scholarly journals MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS

1974 ◽  
Vol 61 (2) ◽  
pp. 514-536 ◽  
Author(s):  
David L. Brown ◽  
G. Benjamin Bouck
Keyword(s):  
Protein Synthesis ◽  
Cell Shape ◽  
Microtubule Assembly ◽  
Short Term ◽  
Protein Pool ◽  
Microtubule Protein ◽  
Kinetics Of ◽  
Mitotic Inhibitor ◽  
Kinetics Of Inhibition

The role of microtubules and microtubule nucleating sites in the unicell, Ochromonas has been examined through the use of two mitotic inhibitors, isopropyl N-phenylcarbamate (IPC) and isopropyl N-3-chlorophenyl carbamate (CIPC). Although IPC and CIPC have little or no effect on intact microtubules, the assembly of three separate sets of microtubules in Ochromonas has been found to be differentially affected by IPC and CIPC. The assembly of flagellar microtubules after mechanical deflagellation is partially inhibited; the reassembly of rhizoplast microtubules after pressure depolymerization is totally inhibited (however, macrotubules may form at the sites of microtubule initiation or elsewhere); and, the reassembly of the beak set of microtubules after pressure depolymerization may be unaffected although similar concentrations of IPC and CICP completely inhibit microtubule regeneration on the rhizoplast. These effects on microtubule assembly, either inhibitory or macrotubule inducing, are fully reversible. The kinetics of inhibition and reversal are found to be generally similar for both flagellar and cell shape regeneration. Incorporation data suggest that neither IPC nor CIPC has significant effects on protein synthesis in short term experiments. Conversely, inhibiting protein synthesis with cycloheximide has little effect on microtubule regeneration when IPC or CIPC is removed. Although the exact target for IPC and CIPC action remains uncertain, the available evidence suggests that the microtubule protein pool or the microtubule nucleating sites are specifically and reversibly affected. Comparative experiments using the mitotic inhibitor colchicine indicate some similarities and differences in its mode of action with respect to that of IPC and CIPC on assembly and disassembly of microtubules in these cells.

scholarly journals Purification and kinetic mechanism of the major glutathione S-transferase from bovine brain

1989 ◽  
Vol 257 (2) ◽  
pp. 541-548 ◽  
Author(s):  
P R Young ◽  
A V Briedis
Keyword(s):  
Decanoic Acid ◽  
Kinetic Mechanism ◽  
Order Rate Constant ◽  
Bovine Brain ◽  
Glutathione S Transferase ◽  
Rate Acceleration ◽  
Hydrophobic Binding ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The major glutathione S-transferase isoenzyme from bovine brain was isolated and purified approx. 500-fold. The enzyme has a pI of 7.39 +/- 0.02 and consists of two non-identical subunits having apparent Mr values of 22,000 and 24,000. The enzyme is uniformly distributed in brain, and kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate suggest a random rapid-equilibrium mechanism. The kinetics of inhibition by product, by GSH analogues and by NADH are consistent with the suggested mechanism and require inhibitor binding to several different enzyme forms. Long-chain fatty acids are excellent inhibitors of the enzyme, and values of 1nKi for hexanoic acid, octanoic acid, decanoic acid and lauric acid form a linear series when plotted as a function of alkyl chain length. A free-energy change of -1900 J/mol (-455 cal/mol) per CH2 unit is calculated for the contribution of hydrophobic binding energy to the inhibition constants. The turnover number of the purified enzyme dimer is approx. 3400/min. When compared with the second-order rate constant for the reaction between CDNB and GSH, the enzyme is providing a rate acceleration of about 1000-fold. The role of entropic contributions to this small rate acceleration is discussed.

scholarly journals Kinetics of GTP hydrolysis during the assembly of chick brain MAP2-tubulin microtubule protein

1991 ◽  
Vol 277 (1) ◽  
pp. 239-243 ◽  
Author(s):  
R G Burns
Keyword(s):  
Initial Rate ◽  
Molar Concentration ◽  
Microtubule Assembly ◽  
Chick Brain ◽  
Gtp Hydrolysis ◽  
Microtubule Protein ◽  
Rate Of Hydrolysis ◽  
Kinetics Of

The kinetics of GTP hydrolysis during microtubule assembly have been examined using chick brain MAP2-tubulin microtubule protein in a NaCl-supplemented buffer. The elongating microtubules terminate in a ‘GTP cap’, since the kinetics of GTP hydrolysis are slower than those of subunit addition. GTP hydrolysis is (a) stoichiometric, (b) occurs as a vectorial wave as the initial rate of hydrolysis is proportional to the molar concentration of microtubule ends and not to the initial rate of subunit addition, and (c) either does not occur, or occurs only at a much lower rate, in the terminal subunits.

scholarly journals Microtubule assembly kinetics. Changes with solution conditions

1987 ◽  
Vol 247 (3) ◽  
pp. 505-511 ◽  
Author(s):  
J S Barton ◽  
D L Vandivort ◽  
D H Heacock ◽  
J A Coffman ◽  
K A Trygg
Keyword(s):  
Activation Energy ◽  
Ionic Strength ◽  
Low Temperature ◽  
Number Concentration ◽  
High Ionic Strength ◽  
Microtubule Assembly ◽  
Microtubule Protein ◽  
Assembly Kinetics ◽  
Low Ionic Strength ◽  
Kinetics Of

The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.

Rapid synthesis and secretion of intestinal apolipoprotein A-IV after gastric fat loading in rats

1997 ◽  
Vol 272 (4) ◽  
pp. R1170-R1177 ◽  
Author(s):  
M. D. Rodriguez ◽  
T. J. Kalogeris ◽  
X. L. Wang ◽  
R. Wolf ◽  
P. Tso
Keyword(s):  
Small Intestine ◽  
Food Intake ◽  
Intragastric Administration ◽  
Short Term ◽  
Apolipoprotein A ◽  
Inhibit Food Intake ◽  
Rapid Stimulation ◽  
Kinetics Of ◽  
Distal Jejunum

To further investigate the possible role of apolipoprotein A-IV (apo A-IV) in the short-term control of food intake, we examined the kinetics of intestinal apo A-IV synthesis and release into lymph and plasma after intragastric delivery of physiological amounts of lipid. Within 30 min of intragastric administration of 0.1 g of triglyceride, plasma and lymph levels of apo A-IV were similar to those produced by exogenous apo A-IV that inhibit food intake. Within 15 min, 5% of gastrically delivered radioactive lipid reached the distal small bowel and cecum; by 30 min radioactivity was evenly distributed throughout the small intestine, with 10-15% of the load in the distal gut. By 30 min, synthesis of apo A-IV was significantly stimulated in proximal and distal jejunum and distal ileum and remained elevated up to 4 h after the delivery of lipid. Our results indicate that the delivery of physiological amounts of lipid into the stomach produces a significant and rapid stimulation of apo A-IV secretion into lymph and plasma, together with a rapid delivery of lipid and increases in mucosal synthesis of apo A-IV along the entire length of the small intestine. The results support a possible role for apo A-IV in the short-term control of food intake and suggest a role for the entire gut in the integrative response of apo A-IV to a fat meal.

scholarly journals Microtubules and nucleoside diphosphate kinase. Comparison of kinetics of GTP- and CTP-induced assembly

1985 ◽  
Vol 232 (3) ◽  
pp. 657-662 ◽  
Author(s):  
K Islam ◽  
R G Burns
Keyword(s):  
Substrate Specificity ◽  
Kinase Activity ◽  
Nucleoside Diphosphate Kinase ◽  
Elongation Rate ◽  
Microtubule Assembly ◽  
Microtubule Protein ◽  
Assembly Kinetics ◽  
Kinetics Of

The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.

scholarly journals The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm

1976 ◽  
Vol 154 (2) ◽  
pp. 501-506 ◽  
Author(s):  
L Bowden ◽  
J. M Lord
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Castor Bean ◽  
Developmental Stages ◽  
Endoplasmic Reticulum Membrane ◽  
Endosperm Tissue ◽  
Cellular Origin ◽  
Lag Period ◽  
Kinetics Of

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.

Abstract 322: HDL Dynamics in Circulation: Complexity of Protein Distribution and Metabolism Across HDL Size

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Sasha Singh ◽  
Allison Andraski ◽  
Brett Pieper ◽  
Wilson Goh ◽  
Frank M Sacks ◽  
...  
Keyword(s):  
Protein Distribution ◽  
Size Fractions ◽  
Immunoaffinity Purification ◽  
Protein Pool ◽  
Parallel Reaction Monitoring ◽  
Single Protein ◽  
Hdl Metabolism ◽  
Kinetics Of ◽  
Distinct Distribution

Introduction: The composition of specific apolipoproteins may determine HDL functions. We present novel mass spectrometry (MS)-based methods that capture the absolute quantities and kinetics of 7 apolipoproteins in 5 HDL size fractions. Methods and Results: Three participants were recruited, infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-containing HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0 by ND-PAGE, and in-gel trypsinized for MS. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE and apoM. Each protein pool size had a distinct distribution across the HDL sizes. ApoE and apoM were enriched in larger HDL, whereas apoC-III and apoA-IV were enriched in smaller HDL sizes. We evaluated the tracer enrichment curves of these 7 proteins in the 5 fractions using high resolution parallel reaction monitoring performed on a quadrupole Orbitrap (Thermo). The enrichment curves for each protein varied from each other by slope and time of peak enrichment (Fig. 1a). In contrast, the enrichment curves across HDL sizes for a single protein showed smaller, but likely meaningful, differences in either slope or time of peak enrichment. Irrespective of the HDL size on which it resides, apoE had the fastest FCR, followed by apoA-IV, and apoC-III. ApoA-I/A-II, apoM, and apoD had slower but similar FCRs (Fig. 1b). Conclusions: This study showed distinct distribution and kinetic behaviors of 7 HDL proteins across 5 HDL size fractions that were conserved in the three participants. These findings may help elucidate the functional role of these proteins and the HDL particles that contain them.

Inhibition of microtubule polymerization by the tubulin–colchicines complex; inhibition of spontaneous assembly

1981 ◽  
Vol 59 (5) ◽  
pp. 361-370 ◽  
Author(s):  
R. A. B. Keates ◽  
G. B. Mason
Keyword(s):  
Total Concentration ◽  
Temperature Shift ◽  
Degree Of Polymerization ◽  
Microtubule Polymerization ◽  
Minor Factor ◽  
Microtubule Protein ◽  
A Minor ◽  
Slow Dissociation ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The inhibition of microtubule polymerization by colchicine requires the formation of tubulin–colchicine complexes, and inhibition of polymerization is proportional to the concentration of tubulin–colchicine complexes rather than to the total concentration of colchicine. Because the formation of such complexes is slow relative to polymerization, the kinetics of complex formation obscure the kinetics of inhibition of polymerization. We have taken defined quantities of preformed tubulin–colchicine complexes, relying on their slow dissociation, and added these to microtubule protein, which was allowed to polymerize by temperature shift to 37 °C. The degree of polymerization was then determined by measurement of turbidity at 400 nm. An appropriate kinetic analysis allowed us to distinguish effects of inhibitor on initiation and elongation phases of polymerization, without resorting to the use of initiation inhibitors. The results are consistent with a reversible association of tubulin–colchicine complex with microtubule ends blocking further elongation (Ki = 0.16 μM). Steady-state measurements suggest that copolymerization of tubulin–colchicine complex is a minor factor under the conditions used. By contrast, little inhibition of initiation was observed, possibly because tubulin–colchicine complex competes with the tubulin dimer, but not with the larger oligomers required for the initiation process.

scholarly journals Uncovering long-term existence of a silent short-term memory trace

2021 ◽  
Author(s):  
Maha E. Wally ◽  
Masanori Nomoto ◽  
Kareem Abdou ◽  
Kaoru Inokuchi
Keyword(s):  
Protein Synthesis ◽  
Memory Trace ◽  
Short Term Memory ◽  
Short Term ◽  
Term Memory ◽  
Aspartate Receptor ◽  
Hippocampal Ca1 ◽  
Term Functions

Active recall of short-term memory (STM) is known to last for a few hours, but whether STM has long-term functions is unknown. Here we show that, STM can be optogenetically retrieved at a time point during which natural recall is not possible, uncovering the long-term existence of a silent STM engram. Moreover, re-training within 3 days led to natural long-term recall, indicating facilitated consolidation. Calcium imaging revealed hippocampal CA1 reactivations of the STM trace during post-learning sleep. Inhibiting offline CA1 activity, N-methyl-D-aspartate receptor activity, or protein synthesis after first exposure to the STM-forming event impaired the future re-exposure-facilitated consolidation, which highlights a role of protein synthesis and sleep in storing a silent STM trace. These results provide evidence that STM is not completely lost within hours and demonstrates a possible two-step STM consolidation, first storage as a silent engram, then transformation into an active state by recurrence within 3 days.

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