• BMB Reports
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• v.33 no.6
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• pp.531-536
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• 2000

Tropomyosin (TM) is an important actin binding protein involved in regulation of muscle contraction. Unacetylated striated tropomyosin failed to bind to actin whereas unacetylated smooth tropomyosin bound well to actin. It has been demonstrated that high actin affinity of unacetylated ${\alpha}-tropomyosin$ was ascribed to the carboxyl terminal amino acid residues. In order to define the role of the carboxyl terminal residues of tropomyosin molecule on actin binding, two mutant tropomyosins were constructed. TM11 is identical to the striated tropomyosin except that the carboxyl terminal last three amino acids was replaced with $^{282}NNM^{284}$ whereas in TM14 $^{276}HA^{277}$ was substituted with smooth specific $^{276}QT^{277}$. TM11 and TM14 were overproduced in Escherichia coli and analyzed for actin affinity. The apparent binding constants (Kapp) of unacetylated tropomyosins were $2.2{\times}10^6M^{-1}$ for sm9, $1.03{\times}10^6M^{-1}$ for TM14, $0.19{\times}10^6M^{-1}$ for TM11, $>0.1{\times}10^6M^{-1}$ for striated, respectively. This result indicated that higher actin affinity of the unacetylated smooth tropomyosin was primarily attributed to the presence of QT residues in the smooth sequence. In case of the Ala-Ser (AS) dipeptide extension of the amino terminus of tropomyosin, Kapp were $21.1{\times}10^6M^{-1}$ for AS-sm9, $8.0{\times}10^6M^{-1}$ for AS-11, $4.7{\times}10^6M^{-1}$ for AS-14, $3.8{\times}10^6M^{-1}$ for AS-striated. AS-TM11 showed considerably higher actin affinity than AS-TM14, implying that interaction of Ala-Ser of the amino terminus with the carboxyl terminal residues. Since Kapp of AS-TM11 was significantly lower than that of AS-sm9, the presence of QT might be required for restoration of high actin affinity of the smooth ${\alpha}-tropomyosin$. These results suggested that the carboxyl terminal amino acid residues Glutamine275-Threonine276 are important for actin affinity of the recombinant smooth ${\alpha}-tropomyosin$, particularly of unacetylated smooth ${\alpha}-tropomyosin$.

• Journal of Life Science
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• v.11 no.1
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• pp.1-6
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• 2001

In order to determine the role of the carboxyl terminal amino acid residues of unacetylated ${\alpha}$-tropomyosin in actin affinity two mutant tropomyosins were constructed by site-directed mutagenesis. TM16 was identical to the striated tropomyosin except that three amino acids in the carboxyl terminal end were altered to $^{282}TNM^{284}$ while in TM17 $^{282}TSI^{284}$ of the striated was replaced with$^{282}NSM^{284}$. TM16 and TM17 were overproduced in Escherichia coli and analyzed for actin affinity by comparing actin affinities of the striated and TM11 $^{282}NNM^{284}$). The apparent binding constants (Kapp) of unacetylated tropomyosins to actin were $5.1{\times}10^4M^{-1}$ for the striated, $1.1{\times}10^5M^{-1}$ for TM11, $1.09{\times}10^5M^{-1}$ for TM16, and $1.03{\times}10^5M^{-1}$ for TM17, respectively. Since the actin affinities of TM11, TM16, and TM17 were very similar, this result suggested that amino acid residues 282 and 283 were insignificant for acting affinity of unacetylated $\alpha$-tropomyosin. However, they all exhibited higher actin affinities than that of the striated, suggesting that Met residue at the carboxyl terminus of unacetylated smooth tropomyosin was rather important for actin affinity, presumably due to the nucleophilic nature of sulfur atom in Met residue.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.204-210
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• 2007

Previous reports indicated that the carboxyl terminal residues, glutamine276-threonine277 in particular, were important for actin affinity of the unacetylated smooth ${\alpha}-tropomyosin$. To determine the role of the glutamine and threonine residues in C-terminal region in actin binding, we constructed mutant striated muscle ${\alpha}-tropomyosin$ (TMs), in which these two residues were individually substituted. These mutant tropomyosins, designated TM18 (HT) and TM19 (QA), were overexpressed in E. coli as an either unacetylated form or Ala-Ser. (AS) dipeptide fusion form, and were analyzed F-actin affinity by cosedimentation. Unacetylated TM19 (QA) bound to actin approximately three times stronger than TM18 (HT) and much stronger than ST (HA). AS/TM19 (QA) showed four times stronger, in actin affinity than AS/ST (HA) while AS/TM14 (QT) bound to actin stronger to some extent than AS/TM18 (HT). These results suggested that the presence of Gln residue at 276 be primarily attributed to higher actin affinity of smooth ${\alpha}-tropomyosin$.

• Journal of Life Science
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• v.19 no.5
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• pp.573-580
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• 2009

It has been previously reported that the carboxyl terminal residues 276 and 277 of ${\alpha}$-tropomyosin are important for actin affinity. In order to investigate actin affinities of these two residues of skeletal (HA) and smooth (QT) muscle ${\alpha}$-tropomyosins, a series of mutant tropomyosins were constructed in which residues at either 276 or 277 were individually replaced with a cysteine residue for chemical modification. These mutants were overexpressed in E. coli as unacetylated and Ala-Ser (AS) dipeptide fusion forms. While actin affinities of unacetylated tropomyosins were considerably low, those of AS/TMs were remarkably higher than those of corresponding unacetylated tropomyosins. However, actin affinities of AS/TM24 (QC) and AS/TM29 (HC) were dramatically lower than those of other AS/TMs and were close to those of unacetylated tropomyosins. In addition, actin affinities of unacetylated TM24 (QC) and TM29 (HC) failed to be restored in the presence of troponin, unlike unacetylated TM10 (HA) and TM23 (CA). These results indicated that the presence of a cysteine residue at 277 caused a drastic decrease in actin affinity, and also that the residue 277 is important for actin affinity of ${\alpha}$-tropomyosin. Since TM23 (CA) showed high actin affinity, it may serve as a valuable tool for chemical modification studies for investigating the interaction of the carboxyl terminal residues of ${\alpha}$-tropomyosin with actin and/or troponin.

• Journal of Life Science
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• v.14 no.5
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• pp.770-777
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• 2004

It has been previously reported that unacetylated a-tropomyosin(TM) produced in E. coli failed to bind to actin while acetylated muscle TM and Ala-Ser dipeptide fusion TM (AS-TM) bound well to actin. In order to determine the structural requirement of the amino terminus for high actin affinity, a recombinant tropomyosin (Ala-TM) that a single Ala residue was added to the amino terminus of Ala-TM was constructed, overexpressed, and purified from E. coli. Actin affinity of Ala-TM was 2.3$\times$$10^{6}$$M^{-1}$, whereas that of unacetylated TM was considerably lower than 0.1$\times$$10^{-6}$$M^{-1}$ indicating that addition of a single Ala residue to the amino terminus drastically increased, at least twenty times, actin affinity of TM. Ala-TM, however, bound to actin about three times weaker than acetylated TM and AS- TM, implying that the addition of an Ala residue was insufficient for complete restoration of high actin affinity. While Ala-TM, AS-TM, and muscle TM showed inhibition and activation of actomyosin Sl ATPase activity depending on myosin Sl concentration, the degree of inhibition and activation was different from each other. AS-TM exhibited the greatest inhibition of the ATPase at low Sl concentration, whereas the greatest activation of the ATPase was observed with muscle TM. These results, together with previous findings, strongly suggested that local structure of the amino terminus is the crucial functional determinant of TM.

• BMB Reports
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• v.31 no.5
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• pp.427-431
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• 1998

In the previous report (Choi et al., 1997), the angiogenin binding peptides identified from a phage-peptide library were analyzed by using the fusion proteins composed of the Escherichia coli maltose binding protein and its corresponding peptides. However, it was difficult to obtain a sufficient amount of the fusion proteins required for further analysis because of the low expression level. We now report a high level expression of the fusion protein and analysis of its anti-angiogenin activity. The use of strong T7 promoter and removal of signal sequence allowed about a 20-fold increase in the expression efficiency of the fusion protein. We were able to obtain about 10 mg of purified fusion protein from one liter of culture. The purified fusion protein showed angiogenin-specific affinity and inhibited the binding of biotinylated actin to human angiogenin at $IC_{50}$ of 0.6 mM. Its anti-angiogenin activity was also revealed by the chorioallantoic membrane assay.

• Journal of Photoscience
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• v.11 no.3
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• pp.121-127
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• 2004

The${\beta}$-glucosidases occur widely in all living organisms and has in general a tendency to form oligomers of varying numbers of subunits or aggregates, although the functional implications of such diverse oligomerization schemes remain unclear. In particular, the assembly mode of the oat ${\beta}$-glucosidase is very unique in that it multimerizes by linear stacking of a hexameric building block to form long fibrillar multimers. Some structural proteins such as actin and tubulin assemble into long fibrils in a helical fashion and several enzymes such as GroEL and Pyrodictium ATPase functional complexes, 20S proteasome of the archaebacterium Thermoplasma acidophilum, and lutamine synthetase fromblue-green algae, assemble into discrete oligomers upto 4 stacked rings to maintain their enzymatic activities. In particular, oat ${\beta}$-glucosidase exists in vivo as a discrete long fibrillar multimer assembly that is a novel structure for enzyme protein. It is assembled by linear stacking of hollow trimeric units. The fibril has a long central tunnel connecting to the outer medium via regularly distributed side fenestrations. The enzyme active sites are located within the central tunnel and multimerization increases enzyme affinity to the substrates and catalytic efficiency of the enzyme. Although it is suggested that oligomerization may contribute to the enzyme stability and catalytic efficiency of ${\beta}$-glycosidases, the functional implications of such diverse oligomerization schemes remain unclear so far.

• Journal of fish pathology
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• v.22 no.2
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• pp.147-153
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• 2009

To study the biological activity of interleukin-$1\beta$(IL-$1\beta$), a proinflammatory cytokine, in nile tilapia, Oreochromis niliticus, the recombinant tilapia IL-$1\beta$ was produced in E. coli cells based on pQE vector. Ni-NTA (nitriloacetic acid) metal affinity chromatography was used to purify recombinant protein. The eluted fractions exhibited a single band of protein with a molecular weight of about 25kDa, which is in close agreement with 25.4 kDa predicted by the cDNA sequence. The biological activity of the purified recombinant tilapia IL-$1\beta$ was tested through its effects on IL-$1\beta$ gene expression, which are known as IL-$1\beta$ inducible genes in mammals and fishes. IL-$1\beta$ gene expression induced by poly I:C, a synthetic double stranded RNA, was also assessed in tilapia head kidney cells. IL-$1\beta$ gene expression was analysed using QPCR (quantitative polymerase chain reaction). The ratio of the indicated gene expression was expressed as the relative mRNA level to $\beta$-actin mRNA level, which is constitutively expressed in macrophages. Consequently, head kidney cells incubated for three hours with rIL-$1\beta$(10, 2, 1 $\mu{g}$/ml) showed a dose dependent increase in IL-$1\beta$ mRNA levels and 1 $\mu{g}$/ml of poly I:C was also able to induce IL-$1\beta$ gene expression in head kidney in tilapia.

• Archives of Plastic Surgery
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• v.33 no.1
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• pp.46-52
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• 2006

High-density micromass culture was needed to take three dimensions culture with ASCs(adipose derived stromal cells) and chondrogenesis. However, the synthetic polymer has hydrophobic character and low affinity to cells and other biomolecules. Therefore, the surface modification without changes of physical and chemical properties is necessary for more suitable condition to cells and biomolecules. This study was performed to investigate the effect of surface modification of poly (lactic-co-glycolic acid)(PLGA) scaffold by plasma treatment (P(+)) on the adhesion, proliferation and chondrogenesis of ASCs, and not plasma treatment (P(-)). ASCs were isolated from human subcutaneous adipose tissue obtained by lipectomy and liposuction. At 1 hour 30 minutes and 3days after cell seeding onto the P(-) group and the P(+) group, total DNA amount of attached and proliferated ASCs markedly increased in the P(+) group (p < 0.05). The changes of the actin under confocal microscope were done for evaluation of cellular affinity, at 1 hour 30 minutes, the shape of the cells was spherical form in all group. At 3rd day, the shape of the cells was fiber network form and finely arranged in P(+) group rather than in P(-) group. RT-PCR analysis of cartilage-specific type II collagen and link protein were expressed in 1, 2 weeks of induction. Amount of Glycoaminoglycan (GAG) markedly increased in P(+) group(p < 0.05). In a week, extracellular matrix was not observed in the Alcian blue and Safranin O staining. However in 2 weeks, it was observed that sulfated proteoglycan increased in P(+) group rather than in P(-) group. In conclusion, we recognized that plasma treatment of PLGA scaffold could increase the hydrophilic property of cells, and provide suitable environment for high-density micromass culture to chondrogenesis

• Biomedical Science Letters
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• v.12 no.2
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• pp.73-79
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• 2006

Nebulin is a giant ($600{\sim}900$ kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin filamints in vertebrate skeletal muscles. Recently, There is an evidence that the nebulin also expressed in non muscle tissue, brain and liver. We identified a new isoform of nebulin from adult brain library by PCR screening. It contains two simple-repeats exon 165, 166 and linker-repeats exon $154{\sim}161$ except exon 159. The nebulin modules M160 to M170 (exon 150 to exon 161) has been shown to bind desmin. In mature striated muscle, desmin intermediate filaments surround Z-discs and link individual myofibrils laterally at their Z-discs and to other intracellular structures, including the costameres and the intercalated discs of the sarcolemma, sarcoplasmic reticulum, mitochondria, T-tubules, and nuclei. Therefore, it is an interesting possibility that the differential splice pathways within the linker region of nebulin modify the affinity of nebulin's interaction with desmin. The specific interactions of nebulin and desmin were confirmed in vivo by yeast two hybrid experiments. To verify in the cellular level the interaction between nebulin isoform and desmin, we transfected COS-7 cell with EGFP-tagged nebulin and DsRed-tagged desmin. Based on evidence showing that despite exon 159 was deleted, the new isoform of nebulin was interact with desmin. This suggest that nebulin in brain may interact with another intermediate filament. The conservation of these ligand-binding capacity in brain and skeletal nebulins suggest that nebulins may have conserved roles in brain and skeletal muscle.