• Bulletin of the Korean Chemical Society
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• v.26 no.4
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• pp.553-557
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• 2005

The formation of striped phases of unsymmetric hexyl octadecyl disulfide ($CH_3(CH_2)_5SS(CH_2)_{17}CH_3$, HOD) and 1-hydroxyundecyl octadecyl disulfide ($CH_3(CH_2)_{17}SS(CH_2)_{11}$OH, HUOD) on Au(111) and graphite has been investigated by scanning tunneling microscopy (STM) to understand the self-assembly processes of dialkyl disulfides. STM imaging clearly shows the formation of striped phases having corrugation periodicities that are nearly consistent with the molecular length of alkanethiolate moieties formed after the S-S bond cleavage of dialkyl disulfide on a gold surface. On the other hand, self-assembled monolayers (SAMs) of dialkyl disulfides on a graphite surface displayed long-range, well-ordered monolayers with one striped pattern that shows periodicity as a function of molecular length via nondissociative adsorption. From a nonoscopic viewpoint, we have clearly demonstrated that dialkyl disulfide SAMs on gold form via S-S bond cleavage of disulfide.

• Biotechnology and Bioprocess Engineering:BBE
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• v.1 no.1
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• pp.13-17
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• 1996

The protein disulfide isomerase(PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody(MAb) refolding and assembly which accompanies disulfide bond formation The MAb in vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb intermediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hybridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant for a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specific MAb productivity exists.

• Archives of Pharmacal Research
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• v.28 no.5
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• pp.561-565
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• 2005

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of sulfate group from a phenyl sulfate ester to a phenolic acceptor. The promoter region and the transcripti on start sites of Enterobacter amnigenus astA have been determined by primer extension analysis. Northern blot analysis resolved two mRNA species with lengths of 3.3 and 2.0 kb, which correspond to the distances between the transcriptional initiation sites and the two inverted repeat sequences (IRSs). By length, the 3.3 kb RNA could comprise the three-gene (astA with dsbA and dsbB) operon. ASST has three highly conserved cysteine residues. Reducing and non-reducing SDS-PAGE and activity staining showed that disulfide bond is needed for the activity of the enzyme. To identify the cysteine residues responsible for the disulfide bond formation, a series of Cys to Ser mutants has been constructed and the enzymatic activity was measured. Based on the results, we assumed that the first cysteine (Cys349) might be involved in disulfide bond mainly with the second cysteine (Cys445) and result in active conformation.

• Biotechnology and Bioprocess Engineering:BBE
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• v.8 no.2
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• pp.59-63
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• 2003

The protein disulfide isomerase (PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody (Mab) refolding and assembly which accompanies disulfide bend formation. The MAb in vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb in-termediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hybridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant fur a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specific MAb productivity exists.

• Journal of Life Science
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• v.11 no.5
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• pp.415-422
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• 2001

Many secreted proteins have disulfide bonds that are important for their structure and function. Protein disulfide isomerase (PDI, EC 5.3.1.4.), an enzyme that catalyzes the formation and rearrangement of thiol/disulfide exchange reactions, is a resident of the endoplasmic reticulum (ER). The subcellular localization and its function as catalyst of disulfide bond formation in the biosynthesis of secretory and cell membrane proteins suggest that PDI plays a key role in the secretory pathway. We have isolated a cDNA encoding protein disulfide isomerase from Bombyx mori(bPDI). It has been characterized under ER stress conditions (dominantly induced by calcium ionophore A23187, tunicamycin and DTT), which is known to cause an accumulation of unfolded proteins in the ER. Furthermore, It has also been examined for tissue distribution(pronounced at the fat body), hormonal regulation (juvenile hormone, insulin and juvenile +transferrin; however, it is not effected by transferrin alone), and the effect of exogenous bacteria (peak at 16 h after infection) on the bPDI mRNA expression. The results suggest that bPDI is a member of the ER stress protein group, and it may play an important role in exogenous bacterial infection in fat body, and that homones regulate its expression.

• Molecules and Cells
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• v.27 no.6
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• pp.673-680
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• 2009

Conversion of the normal soluble form of prion protein, PrP ($PrP^C$), to proteinase K-resistant form ($PrP^{Sc}$) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from ${\alpha}$-helix to ${\beta}$-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1475-1481
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• 2008

B3 antibody specifically binds the $Lewis^Y$-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab-PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, $(Fab-PE38fl)_2$. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences hi, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers $(Fabh1-PE38fl)_2$, $(Fabh2-PE38fl)_2$, and $(Fabh3-PE38fl)_2$. The refolding yields of these dimers were 5-16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment $(Fabh2-PE38)_2$ [8]. Our data suggest that the steric repulsion between the two PE38s in $(Fabh1-PE38)_2$ during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.62-63
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• 2005

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.235-242
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• 1999

We investigated the growth-inhibitory mechanism of Helicobacter pylori by omeprazole (OMP) and its activated sulfenamide (OAS). Using dithiothreitol (DTT) and 5,5'-dithio-bis[2-nitrobenzoic acid] (DTNB; Ellman's reagent), we first determined the relationship between the binding capacity of these compounds to H. pylori membrane and its significance to membrane P-type ATPase activity. After incubation of the intact H. pylori cells with either OMP or OAS, the residual quantity of free SH-groups on the cell membrane was measured, and, the resulting values were plotted as a function of time. From this experiment, we found that there was a considerable difference in the membrane-binding rates between OMP and OAS. At neutral pH, the disulfide bond formation on H. pylori membrane was completed within 2 min of incubation of the intact cells with OAS. By OMP, however, it was gradually formed, exceeding 10 min of incubation for completion, whereby, the extent of P-type ATPase inhibition appeared to be proportional to the disulfide forming rate. From this data, it was suggested that the disulfide formation might directly affect enzyme activity. Since OMP per se cannot yield a disulfide bond with cysteine, it is predicted that the enzyme inactivation must be caused by the OAS form. Accordingly, we postulated that, under the neutral pH, OMP could be converted to OAS in the course of transport. By extrapolating the inhibitory slopes, we could evaluate K₁ values, relating to their minimal inhibitory concentrations (MICs) for H. pylori growth. In these MIC ranges, H. pylori uptake or vesicular export of nutrients such as peptides were totally prohibited, but their effect in Escherichia coli were negligible. From these observations, we strongly suggest that the P-type ATPase activity is essential for the survival of H. pylori cells in particular.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2607-2612
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• 2010

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.