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101.
Two mutants of the Desulfovibrio vulgaris flavodoxin, T12H and N14H, were generated which, for the first time, place a basic residue within the normally neutral 5'-phosphate binding loop of the flavin mononucleotide cofactor binding site found in all flavodoxins. These histidine residues were designed to form an ion pair with the dianionic 5'-phosphate, either altering its ionization state or offsetting its negative charge to allow evaluation of the magnitude of its electrostatic effect on the redox properties of the cofactor. The midpoint potential for the oxidized/semiquinone couple was not significantly altered in either mutant. However, the midpoint potentials for the semiquinone/hydroquinone couple (Esq/hq) were less negative than that of the wild type, increasing by 28 and 15 mV relative to that of the wild type for the T12H and N14H mutants, respectively, at pH 6. 31P NMR spectroscopy suggests that, just as for wild type, the phosphate group in each mutant does not change its ionization state between pH 6 and 8. Therefore, the small increases in midpoint potential must be linked to the protonation of the histidine residues, either through favorable interactions with the anionic hydroquinone or by the partial compensation of the charge on the 5'-phosphate. Values for the pKa of His12 and His14 in the oxidized flavodoxin were determined by 1H NMR spectroscopy to be 6.71 and 6.93, respectively, which are only modestly elevated relative to the average value for histidines in proteins. This suggests that the histidines do not form strong ion-pairing interactions with the phosphate and/or that the effective charge on the 5'-phosphate may be substantially less than the reported formal dianionic charge. Either way, the data provide evidence for the rather weak electrostatic interaction between a charged group at this site and the anionic flavin hydroquinone. In contrast, Esq/hq reported for the apoflavodoxin-riboflavin complex, which lacks the 5'-phosphate group, is 180 mV less negative than that of the native flavodoxin. The re-evaluation of the redox and cofactor binding properties of the riboflavin complex generated values for the dissociation constants for the riboflavin complex in the oxidized, semiquinone, and hydroquinone oxidation states that are 2100-, 63000-, and 54-fold higher, respectively, than that for the naturally occurring flavin mononucleotide complex. The large redox potential shifts observed for both redox couples in the riboflavin complex are primarily the consequence of a decreased stabilization of the semiquinone rather than the result of the absence of the negative charge of the 5'-phosphate. It is concluded from this study that the negative charge on the phosphate group of the cofactor does not play a disproportionate role in decreasing Esq/hq, at most contributing equivalently with the acidic amino acid residues clustered around the flavin to an unfavorable electrostatic environment for the formation of the flavin hydroquinone anion. 相似文献
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SR Johnston BP Haynes NP Sacks JA McKinna LJ Griggs M Jarman M Baum IE Smith M Dowsett 《Canadian Metallurgical Quarterly》1993,28(3):241-250
Chronic in utero methamphetamine treatment, throughout gestation in rats, resulted in alterations in both behavior and brain monoamine function in the adult offspring. The higher dose of methamphetamine (10 mg/kg/b.i.d.) caused a significant decrease in square crossing and rearing in an open field, as well as a regional increase of serotonin and dopamine uptake sites. In contrast, the lower dose of in utero methamphetamine (2 mg/kg/b.i.d.) resulted in a significant decrease in regional densities of serotonin and dopamine uptake sites, and only decreased rearing behavior. Across treatment groups, there were significant correlations between open-field square crossing activity and the number of uptake sites in specific brain areas. Other measured behaviors, such as the neonate righting reflex and the adult Morris water maze performance, were unaffected by either in utero drug regimen. These results are discussed in terms of the known neurotoxicity of amphetamines and the ability of the immature nervous system to compensate for fetal exposure to methamphetamine. 相似文献
106.
IE Goldberg GC Rossi SR Letchworth JP Mathis J Ryan-Moro L Leventhal W Su D Emmel EA Bolan GW Pasternak 《Canadian Metallurgical Quarterly》1998,286(2):1007-1013
The recently isolated peptides endomorphin-1 and endomorphin-2 have been suggested to be the endogenous ligands for the mu receptor. In traditional opioid receptor binding assays in mouse brain homogenates, both endomorphin-1 and endomorphin-2 competed both mu1 and mu2 receptor sites quite potently. Neither compound had appreciable affinity for either delta or kappa1 receptors, confirming an earlier report. However, the two endomorphins displayed reasonable affinities for kappa3 binding sites, with Ki values between 20 and 30 nM. Both endomorphins competed 3H-[D-Ala2, MePhe4,Gly(ol)5] enkephalin binding to MOR-1 receptors expressed in CHO cells with high affinity. In mouse brain homogenates 125I-endomorphin-1 and 125I-endomorphin-2 binding was selectively competed by mu ligands. 125I-Endomorphin-1 and 125I-endomorphin-2 also labeled MOR-1 receptors expressed in CHO cells with high affinity. Autoradiography of the two 125I-labeled endomorphins demonstrated regional patterns in the brain similar to those previously observed for mu drugs. Pharmacologically, the endomorphins were potent analgesics. Although they were equipotent supraspinally, endomorphin-1 was more potent spinally. Endomorphin analgesia was effectively blocked by naloxone, as well as the mu-selective antagonists beta-funaltrexamine and naloxonazine. In CXBK mice, which are insensitive to supraspinal morphine, neither endomorphin was active, consistent with a mu mechanism of action. Finally, the endomorphins inhibited gastrointestinal transit. In conclusion, these results support the mu selectivity of these agents. 相似文献
107.
IE Smith 《Canadian Metallurgical Quarterly》1997,57(11):573-574
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IE Katina VI Luk'ianenko DA Terent'ev NV Tomilin GA Nasledov 《Canadian Metallurgical Quarterly》1997,83(7):85-95
Quantitative changes of sodium and potassium ionic currents were studied in the frog embryonic skeletal myocytes cultures under conditions preventing cell fusion and division. During 7 days of culturing the mean values of the sodium and potassium currents densities were 7- and 1.8-fold increased, resp. In currents clamp records the APs were observed only after 5 days of culturing, and in cells with proportion of sodium and potassium conductances above the critical level. 相似文献
110.