scholarly journals Functional implications of cold-stable microtubules in kinetochore fibers of insect spermatocytes during anaphase.

1980 ◽  
Vol 85 (3) ◽  
pp. 853-865 ◽  
Author(s):  
E D Salmon ◽  
D A Begg
Keyword(s):  
Dynamic Equilibrium ◽  
Chromosome Movement ◽  
Temperature Dependent ◽  
Abrupt Changes ◽  
Functional Implications ◽  
Early Anaphase ◽  
Physiological Pathways ◽  
Cold Stability ◽  
Rate Limiting ◽  
Steady State Balance

In normal anaphase of crane fly spermatocytes, the autosomes traverse most of the distance to the poles at a constant, temperature-dependent velocity. Concurrently, the birefringent kinetochore fibers shorten while retaining a constant birefringent retardation (BR) and width over most of the fiber length as the autosomes approach the centrosome region. To test the dynamic equilibrium model of chromosome poleward movement, we abruptly cooled or heated primary spermatocytes of the crane fly Nephrotoma ferruginea (and the grasshopper Trimerotropis maritima) during early anaphase. According to this model, abrupt cooling should induce transient depolymerization of the kinetochore fiber microtubules, thus producing a transient acceleration in the poleward movement of the autosomal chromosomes, provided the poles remain separated. Abrupt changes in temperature from 22 degrees C to as low as 4 degrees C or as high as 31 degrees C in fact produced immediate changes in chromosome velocity to new constant velocities. No transient changes in velocity were observed. At 4 degrees C (10 degrees C for grasshopper cells), chromosome movement ceased. Although no nonkinetochore fiber BR remained at these low temperatures, kinetochore fiber BR had changed very little. The cold stability of the kinetochore fiber microtubules, the constant velocity character of chromosome movement, and the observed Arrhenius relationship between temperature and chromosome velocity indicate that a rate-limiting catalyzed process is involved in the normal anaphase depolymerization of the spindle fiber microtubules. On the basis of our birefringence observations, the kinetochore fiber microtubules appear to exist in a steady-state balance between comparatively irreversible, and probably different, physiological pathways of polymerization and depolymerization.

scholarly journals The Dynamics of Subunit Rotation in a Eukaryotic Ribosome

Biophysica ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 204-221
Author(s):  
Frederico Campos Freitas ◽  
Gabriele Fuchs ◽  
Ronaldo Junio de Oliveira ◽  
Paul Charles Whitford
Keyword(s):  
Free Energy ◽  
Large Scale ◽  
Small Subunit ◽  
Free Energy Barrier ◽  
Temperature Dependent ◽  
Molecular Flexibility ◽  
Subunit Rotation ◽  
Biological Kinetics ◽  
Rate Limiting ◽  
Reversible Rotation

Protein synthesis by the ribosome is coordinated by an intricate series of large-scale conformational rearrangements. Structural studies can provide information about long-lived states, however biological kinetics are controlled by the intervening free-energy barriers. While there has been progress describing the energy landscapes of bacterial ribosomes, very little is known about the energetics of large-scale rearrangements in eukaryotic systems. To address this topic, we constructed an all-atom model with simplified energetics and performed simulations of subunit rotation in the yeast ribosome. In these simulations, the small subunit (SSU; ∼1 MDa) undergoes spontaneous and reversible rotation events (∼8∘). By enabling the simulation of this rearrangement under equilibrium conditions, these calculations provide initial insights into the molecular factors that control dynamics in eukaryotic ribosomes. Through this, we are able to identify specific inter-subunit interactions that have a pronounced influence on the rate-limiting free-energy barrier. We also show that, as a result of changes in molecular flexibility, the thermodynamic balance between the rotated and unrotated states is temperature-dependent. This effect may be interpreted in terms of differential molecular flexibility within the rotated and unrotated states. Together, these calculations provide a foundation, upon which the field may begin to dissect the energetics of these complex molecular machines.

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

2021 ◽  
Author(s):  
Biswash Thakuri ◽  
Bruce O'Rourke ◽  
Amanda Graves ◽  
Matthew Liptak
Keyword(s):  
Dynamic Equilibrium ◽  
Ionization Mass ◽  
Esi Ms ◽  
Enzymatic Mechanism ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Planar Substrate ◽  
Out Of Plane ◽  
Substrate Conformation ◽  
Rate Limiting

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a refined enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

Ischemia in isolated interventricular septa: mechanical events

1976 ◽  
Vol 231 (4) ◽  
pp. 1225-1232 ◽  
Author(s):  
KI Shine ◽  
AM Douglas ◽  
N Ricchiuti
Keyword(s):  
Nitrogen Atmosphere ◽  
Ischemia And Reperfusion ◽  
Mechanical Function ◽  
Maximal Rate ◽  
Temperature Dependent ◽  
Tension Development ◽  
Time To Peak ◽  
Rate Of Decline ◽  
Rate Limiting ◽  
Mechanical Recovery

Isolated blood-perfused rabbit interventricular septa were adapted for studies of global ischemia by enclosure in a constant-humidity nitrogen atmosphere. During ischemia, developed tension (DT) and maximal rate of relaxation (-dP/dt) declined monoexponentially, lambda = 0.39 min-1 at 37 degrees C and 72 beats/min with a Q10 of 1.4 for DT and a Q10 of 1.9 for -dP/dt. After a 60- to 90-s delay the maximal rate of tension development (+dP/dt) declined at the same rate as DT. Time-to-peak tension (TPT) shortened immediately with ischemia but action potential duration shortened after 60-90 s. Calcium at a concentration of 5 mM slowed the rate of decline of +dP/dt to lambda = 0.26 min-1. Upon reperfusion after 10 min of ischemia the rates of recovery of DT, +dP/dt, and -dP/dt were similar, lambda = 0.21-0.23 min-1, and were not temperature dependent. The magnitude of recovery was 10-17% less at 37 degrees C than 28 degrees C. Potassium at a concentration of 10 mM did not alter the rate of decline of mechanical function, but significantly (P less than 0.01) increased the magnitude of mechanical recovery. The results suggest depletion and/or repletion of single compartments as the rate-limiting steps in ischemia and reperfusion.

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

2020 ◽  
Author(s):  
Biswash Thakuri ◽  
Bruce O'Rourke ◽  
Amanda Graves ◽  
Matthew Liptak
Keyword(s):  
Dynamic Equilibrium ◽  
Ionization Mass ◽  
Esi Ms ◽  
Enzymatic Mechanism ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Planar Substrate ◽  
Out Of Plane ◽  
Substrate Conformation ◽  
Rate Limiting

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

Behaviour of kinetochore fibres in Haemanthus katherinae during anaphase movements of chromosomes

1977 ◽  
Vol 27 (1) ◽  
pp. 47-56
Author(s):  
R. Hard ◽  
R.D. Allen
Keyword(s):  
Light Source ◽  
Laser Light ◽  
Living Cells ◽  
Differential Interference Contrast ◽  
Chromosome Movement ◽  
Lateral Interactions ◽  
Parallel Orientation ◽  
Early Anaphase ◽  
Laser Light Source ◽  
Dic Microscopy

A laser light source along with a new method of preparing endosperm cells of Haemanthus katherinae for differential interference contrast (DIC) microscopy has led to increased visibility of kinetochore fibres. Little information is available concerning the behaviour of these fibres during anaphase in living cells. In metaphase, kinetochore fibres are seen as distinct bundles of microtubules, here referred to as ‘filaments’, extending from the kinetochore to the ‘diffuse’ pole. They possess an apparent globular substructure which corresponds to the moving ‘particles or states’ described previously from cine films. In early anaphase, the filaments of each kinetochore fibre lose their parallel orientation characteristic of metaphase and splay out so that the more peripheral filaments intermingle with those of other kinetochore fibres. This process begins at the poles and proceeds as a wave toward the kinetochores as chromosomal movement progresses. This behaviour has been examined in relation to a number of proposed models for the mechanism of chromosome movement and has been found to place some constraints on some models but to be consistent with any model that hypothesizes that chromosomes move as a consequence of cumulative cohesive lateral interactions of microtubules.

Temperature dependent pairing gap in nuclei

2018 ◽  
Vol 27 (01) ◽  
pp. 1850003 ◽  
Author(s):  
A. Rahmatinejad ◽  
R. Razavi ◽  
T. Kakavand
Keyword(s):  
Phase Transition ◽  
Critical Temperature ◽  
Theoretical Models ◽  
Thermal Fluctuations ◽  
Gap Function ◽  
Temperature Dependent ◽  
Model Calculations ◽  
Soft Phase ◽  
Abrupt Changes ◽  
Size Of Nuclei

Based on theoretical models and experimental evidences of a soft phase transition, with significant, but not abrupt changes in pairing gap of nuclei, a modified pairing gap function was suggested and examined for some of nuclei in our previous publications. Now, we propose a new method to extract the parameters, which exist in the modified pairing gap function. This method is based on phenomenological models, which consider the small size of nuclei and the effect of thermal fluctuations, at the vicinity of the critical temperature. The obtained pairing gap function by this method is easily applicable in the microscopic model calculations within an entire temperature range, from zero to temperatures above the critical point.

High Rate Superplastic Deformation Mechanisms in IN90211 Mechanically Alloyed Aluminum

1990 ◽  
Vol 196 ◽  
Author(s):  
Thomas R. Bieler ◽  
Amiya K. Mukherjee
Keyword(s):  
Strain Rate ◽  
Superplastic Deformation ◽  
Rate Sensitivity ◽  
High Rate ◽  
Temperature Dependent ◽  
Mechanically Alloyed ◽  
Rate Limiting Step ◽  
Orowan Stress ◽  
Apparent Strain ◽  
Rate Limiting

ABSTRACTIN90211 has exhibited superplastic elongations above 500% at high homologous temperatures (0.76–0.82 Tm). A high strain rate and flow stress for optimum elongation was measured (1–5/sec, 20–60 MPa, 425–485 °C). The apparent strain rate sensitivity of m≈0.25 differs from the usual m≈0.5 observations of superplastic deformation. An analysis of the data at several strains indicates a highly temperature dependent threshold stress is present, with either a n=2 or n=3 assumption for the stress exponent. The magnitude of the threshold stresses in IN90211 are smaller than usually observed in a dispersion strengthened matrix (1–20% instead of ≈50% of the Orowan stress). Experimental evidence from creep experiments supports the n=3 deformation mechanism as the rate limiting step of deformation.

scholarly journals A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

2020 ◽  
Author(s):  
Biswash Thakuri ◽  
Bruce O'Rourke ◽  
Amanda Graves ◽  
Matthew Liptak
Keyword(s):  
Dynamic Equilibrium ◽  
Ionization Mass ◽  
Esi Ms ◽  
Enzymatic Mechanism ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Planar Substrate ◽  
Out Of Plane ◽  
Substrate Conformation ◽  
Rate Limiting

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a revised enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a revised enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

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