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.

scholarly journals Mechanism of retraction of the trailing edge during fibroblast movement.

1981 ◽  
Vol 90 (1) ◽  
pp. 187-200 ◽  
Author(s):  
W T Chen
Keyword(s):  
Slow Component ◽  
Fast Component ◽  
Slow Phase ◽  
Main Body ◽  
Elastic Recoil ◽  
Forward Movement ◽  
Trailing Edge ◽  
Fast Phase ◽  
Focal Contacts

Retraction of the taut, trailing portion of a moving chick heart fibroblast in vitro is an abrupt dynamic process. Upon retraction, the fibroblast tail always ruptures, leaving a small amount of itself attached to the substratum by focal contacts. Time-lapse cinemicrography shows that retraction produces a sudden, massive movement of both surface and cytoplasmic material toward a cluster of focal contacts near the main body of the cell. The appearance of folds on the upper cell surface at this time and the absence of endocytotic vesicles are consistent with this forward movement. Retraction of the trailing edge, either occurring naturally or produced artificially with a microneedle, consists of an initial fast component followed and overlapped by a slow component. Upon artificial detachment in the presence of iodoacetate, dinitrophenol, and sodium fluoride, and at 4 degrees C, the slow component is strongly inhibited and the fast one only slightly inhibited. Moreover of the bundles of microfilaments oriented parallel to the long axis of the tail seen in TEM. Most of the birefringence is lost during the fast phase and the rest during the slow phase of retraction. Concurrently, the bundles of microfilaments disappear during the fast phase of retraction and are replaced by a microfilament meshwork. All of these results are consistent with the hypothesis that the initial fast component of retraction is a passive elastic recoil, associated with the oriented bundles of microfilaments, and that the slow component of retraction is an active contraction, associated with a meshwork of microfilaments.

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 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 Temperature-dependent Self assembly of biofilaments during red blood cell sickling

2021 ◽  
Author(s):  
Arabinda Behera ◽  
Oshin Sharma ◽  
Debjani Paul ◽  
Anirban Sain
Keyword(s):  
Self Assembly ◽  
Kinetic Monte Carlo ◽  
Lag Time ◽  
Vital Role ◽  
Temperature Dependent ◽  
Opposite Case ◽  
Assembly Kinetics ◽  
The Mean ◽  
Kinetics Of

Molecular self-assembly plays vital role in various biological functions. However, when aberrant molecules self-assemble to form large aggregates, it can give rise to various diseases. For example, the sickle cell disease and Alzheimer’s disease are caused by self-assembled hemoglobin fibers and amyloid plaques, respectively. Here we study the assembly kinetics of such fibers using kinetic Monte-Carlo simulation. We focus on the initial lag time of these highly stochastic processes, during which self-assembly is very slow. The lag time distributions turn out to be similar for two very different regimes of polymerization, namely, a) when polymerization is slow and depolymerization is fast, and b) the opposite case, when polymerization is fast and depolymerization is slow. Using temperature dependent on- and off-rates for hemoglobin fiber growth, reported in recent in-vitro experiments, we show that the mean lag time can exhibit non-monotonic behaviour with respect to change of temperature.

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 Small-Molecule Effectors of Hepatitis B Virus Capsid Assembly Give Insight into Virus Life Cycle

2008 ◽  
Vol 82 (20) ◽  
pp. 10262-10270 ◽  
Author(s):  
Christina Bourne ◽  
Sejin Lee ◽  
Bollu Venkataiah ◽  
Angela Lee ◽  
Brent Korba ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Small Molecule ◽  
Self Assembly ◽  
Binding Pocket ◽  
Hbv Replication ◽  
B Virus ◽  
In Vitro Assembly ◽  
Kinetics Of

ABSTRACT The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors.

scholarly journals The release of toxic oligomers from α-synuclein fibrils induces dysfunction in neuronal cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Roberta Cascella ◽  
Serene W. Chen ◽  
Alessandra Bigi ◽  
José D. Camino ◽  
Catherine K. Xu ◽  
...  
Keyword(s):  
Self Assembly ◽  
Inclusion Bodies ◽  
Neuronal Damage ◽  
Cellular Systems ◽  
The Self ◽  
Brain Areas ◽  
Rapid Release ◽  
Neuronal Membranes ◽  
Kinetics Of

AbstractThe self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson’s disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.

scholarly journals Taxol-induced bundling of brain-derived microtubules.

1984 ◽  
Vol 99 (3) ◽  
pp. 940-946 ◽  
Author(s):  
P F Turner ◽  
R L Margolis
Keyword(s):  
Electron Microscopy ◽  
Rat Brain ◽  
Crude Extract ◽  
Electrophoretic Analysis ◽  
Preliminary Characterization ◽  
Crude Extracts ◽  
Two Samples ◽  
Microtubule Protein

Taxol has two obvious effects in cells. It stabilizes microtubules and it induces microtubule bundling. We have duplicated the microtubule-bundling effect of taxol in vitro and report preliminary characterization of this bundling using electron microscopy, sedimentation, and electrophoretic analyses. Taxol-bundled microtubules from rat brain crude extracts were seen as massive bundles by electron microscopy. Bundled microtubules sedimented through sucrose five times faster than control microtubules. Electrophoretic analysis of control and taxol-bundled microtubules pelleted through sucrose revealed no striking differences between the two samples except for a protein doublet of approximately 100,000 daltons. Taxol-induced microtubule bundling was not produced by using pure tubulin or recycled microtubule protein; this suggested that taxol-induced microtubule bundling was mediated by a factor present in rat brain crude extracts. Taxol cross-linked rat brain crude extract microtubules were entirely labile to ATP in the millimolar range. This ATP-dependent relaxation was also demonstrated in a more purified system, using taxol-bundled microtubules pelleted through sucrose and gently resuspended. Although the bundling factor did not recycle with microtubule protein, it was apparently retained on isolated taxol-stabilized microtubules. The bundling factor was salt extracted from taxol-stabilized microtubules and its retained activity was demonstrated in an add-back experiment with assembled phosphocellulose-purified tubulin.

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.

Concentrations of trichloroethylene and its metabolites in blood and urine after acute poisoning by ingestion

1996 ◽  
Vol 15 (3) ◽  
pp. 254-258 ◽  
Author(s):  
M. Yoshida ◽  
S. Fukabori ◽  
K. Hara ◽  
H. Yuasa ◽  
K. Nakaaki ◽  
...  
Keyword(s):  
Half Life ◽  
Trichloroacetic Acid ◽  
Inhalation Exposure ◽  
Acute Poisoning ◽  
Deep Coma ◽  
Slow Phase ◽  
Half Time ◽  
Chemical Burns ◽  
Fast Phase ◽  
Kinetics Of

A 58-year-old man fell into a trichloroethylene reservoir bath head first, during a maintenance degreasing bath and accidentally ingested the solvent. Although he showed deep coma, chemical burns and pneumonia on admission, these symptoms gradually subsided. The concentrations of trichloroethylene (TRI) and its metabolites, trichloroethanol (TCE) and trichloroacetic acid (TCA) in blood and urine were measured during hospitalization. Eight hours after the accident, the concentrations ofTRI and its metabolites in serum were 31.4 μ g/ml TRI, 16.5 μg/ ml TCE and 79.5 μg/ml TCA. The serum TRI concentration decreased to 4.3 μg/ml on the following day. Elimination of TCE and TCA from serum occurred biphasically, the estimated half-lives of each metabolites being about 52.6 and 50.4 h in an initial fast phase and 268.3 and 277.2 h in a subsequent slow phase, respectively. Urinary TRI excretion persisted for the first 2 days. The urinary TCE and TCA excretions were longer than that of TRI with a biphasic decrease and the total amount of TCE excreted during the first 2 days was about two times that of TCA. The half-life of urinary TCE excretion (t½ 25.7 h) was shorter than that of TCA (t½ 52.1 h) in the fast phase but did no difference during the slow phase, with each half-time being about 166.3 h. The kinetics of TRI metabolites in blood and urine in this case were in slight agreement with the results following inhalation exposure previously reported in the literature.

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