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1.
Long-lived tryptophan fluorescence in phosphoglycerate mutase 总被引:1,自引:0,他引:1
Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed. 相似文献
2.
Inhibition of proteinase K by phosphorylated sugars. 总被引:1,自引:0,他引:1
Proteolysis of lactate dehydrogenase, aldolase and the synthetic substrate N-succinylalanylalanylalanyl-p-nitroanilide by proteinase K is inhibited by glucose-6-phosphate and fructose-1,6-biphosphate. Analysis of the kinetic data obtained with the synthetic substrate indicates that the inhibition is a mixed-type and that more than one inhibitor molecule binds to proteinase K. Glucose and fructose are ineffective as inhibitors. In the presence of 0.2-4 mM fructose-1,6-biphosphate, aldolase becomes more susceptible to proteolysis, probably as a result of a conformational change induced by the substrate. 相似文献
3.
The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements. Equilibrium experiments at 25 degrees C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M. Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps. The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration. This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration. Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx. 3-times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution. 相似文献
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5.
Isolation of a kaurene synthetase inhibitor from castor bean seedlings and cell suspension cultures 总被引:2,自引:2,他引:0
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Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures and seedlings of castor bean. Both cell-free extracts contain an inhibitor of kaurene synthetase. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and has little or no effect on the further cyclization of copalyl pyrophosphate to ent-kaurene (activity B) in both castor bean and Fusarium moniliforme cell-free enzyme preparations. In castor bean cell suspension cultures, the inhibitor diffuses out of the cells to the growth medium. The inhibitor is stable to 100 C heat treatment for 10 minutes and exposure to pH values of 2.0 or 13.0, and it diffuses through a dialysis bag (104-dalton cutoff). Gel filtration chromatography of the inhibitor on a calibrated Bio-Gel P-10 column indicated a molecular weight of 7,500. Kinetic studies indicate that the inhibition of activity of A of kaurene synthetase is noncompetitive and reversible. 相似文献
6.
Processing of spatio-temporal information in the human visual system has been investigated thoroughly during the past decade, but is still far from being properly understood. Moreover, the theory of separation of information by means of sustained and transient channels already at the retinal level is not satisfactory, as experimental results indicate that these two types of channels span a continuum of temporal characteristics. It is however obvious, that the process of pattern recognition and velocity perception calls for their separation at some level of the hierarchy. In this communication, we extend our model of three-dimensional spatio-temporal frequency expansion in the visual system (Gafni and Zeevi, 1977) to show how velocity-information extraction channels, sensitive to direction and velocity exclusively, can be formed by simple summation of signals from well-defined sets of channels representing points in the frequency space. Correspondence of these channels to characteristics of the cortical neurons is discussed. 相似文献
7.
The apparent microviscosity of intact rat plasma very low density lipoprotein (VLDL) and post-lipolysis very low density lipoprotein was determined by fluorescence depolarization measurements and flurorescence decay measurements using 1, 6-diphenylhexatriene. Post-lipolysis very low density lipoprotein was prepared in vitro after incubation of the intact lipoprotein with either purified bovine milk lipoprotein lipase or lipoprotein lipase rich (post-heparin) plasma. During lipolysis, an average of 88% of the triglycerides were hydrolyzed, and the lipoprotein became depleted in phospholipids, cholesterol and apolipoprotein C. The apparent microviscosity of the lipoprotein increased by three-fold from 0.63 to 1.88 poise. It is concluded that the compositional changes occurring during lipolysis affect the physical properties of the lipoprotein, as measured here by the fluidity (microviscosity) of the particles. 相似文献
8.
BACKGROUND: Fractionated plasma metanephrine measurements are commonly used in biochemical testing in search of pheochromocytoma. METHODS: We aimed to critically appraise the diagnostic efficacy of fractionated plasma free metanephrine measurements in detecting pheochromocytoma. Nine electronic databases, meeting abstracts, and the Science Citation Index were searched and supplemented with previously unpublished data. Methodologic and reporting quality was independently assessed by two endocrinologists using a checklist developed by the Standards for Reporting of Diagnostic Studies Accuracy Group and data were independently abstracted. RESULTS: Limitations in methodologic quality were noted in all studies. In all subjects (including those with genetic predisposition): the sensitivities for detection of pheochromocytoma were 96%-100% (95% CI ranged from 82% to 100%), whereas the specificities were 85%-100% (95% CI ranged from 78% to 100%). Statistical heterogeneity was noted upon pooling positive likelihood ratios when those with predisposition to disease were included (p < 0.001). However, upon pooling the positive or negative likelihood ratios for patients with sporadic pheochromocytoma (n = 191) or those at risk for sporadic pheochromocytoma (n = 718), no statistical heterogeneity was noted (p = 0.4). For sporadic subjects, the pooled positive likelihood ratio was 5.77 (95% CI = 4.90, 6.81) and the pooled negative likelihood ratio was 0.02 (95% CI = 0.01, 0.07). CONCLUSION: Negative plasma fractionated free metanephrine measurements are effective in ruling out pheochromocytoma. However, a positive test result only moderately increases suspicion of disease, particularly when screening for sporadic pheochromocytoma. 相似文献
9.
Karin Rand Irina Kobrinsky-Aaronowitz Yael Levy Orit Shaul Roni Aloni Yedidya Gafni 《Plant signaling & behavior》2011,6(6):815-820
Macromolecules may transfer between the cytoplasm and the nucleus only through specific gates—the nuclear pore complexes (NPCs). Translocation of nucleic acids and large proteins requires the presence of a nuclear localization signal (NLS) within the transported molecule. This NLS is recognized by a class of soluble transport receptors termed karyopherins α and β. We previously characterized the expression pattern of the tomato karyopherin α1 (LeKAPα1) promoter in transformed tobacco plants. Expression of LeKAPα1 was mainly observed in growing tissues where cell division and extension is rapid. The expression pattern of LeKAPα1 resembled that of auxin-responsive genes. This led us to suggest that auxin participates in the regulation of LeKAPα1 expression. Here we characterized the correlation between auxin level and the activity of the LeKAPα1 promoter. To this end, transgenic tobacco plants carrying the GUS reporter gene under the control of the LeKAPα1 promoter were treated with various levels of exogenous auxin. We also studied transgenic plants in which we increased the endogenous levels of auxin. For this, we expressed in plants both the LeKAPα1 promoter-GUS reporter and the Agrobacterium tumefaciens iaaM gene, which increases the endogenous levels of auxin. The results indicate that the auxin indole-3-acetic acid (IAA) can induce LeKAPα1 expression. We also identified that the sites and levels of LeKAPα1 expression correlated with the endogenous pathways of polar auxin transport.Key words: auxin, karyopherin α1, nuclear pore complex, TYLCV, plant virus 相似文献
10.
In this overview to biomedical computing in the cloud, we discussed two primary ways to use the cloud (a single instance or cluster), provided a detailed example using NGS mapping, and highlighted the associated costs. While many users new to the cloud may assume that entry is as straightforward as uploading an application and selecting an instance type and storage options, we illustrated that there is substantial up-front effort required before an application can make full use of the cloud's vast resources. Our intention was to provide a set of best practices and to illustrate how those apply to a typical application pipeline for biomedical informatics, but also general enough for extrapolation to other types of computational problems. Our mapping example was intended to illustrate how to develop a scalable project and not to compare and contrast alignment algorithms for read mapping and genome assembly. Indeed, with a newer aligner such as Bowtie, it is possible to map the entire African genome using one m2.2xlarge instance in 48 hours for a total cost of approximately $48 in computation time. In our example, we were not concerned with data transfer rates, which are heavily influenced by the amount of available bandwidth, connection latency, and network availability. When transferring large amounts of data to the cloud, bandwidth limitations can be a major bottleneck, and in some cases it is more efficient to simply mail a storage device containing the data to AWS (http://aws.amazon.com/importexport/). More information about cloud computing, detailed cost analysis, and security can be found in references. 相似文献