant of 1.1 × 10(4)M-1-S-1, but reaches a rate limit at high concentration of reductant; the slower phase is concentration-independent with a rate of 0.3S-1. The activation energies of the fast and the slow processes are 35 and 71 kJ/mol respectively. The reduction kinetics of the mixed-valence CO complex and the cyanide-inhibited enzyme were compared with those of the fully oxidized forms: both the liganded species have a fast phase identical with that found in the oxidized oxidase. A comparison of the kinetic difference spectra obtained for the fast phase of reduction of oxidized oxidase with those obtained on reduction of the liganded species suggests that the rapid phase arises from the reduction ofhaem a, and the slow phase from the reduction of haem a3.

scholarly journals The assembly of microtubule protein in vitro. The kinetic role in microtubule elongation of oligomeric fragments containing microtubule-associated proteins

1985 ◽  
Vol 227 (2) ◽  
pp. 439-455 ◽  
Author(s):  
P M Bayley ◽  
F M M Butler ◽  
D C Clark ◽  
E J Manser ◽  
S R Martin
Keyword(s):  
Self Assembly ◽  
Protein Concentration ◽  
Wavelength Dependence ◽  
Electrophoretic Analysis ◽  
Slow Phase ◽  
Condensation Polymerization ◽  
Fast Phase ◽  
Microtubule Protein ◽  
Kinetics Of

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.

Determinants of relaxation rate in skinned frog skeletal muscle fibers

1998 ◽  
Vol 274 (6) ◽  
pp. C1608-C1615 ◽  
Author(s):  
Philip A. Wahr ◽  
J. David Johnson ◽  
Jack. A. Rall
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Slow Phase ◽  
Frog Skeletal Muscle ◽  
Force Of Contraction ◽  
Thin Filaments ◽  
Fast Phase ◽  
Skeletal Muscle Fibers ◽  
Kinetics Of ◽  
Overall Kinetics

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10°C. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

scholarly journals Light Adaptation in the Ventral Photoreceptor of Limulus

1974 ◽  
Vol 64 (2) ◽  
pp. 166-185 ◽  
Author(s):  
Richard Srebro ◽  
Michael Behbehani
Keyword(s):  
Time Course ◽  
Light Adaptation ◽  
Light Response ◽  
Slow Phase ◽  
Rapid Phase ◽  
Short Delay ◽  
Light Intensities ◽  
Lateral Eye ◽  
Complex Process ◽  
Two Phases

Light adaptation in both the ventral photoreceptor and the lateral eye photoreceptor is a complex process consisting of at least two phases. One phase, which we call the rapid phase of adaptation, occurs whenever there is temporal overlap of the discrete waves that compose a light response. The recovery from the rapid phase of adaptation follows an exponential time-course with a time constant of approximately 75 ms at 21°C. The rapid phase of adaptation occurs at light intensities barely above discrete wave threshold as well as at substantially higher light intensities with the same recovery time-course at all intensities. It occurs in voltage-clamped and unclamped photoreceptors. The kinetics of the rapid phase of adaptation is closely correlated to the photocurrent which appears to initiate it after a short delay. The rapid phase of adaptation is probably identical to what is called the "adapting bump" process. At light intensities greater than about 10 times discrete wave threshold another phase of light adaptation occurs. It develops slowly over a period of ½ s or so, and decays even more slowly over a period of several seconds. It is graded with light intensity and occurs in both voltage-clamped and unclamped photoreceptors. We call this the slow phase of light adaptation.

Kinetics Of Thrombin, Antithrombin III And Heparin Interactions

1981 ◽  
Author(s):  
S T Olson ◽  
S A Evans ◽  
J D Shore
Keyword(s):  
Rate Constant ◽  
Antithrombin Iii ◽  
Heart Association ◽  
Slow Phase ◽  
Rapid Phase ◽  
Bimolecular Rate Constant ◽  
At Iii ◽  
Active Site Probe ◽  
Initial Binding ◽  
Kinetics Of

The rates of thrombin inactivation by AT III were studied by stopped-flow fluorimetry using p-aminobenzami- dine as an active site probe. p-Aminobenzamidine binds to thrombin with a of KD of 35 μM and a 50-fold fluorescence enhancement. The bimolecular rate constant for the thrombin-AT III reaction was 8.7 × 103 M-1 s-1 at 25° C and pH 7.4, with no diminished amplitude or evidence for a limiting rate at AT III concentrations as high as 10-4 M. Using lower AT III concentrations, an identical bimolecular rate constant was obtained by discontinuous assay of residual thrombin activity. Inactivation of thrombin by antithrombin III complexed with stoichiometric high affinity heparin at concentrations between 2.5 to 20 μM resulted in biphasic reactions: a rapid phase complete in less than 10 milliseconds and a slow phase with a rate constant of 4-6 s-1 . Relative amplitudes and slow phase rate constants showed slight changes over this AT III-heparin concentration range. These results are consistent with weak initial binding of AT III to thrombin in the absence of heparin (KD> 10-4 M), with a major part of the heparin rate enhancement due to a substantial decrease in the KD for the initial binding step. Supported by Michigan Heart Association and USPHS grant F32 HL06171-01.

The Kinetics of Sodium Transfer in the Central Nervous System of the Cockroach, Periplaneta Americana L

1961 ◽  
Vol 38 (4) ◽  
pp. 737-746
Author(s):  
J. E. TREHERNE
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Periplaneta Americana ◽  
Slow Phase ◽  
Second Phase ◽  
Sodium Ions ◽  
Rapid Phase ◽  
Fast Phase ◽  
The Central Nervous System ◽  
Sodium Extrusion

1. The exchange of sodium ions in the cockroach central nervous system has been studied by following the escape of 24Na from isolated abdominal nerve cords, single connectives and ganglia. Particular attention was paid to the initial rapid exchanges of sodium. 2. The escape of sodium ions occurred as a two-stage process, an initial rapid phase eventually giving way to a slower exponential phase of sodium loss. The fast phase of efflux was not affected by the presence of 2:4-dinitrophenol, although this poison significantly reduced the second slow phase of sodium extrusion. 3. The initial fast phase is attributed to a rapid diffusion from an extracellular space, demonstrated by 14C-inulin; the second phase is identified as the slower extrusion from the cellular components of the central nervous system.

scholarly journals Studies on partially reduced mammalian cytochrome oxidase. Reactions with carbon monoxide and oxygen

1974 ◽  
Vol 137 (2) ◽  
pp. 205-215 ◽  
Author(s):  
Colin Greenwood ◽  
Michael T. Wilson ◽  
Maurizio Brunori
Keyword(s):  
Cytochrome Oxidase ◽  
Redox State ◽  
Mixed Valence ◽  
Order Rate Constant ◽  
Low Oxygen ◽  
Difference Spectra ◽  
Ferrocytochrome C ◽  
Kinetics Of ◽  
Enzyme Species ◽  
Binding Behaviour

A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c–oxygen oxidoreductase) in which only cytochrome a3 is reduced and in combination with CO. The kinetics of CO binding by cytochrome a32+ in this species is significantly different from that exhibited by cytochrome a32+ in the fully reduced enzyme. The second-order rate constant for combination was 5X104m−1·s−1 and the ‘off’ constant was 3X10−2s−1. The kinetic difference spectra cytochrome a32+–cytochrome a32+–CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a32+ and oxygen in the mixed-valence species was followed in flow–flash experiments and reveals a fast, oxygen-dependent (8X107m−1·s−1 at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O2]. The fast oxygen-dependent reaction yields as the first product the so-called ‘oxygenated’ enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a3 depends on the redox state of its partners, a fact which represents clear evidence for site–site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a3, is reduced represents clear and unequivocal evidence that this is indeed the O2-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.

scholarly journals Two Phases of Actin Polymerization Display Different Dependencies on PI(3,4,5)P3 Accumulation and Have Unique Roles during Chemotaxis

2003 ◽  
Vol 14 (12) ◽  
pp. 5028-5037 ◽  
Author(s):  
Lingfeng Chen ◽  
Chris Janetopoulos ◽  
Yi Elaine Huang ◽  
Miho Iijima ◽  
Jane Borleis ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Slow Phase ◽  
Wild Type ◽  
Rapid Phase ◽  
Pi3k Inhibitors ◽  
Pi3k Inhibition ◽  
Directional Movement ◽  
Temporal And Spatial ◽  
Two Phases ◽  
Highly Correlated

The directional movement of cells in chemoattractant gradients requires sophisticated control of the actin cytoskeleton. Uniform exposure of Dictyostelium discoideum amoebae as well as mammalian leukocytes to chemoattractant triggers two phases of actin polymerization. In the initial rapid phase, motility stops and the cell rounds up. During the second slow phase, pseudopodia are extended from local regions of the cell perimeter. These responses are highly correlated with temporal and spatial accumulations of PI(3,4,5)P3/PI(3,4)P2 reflected by the translocation of specific PH domains to the membrane. The slower phase of PI accumulation and actin polymerization is more prominent in less differentiated, unpolarized cells, is selectively increased by disruption of PTEN, and is relatively more sensitive to perturbations of PI3K. Optimal levels of the second responses allow the cell to respond rapidly to switches in gradient direction by extending lateral pseudopods. Consequently, PI3K inhibitors impair chemotaxis in wild-type cells but partially restore polarity and chemotactic response in pten- cells. Surprisingly, the fast phase of PI(3,4,5)P3 accumulation and actin polymerization, which is relatively resistant to PI3K inhibition, can support inefficient but reasonably accurate chemotaxis.

The Effects of Externally Applied Lithium and Strontium on the Arsenazo-Monitored Cytosolic Calcium Signal of the Limulus Ventral Photoreceptor

1994 ◽  
Vol 49 (5-6) ◽  
pp. 372-392 ◽  
Author(s):  
Hennig Stieve ◽  
Gabriele Rüsing ◽  
Weijia Yuan ◽  
H. Thomas Hennig
Keyword(s):  
Receptor Potential ◽  
Physiological Saline ◽  
Cytosolic Calcium ◽  
Slow Phase ◽  
Calcium Signal ◽  
Complete Suppression ◽  
Uptake System ◽  
Fast Phase ◽  
Intracellular Calcium Level ◽  
Two Phases

Abstract The intracellular arsenazo signal indicating the transient light-evoked change in cytosolic Ca2+ (or Sr2+) concentration was measured in Limulus ventral photoreceptor simultaneously with the receptor potential at 15 °C. The decline of the arsenazo signal has two phases (D1 and D2) when the photoreceptor is bathed in physiological saline. 1. When calcium is replaced by strontium in the superfusate both receptor potential and arsenazo signal are markedly increased in amplitude and the membrane potential is hyper­ polarized. The decline of the arsenazo signal is prolonged and becomes monophasic; the fast phase D1 of the decline disappears. 2. In strontium saline under voltage clamp conditions the slope of the monophasic decline of the arsenazo signal is the steeper the more negative the membrane voltage. 3. After replacing sodium by lithium in the superfusate the rise of the receptor potential and of the arsenazo signal are not much altered. The decline of the arsenazo signal, however, is slowed down more than 3-fold; this is due to the complete suppression of the fast phase D1 and the retardation of the slow phase D2 of the calcium re-decline. Interpretation: 1. The Na-C a exchanger can accept strontium as a calcium substitute. Strontium has a weaker desensitizing action than calcium. Strontium is not-or only very little -taken up by the endoplasmic cisternae. 2. In sodium-free lithium saline the Na-C a exchanger, the Na-K ATPase and the calcium uptake system of the endoplasmic cisternae do not function. Therefore the intracellular calcium level rises.

scholarly journals Kinetics of reaction of [nitrotyrosyl]cytochrome c with ligands

1976 ◽  
Vol 157 (1) ◽  
pp. 217-220
Author(s):  
A José do Nascimento ◽  
K Hishida do Nascimento
Keyword(s):  
Cytochrome C ◽  
Slow Phase ◽  
Stopped Flow ◽  
Order Process ◽  
First Order ◽  
Fast Phase ◽  
Kinetics Of Reaction ◽  
Kinetics Of ◽  
Flow Techniques

The reaction of [nitrotyrosyl]cytochrome c with ligands was studied by stopped-flow techniques. At pH 7.0 the reaction with imidazole shows two distinct phases, one fast phase being concentration-dependent and a slow phase being concentration-independent. The results are consistent with the existence of two forms of [nitrotyrosyl]cytochrome c in solutions [Schejter et al. (1970) Biochemistry 9, 5118-5122]; form I, the smaller fraction, seems to be responsible for the slow first-order process.

scholarly journals Kinetics of removal of intravenous testosterone pulses in normal men

2010 ◽  
Vol 162 (4) ◽  
pp. 787-794 ◽  
Author(s):  
Johannes D Veldhuis ◽  
Daniel M Keenan ◽  
Peter Y Liu ◽  
Paul Y Takahashi
Keyword(s):  
Sampling Strategy ◽  
Free Testosterone ◽  
Slow Phase ◽  
Total Testosterone ◽  
Rapid Phase ◽  
Deconvolution Analysis ◽  
Normal Men ◽  
Frequent Sampling ◽  
Kinetics Of

BackgroundTestosterone is secreted into the bloodstream episodically, putatively distributing into total, bioavailable (bio) nonsex hormone-binding globulin (nonSHBG-bound), and free testosterone moieties. The kinetics of total, bio, and free testosterone pulses are unknown.DesignAdrenal and gonadal steroidogenesis was blocked pharmacologically, glucocorticoid was replaced, and testosterone was infused in pulses in four distinct doses in 14 healthy men under two different paradigms (a total of 220 testosterone pulses).MethodsTestosterone kinetics were assessed by deconvolution analysis of total, free, bioavailable, SHBG-bound, and albumin-bound testosterone concentration–time profiles.ResultsIndependently of testosterone dose or paradigm, rapid-phase half-lives (min) of total, free, bioavailable, SHBG-bound, and albumin-bound testosterone were comparable at 1.4±0.22 min (grand mean±s.e.m. of geometric means). Slow-phase testosterone half-lives were highest for SHBG-bound testosterone (32 min) and total testosterone (27 min) with the former exceeding that of free testosterone (18 min), bioavailable testosterone (14 min), and albumin-bound testosterone (18 min; P<0.001). Collective outcomes indicate that i) the rapid phase of testosterone disappearance from point sampling in the circulation is not explained by testosterone dose; ii) SHBG-bound testosterone and total testosterone kinetics are prolonged; and iii) the half-lives of bioavailable, albumin-bound, and free testosterone are short.ConclusionA frequent-sampling strategy comprising an experimental hormone clamp, estimation of hormone concentrations as bound and free moieties, mimicry of physiological pulses, and deconvolution analysis may have utility in estimating the in vivo kinetics of other hormones, substrates, and metabolites.

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