• BMB Reports
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• v.41 no.7
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• pp.555-560
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• 2008

The interaction of the rod GTP binding protein, Transducin ($G_t$), with bleached Rhodopsin ($R^*$) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound Gt by reconstituting $G_t$ containing bound GDP ($G_t$-GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate ($G_t$-p[NH]ppG) with $R^*$ under physiological conditions. Release of GDP and p[NH]ppG from $G_t$ occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of $G_t$ without bound nucleotide ($G_{t^-}$) were generated. When ROS containing bleached rhodopsin ($R^*$) were centrifuged in low ionic strength buffer, $G_{t^-}$ remained associated with the membrane fraction, whereas $G_t$-GDP remained in the soluble fraction. These results suggest that $G_t$-GDP and $G_t$-p[NH]ppG have similar affinities for $R^*$. The results also suggest that $G_{t^-}$, rather than $G_t$-GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin.

• BMB Reports
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• v.39 no.3
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• pp.319-328
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• 2006

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

• Parasites, Hosts and Diseases
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• v.53 no.3
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• pp.335-339
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• 2015

Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of $Ca^{2+}$, $Mg^{2+}$, $K^+$, and $HCO_3{^-}$ in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

• Journal of the Korean Chemical Society
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• v.28 no.3
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• pp.185-193
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• 1984

At the spermine concentration to cover the number of the binding site of spermine 0.016 per nucleotide, the Hill coefficient of the ethidium binding to the calf thymus DNA was 1.7, while the value was 0.38 in the absence of the spermine. On the basis of the data, together with other present data on the viscometric titration of the DNA with spermine and anomalous absorbance-temperature profile at 260nm and viscosity-temperature profile, it can be speculated that allosteric propagation of the conformational transition induced by the binding of the spermine may be involved in the monomolecular collapse of the DNA to a condensed structure.

• Biomedical Science Letters
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• v.13 no.2
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• pp.127-133
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• 2007

Heterotrimeric GTP binding proteins (G proteins) mediate signal generated by neurotransmitter and hormones. Among all G proteins, Go is the most abundant in brain but its role in brain is not clearly understood. To determine the function of the alpha subunit of Go (Go$\alpha$), we search for the interacting partner of Go$\alpha$ in brain using yeast two-hybrid system. A resistant to inhibitor of cholinesterase (Ric-8B) was identified as a Go$\alpha$ interacting protein. We confirmed interaction between Go$\alpha$ and Ric-8b employing in vitro affinity binding assay and showed that the Ric-8b increased the function of Go$\alpha$. Our findings indicate that Ric-8b is possible guanine nucleotide exchange factor for Go$\alpha$.

• Journal of the Korean Magnetic Resonance Society
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• v.16 no.1
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• pp.67-77
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• 2012

The small molecule binding to the c-Jun N-terminal kinase 3 (JNK3) was examined by the measurements of saturation transfer difference (STD) NMR experiments. The STD NMR experiment of ATP added to JNK3 clearly showed the binding of the nucleotide to the kinase. The STD NMR spectrum of dNTPs added to JNK3 discriminated the kinase-bound nucleotide from the unbound ones. After the five-fold addition of ATP to the dNTPs and JNK3 mixture, only signals of the cognate substrate of JNK3, ATP, were observed from the STD NMR experiment. These results signify that by the STD NMR the small molecules bound to JNK3 can be discriminated from the pool of the unbound molecules. Furthermore the binding mode of the small molecule to JNK3 can be determined by the competition experiments with ATP.

• BMB Reports
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• v.35 no.4
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• pp.437-441
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• 2002

Dihydrolipoamide dehydrogenase (E3) is a member of the pyridine nucleotide-disulfide oxidoreductase family. Thr residues are highly conserved. They are at the active site disulfide-bond regions of most E3s and other oxidoreductases,. The crystal structure of Azotobacter vinelandii E3 suggests that the hydroxyl group of Thr that are involved in the FAD binding interact with the adenosine phosphate of FAD. However, several prokaryotic E3s have Val instead of Thr. To investigate the meaning and importance of the Thr conservation in many E3s, the corresponding residue, Thr-44, in human E3 was substituted to Val by site-directed mutagenesis. The mutant’s E3 activity showed about a 2.2-fold decrease. Its UV-visible and fluorescence spectra indicated that the mutant might have a slightly different microenvironment at the FAD-binding region.

• Archives of Pharmacal Research
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• v.15 no.2
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• pp.146-151
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• 1992

The aim of this study was to evaluate capability of pertussis toxin (PT) to active mouse macrophages. The investigations were undertaken to determine whether the role played by this toxin required the A-protomer of the toxin to ADP-ribosylate a guanine nucleotide binding protein (a class I activity) or was dependent on the binding of B-oligomer of the toxin to the surface of target cells (a Class II activity). The results of these experiments have established that the mechanism of macrophage activation with PT seems to be dependent upon a Class II activity of the toxin.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.8
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• pp.3601-3606
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• 2014

Single nucleotide polymorphisms located at microRNA (miRNA)-binding sites are likely to affect the expression of miRNA targets and may contribute to the susceptibility of humans to common diseases. Here 335 candidate lung cancer-related inflammatory genes were selected according to the existing literature and database. We identified putative miRNA-binding sites of 149 genes by specialised algorithms and screened SNPs in the 3'UTRs of these genes. By calculating binding free energy, we sorted 269 SNPs on the basis of the possibility of prediction. The proposed approach could help to easy the identification of functionally relevant SNPs and minimize the workflow and the costs.

• BMB Reports
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• v.31 no.1
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• pp.44-47
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• 1998

Asymmetry amongst nucleotide binding sites of Escherichia coli $F_1$-ATPase was examined using $^{19}F$ NMR signal from fluorinated analogs of adenine nucleotides bound to nucleotide binding sites. ADP-$CF_2-{PO_3}^{2-}$ showed no inhibitory effect to $F_1$-ATPase. But ADP-CHF-${PO_3}^{2-}$ (racemic mixture) showed competitive inhibition of $F_1$-ATPase with $K_i$ of $60\;{\mu}m$. ADP-CHF-${PO_3}^{2-}$ shows only negligible binding to $EF_1$ in the absence of $Mg^2+$. With the addition of $Mg^2+$ to the medium, the $^{19}F$ resonance of free ADP-CHF-${PO_3}^{2-}$ disappeared and the new broad resonances appeared. Appearance of more than two new asymmetric resonances following the binding of ADP-CHF-${PO_3}^{2-}$ to $EF_1$ may indicate that at least one of the isomers showed split resonances. This may suggest that the region between ${\alpha}$-and ${\beta}$-phosphate of ADP-CHF-${PO_3}^{2-}$ which is bound to catalytic sites is experiencing a different environment at different sites.