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61.
62.
Human cystathionine beta-synthase is one of two key enzymes involved in intracellular metabolism of homocysteine. It catalyzes a beta-replacement reaction in which the thiolate of homocysteine replaces the hydroxyl group of serine to give the product, cystathionine. The enzyme is unusual in its dependence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensation reaction that is catalyzed; however the function of the heme in this protein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Soret peak of ferric cystathionine beta-synthase is at 428 nm and shifts to approximately 395 nm upon addition of the thiol chelator, mercuric chloride. This is indicative of 6-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride treatment of the enzyme results in conversion of the rhombic EPR signal to a g = 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine beta-synthase is 6-coordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consistent with the presence of 5 nitrogens and 1 sulfur ligand. Together, these data support assignment of the axial ligands as cysteinate and imidazole in ferric cystathionine beta-synthase. 相似文献
63.
Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes. 相似文献
64.
W H Cooke I V Ames JE A A Crossman J F Cox T A Kuusela K U Tahvanainen L B Moon J Drescher F J Baisch T Mano B D Levine C G Blomqvist D L Eckberg 《Journal of applied physiology》2000,89(3):1039-1045
We studied three Russian cosmonauts to better understand how long-term exposure to microgravity affects autonomic cardiovascular control. We recorded the electrocardiogram, finger photoplethysmographic pressure, and respiratory flow before, during, and after two 9-mo missions to the Russian space station Mir. Measurements were made during four modes of breathing: 1) uncontrolled spontaneous breathing; 2) stepwise breathing at six different frequencies; 3) fixed-frequency breathing; and 4) random-frequency breathing. R wave-to-R wave (R-R) interval standard deviations decreased in all and respiratory frequency R-R interval spectral power decreased in two cosmonauts in space. Two weeks after the cosmonauts returned to Earth, R-R interval spectral power was decreased, and systolic pressure spectral power was increased in all. The transfer function between systolic pressures and R-R intervals was reduced in-flight, was reduced further the day after landing, and had not returned to preflight levels by 14 days after landing. Our results suggest that long-duration spaceflight reduces vagal-cardiac nerve traffic and decreases vagal baroreflex gain and that these changes may persist as long as 2 wk after return to Earth. 相似文献
65.
MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method
for RNA structure prediction. The MWM method is capable of detecting
pseudoknots and other tertiary base-pairing interactions in a
computationally efficient manner (Cary and Stormo, Proceedings of the Third
International Conference on Intelligent Systems for Molecular Biology, pp.
75-80, 1995). Here we report on the results of our efforts to improve the
MWM method's predictive accuracy, and show how the method can be extended
to detect base interactions formerly inaccessible to automated RNA modeling
techniques. RESULTS: Improved performance in MWM structure prediction was
achieved in two ways. First, new ways of calculating base pair likelihoods
have been developed. These allow experimental data and combined statistical
and thermodynamic information to be used by the program. Second, accuracy
was improved by developing techniques for filtering out spurious base pairs
predicted by the MWM program. We also demonstrate here a means by which the
MWM folding method may be used to detect the presence of base triples in
RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html
CONTACT: tabaska@cshl.org
相似文献
66.
Single-copy nuclear DNAs (scnDNAs) of eight species of arvicoline and six
species of murine rodents were compared using DNA-DNA hybridization. The
branching pattern derived from the DNA comparisons is congruent with the
fossil evidence and supported by comparative biochemical, chromosomal, and
morphological studies. The recently improved fossil record for these
lineages provides seven approximate divergence dates, which were used to
calibrate the DNA-hybridization data. The average rate of scnDNA divergence
was estimated as 2.5%/Myr. This is approximately 10 times the rate in the
hominoid primates. These results agree with previous reports of accelerated
DNA evolution in muroid rodents and extend the DNA-DNA hybridization data
set of Brownell.
相似文献
67.
Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line 总被引:3,自引:1,他引:2
Brickman YG; Nurcombe V; Ford MD; Gallagher JT; Bartlett PF; Turnbull JE 《Glycobiology》1998,8(5):463-471
Heparan sulfate (HS) glycosaminoglycans are essential modulators of
fibroblast growth factor (FGF) activity both in vivo and in vitro, and
appear to act by cross-linking particular forms of FGF to appropriate FGF
receptors. We have recently isolated and characterized two separate HS
pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D
cells: one from cells in log growth phase, which greatly potentiates the
activity of FGF-2, and the other from cells undergoing contact-inhibition
and differentiation, which preferentially activates FGF-1. These two pools
of HS have very similar functional activities to those species isolated
from primary neuroepithelial cells at corresponding stages of active
proliferation or differentiation. We present here a structural comparison
between these cell line HS species to establish the nature of the changes
that occur in the biosynthesis of HS. A combination of chemical and
enzymatic cleavage, low pressure chromatography and strong anion-exchange
HPLC were used to generate full chain models of each species. Overall, the
HS pools synthesized in the dividing cell line pools possessed less complex
sulfation than those derived from more differentiated, growth arrested
cells.
相似文献
68.
Carbohydrates have been suggested to account for some IgE cross- reactions
between various plant, insect, and mollusk extracts, while some IgG
antibodies have been successfully raised against plant glycoproteins. A rat
monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23)
and rabbit polyclonal antiserum against horseradish peroxidase were
screened for reactivity in enzyme-linked immunosorbent assay against a
range of plant glycoproteins and extracts as well as neoglycoproteins, bee
venom phospholipase, and several animal glycoproteins. Of the
oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish
peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23
and anti- horseradish peroxidase to native horseradish peroxidase
glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase
against Sophora japonica lectin was most inhibited by a neoglycoconjugate
of bromelain glycopeptide cross-linked to bovine serum albumin, while the
defucosylated form of this conjugate was inactive as an inhibitor. A wide
range of plant extracts was found to react against YZ1/2.23 and
anti-horseradish peroxidase, with particularly high reactivities recorded
for grass pollen and nut extracts. All these reactivities were inhibitable
with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom
phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more
strongly with anti-horseradish peroxidase in a manner inhibitable with the
bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin
from Helix pomatia reacted poorly with YZ1/2.23 but did react with
anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose
residue linked to the chitobiose core of plant glycoproteins is the most
important residue in the epitope recognized by the two antibodies studied,
but that the polyclonal anti-horseradish peroxidase antiserum also contains
antibody populations that recognize the xylose linked to the core mannose
of many plant and gastropod N-linked oligosaccharides.
相似文献
69.
70.