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1.
In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner. 相似文献
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Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomerulosa cells measured by digital imaging techniques 总被引:7,自引:0,他引:7
The response of cytosolic calcium [Ca2+]i to angiotensin II (AII) and potassium (K+) in individual rat glomerulosa cells was determined using the calcium-sensitive fluorescent dye, fura-2 and digital imaging. Control (4 mM K+) cytosolic calcium levels were generally in the 80-120 nM range and increased monotonically as [K+] was increased from 4 to 12 mM. There was no delay in the onset of the response. In most cells the [Ca2+]i decreased from its peak after 3-4 min, even in the presence of superfusate containing elevated K+. The time course of the change in [Ca2+]i in response to AII stimulation, on the other hand, was more variable. It was most often characterized by an early decrease followed by a large delayed increase. The response also was observed to decline during sustained AII stimulation. The majority of the cells showed some response to one or the other secretagogue with a sizeable minority (25%) having an increase in [Ca2+]i in excess of 200%. While the majority showed a response, the cell to cell variation was substantial. Finally, the pattern of cytosolic calcium increase sometimes showed a marked dependence on the secretagogue used, with different regions of the same cell being more strongly affected by one agent or the other. A few cells (10%) responded to AII only at one pole, establishing a large concentration gradient of calcium across the cell. Because of differences in time course, pattern, and degree of responsiveness, it is likely that the mechanisms underlying the Ca2+ elevation with K+ and AII are different. 相似文献
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Victoria V. Hargreaves Scarlet S. Shell Dan J. Mazur Martin T. Hess Richard D. Kolodner 《The Journal of biological chemistry》2010,285(12):9301-9310
Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps. 相似文献
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Cynthia L. Deitrick Richard E. Katholi David J. Huddleston Kathy Hardiek Lucienne Burrus 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,751(2)
Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation. 相似文献
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Richard D. Gregory 《Ecography》1998,21(1):92-96
10.
Concentration factor and biological half-life of 54Mn were determined in three species representing an ecologically and economically important food chain. Green algae (Chlorella spp.), Daphnia magna and yellow perch (Perca flavescens) were exposed to 54Mn in water and assayed for 54Mn uptake. Steady state concentration factors computed from the laboratory data for algae, Daphnia and perch were 4230, 17 000 and 11, respectively. Respective biological half-lives were 1.6, 1.2 and 8.3 days. 相似文献