scholarly journals NAD(P)H: FMN-oxidoreductase functioning under macromolecular crowding: in vitro modeling

2019 ◽  
Vol 486 (4) ◽  
pp. 500-503
Author(s):  
A. E. Govorun ◽  
E. N. Esimbekova ◽  
V. A. Kratasyuk
Keyword(s):  
Rate Constants ◽  
Vibrio Fischeri ◽  
Macromolecular Crowding ◽  
Dissociation Rate ◽  
Common Belief ◽  
Native Conformation ◽  
The Common ◽  
Dissociation Rate Constants

The functioning of Vibrio fischeri NAD(P)H: FMN-oxidoreductase (Red) under conditions of macromolecular crowding (MMC) modeled in vitro by adding biopolymers (starch and gelatin) was studied. The dissociation rate constants and the activation energies of dissociation of Red to the subunits were calculated; the process of denaturation of Red was analyzed. It was shown that the functioning of Red both under conditions of MMC and diluted solutions is the same. The result refutes the common belief that due to MMC the stabilization of enzymes’ native conformation occurs in vivo when compared to in vitro.

scholarly journals Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly.

1980 ◽  
Vol 84 (1) ◽  
pp. 141-150 ◽  
Author(s):  
L G Bergen ◽  
G G Borisy
Keyword(s):  
Electron Microscope ◽  
Rate Constants ◽  
Dissociation Constants ◽  
Dissociation Rate ◽  
Association Constants ◽  
Directional Growth ◽  
Microtubule Protein ◽  
Electron Microscope Analysis ◽  
Dissociation Rate Constants

Microtubules are polar structures, and this polarity is reflected in their biased directional growth. Following a convention established previously (G. G. Borisy, 1978, J. Mol. Biol. 124:565--570), we define the plus (+) and minus (-) ends of a microtubule as those equivalent in structural orientation to the distal and proximal ends, respectively, of the A subfiber of flagellar outer doublets. Rates of elongation were obtained for both ends using flagellar axonemes as seeds and porcine brain microtubule protein as subunits. Since the two ends of a flagellar seed are distinguishable morphologically, elongation of each end may be analyzed separately. By plotting rates of elongation at various concentrations of subunit protein, we have determined the association and dissociation rate constants for the plus and minus ends. Under our conditions at 30 degrees C, the association constants were 7.2 X 10(6) M-1 s-1 and 2.25 X 10(6) M-1 s-1 for the plus and minus ends, respectively, and the dissociation constants were 17 s-1 and 7 s-1. From these values and Wegner's equations (1976, J. Mol. Biol. 108:139--150), we identified the plus end of the microtubule as its head and calculated "s," the head-to-tail polymerization parameter. Surprisingly small values (s = 0.07 +/- 0.02) were found. The validity of models of mitosis based upon head-to-tail polymerization (Margolis et al., 1978, Nature (Lond.) 272:450--452) are discussed in light of a small value for s.

In vivo apparent peptide-receptor dissociation rate constants for arginine vasopressin analogs estimated from inhibition of rat pressor responses

1987 ◽  
Vol 65 (10) ◽  
pp. 2099-2103
Author(s):  
Diana Gazis
Keyword(s):  
Blood Pressure ◽  
Arginine Vasopressin ◽  
Rate Constants ◽  
Dissociation Rate ◽  
Arginine Vasotocin ◽  
Moderate Dose ◽  
Peptide Receptor ◽  
Pressor Responses ◽  
Dissociation Rate Constants

Apparent pressor receptor dissociation rate constants for arginine vasopressin, arginine vasotocin, oxytocin, oxypressin, and [1-deamino, 9-D-alanineamide]arginine vasopressin were estimated by the following method. Two minutes after injection of a moderate dose of agonist into urethane-anesthetized rats prepared for recording mean blood pressure, a large dose of inhibitor was injected. Under these conditions, in the first few moments after inhibitor injection, there should be no rebinding of the agonist after it dissociates, because vacant receptors should be immediately occupied by inhibitor. The rate of the blood pressure drop at the initiation of inhibition was calculated and used as an estimate of the dissociation rate of the agaonist. Apparent dissociation rate constants thus estimated were 1.1, 1.1, 6.9, 5.8, and 13.9 min−1 for arginine vasopressin, arginine vasotocin, oxytocin, oxypressin, and [1-deamino, 9-D-alanineamide]arginine vasopressin, respectively. These rate constants were inversely related to the pressor potencies (435, 250, 5, 3, and 0.7 U/mg, respectively) of these five compounds. Such a relationship is to be expected if decreased potency is in part due to a faster "off" rate from pressor receptors.

Mechanism of the regulation of type IB phosphoinositide 3OH-kinase byG-protein βγ subunits

2002 ◽  
Vol 362 (3) ◽  
pp. 725-731 ◽  
Author(s):  
Sonja KRUGMANN ◽  
Matthew A. COOPER ◽  
Dudley H. WILLIAMS ◽  
Phillip T. HAWKINS ◽  
Len R. STEPHENS
Keyword(s):  
Plasma Membrane ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Lipid Vesicles ◽  
Dissociation Rate ◽  
Lipid Monolayers ◽  
Dissociation Rate Constants

Type IB phosphoinositide 3OH-kinase (PI3K) is activated by G-protein βγ subunits (Gβγs). The enzyme is soluble and largely cytosolic in vivo. Its substrate, PtdIns(4,5)P2, and the Gβγs are localized at the plasma membrane. We have addressed the mechanism by which Gβγs regulate the PI3K using an in vitro approach. We used sedimentation assays and surface plasmon resonance to determine association of type IB PI3K with lipid monolayers and vesicles of varying compositions, some of which had Gβγs incorporated. Association and dissociation rate constants were determined. Our results indicated that in an assay situation in vitro the majority of PI3K will be associated with lipid vesicles, irrespective of the presence or absence of Gβγs. In line with this, a constitutively active membrane-targeted PI3K construct could still be activated substantially by Gβγs in vitro. We conclude that Gβγs activate type IB PI3K by a mechanism other than translocation to the plasma membrane.

Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish

2001 ◽  
Vol 280 (4) ◽  
pp. R1123-R1133 ◽  
Author(s):  
David J. Marcinek ◽  
Joseph Bonaventura ◽  
Jonathan B. Wittenberg ◽  
Barbara A. Block
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rate Constants ◽  
Oxygen Affinity ◽  
Dissociation Rate ◽  
Low Oxygen ◽  
Dissociation Kinetics ◽  
Oxygen Dissociation ◽  
Dissociation Rate Constants

Myoglobin (Mb) buffers intracellular O2 and facilitates diffusion of O2 through the cell. These functions of Mb will be most effective when intracellular Po 2 is near the partial pressure of oxygen at which Mb is half saturated (P50) of the molecule. We test the hypothesis that Mb oxygen affinity has evolved such that it is conserved when adjusted for body temperature among closely related animals. We measure oxygen P50s tonometrically and oxygen dissociation rate constants with stopped flow and generate amino acid sequence from cDNA of Mbs from fish with different body temperatures. P50s for the endothermic bluefin tuna, skipjack tuna, and blue marlin at 20°C were 0.62 ± 0.02, 0.59 ± 0.01, 0.58 ± 0.04 mmHg, respectively, and were significantly lower than those for ectothermic bonito (1.03 ± 0.07 mmHg) and mackerel (1.39 ± 0.03 mmHg). Because the oxygen affinity of Mb decreases with increasing temperature, the above differences in oxygen affinity between endothermic and ectothermic fish are reduced when adjusted for the in vivo muscle temperature of the animal. Oxygen dissociation rate constants at 20°C for the endothermic species ranged from 34.1 to 49.3 s−1, whereas those for mackerel and bonito were 102 and 62 s−1, respectively. Correlated with the low oxygen affinity and fast dissociation kinetics of mackerel Mb is a substitution of alanine for proline that would likely result in a more flexible mackerel protein.

The Application of the Cold Atmospheric Plasma-Activated Solutions in Cancer Treatment

2018 ◽  
Vol 18 (6) ◽  
pp. 769-775 ◽  
Author(s):  
Dayun Yan ◽  
Jonathan H. Sherman ◽  
Michael Keidar
Keyword(s):  
Cancer Treatment ◽  
Atmospheric Plasma ◽  
Current Status ◽  
Cold Atmospheric Plasma ◽  
Anti Cancer ◽  
Long Time ◽  
Key Topics ◽  
The Common

Background: Over the past five years, the cold atmospheric plasma-activated solutions (PAS) have shown their promissing application in cancer treatment. Similar as the common direct cold plasma treatment, PAS shows a selective anti-cancer capacity in vitro and in vivo. However, different from the direct cold atmospheric plasma (CAP) treatment, PAS can be stored for a long time and can be used without dependence on a CAP device. The research on PAS is gradually becoming a hot topic in plasma medicine. Objectives: In this review, we gave a concise but comprehensive summary on key topics about PAS including the development, current status, as well as the main conclusions about the anti-cancer mechanism achieved in past years. The approaches to make strong and stable PAS are also summarized.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

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