• 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 ${\alpha}-tropomyosin$.

• Parasites, Hosts and Diseases
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• v.34 no.4
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• pp.215-224
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• 1996

actin and some actin binding proteins such as tropomyosin, α-actinin and troponin T were localized by simultaneous double immunogold labeling in several developmental stages of Cryptosporidium parvum. All of the observed developmental stages have many paricles of tropomyosin and actin around pellicle and cytoplasm. Tropomyosin was labeled much more than the actin when these two proteins were labeled simultaneously. And α-actinin was labeled mostly in the pellicle, but troponin T labeling weas very rarely observed. From this study it was suggested that tropomyosin seemed to be one of the major proteins of C. parvum, so it must be playing important roles in C. parvum.

• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.285-292
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• 1993

C. sinensis total RMh was containing large amount of 185 rRNA but little 285 rRNA. The size of the double-stranded cDNA synthesized from poly $(A)^{+}$ mRNA was 0.4-4.2 kb long with tapering unto 9.5 kb. Degenerated oligonucleotides (as 2 sense and 3 antisense Primers) were designed on the conserved regions of the known tropomyosin amino acid sequences. From one out of the PCR amplifications using total CDNA and matrix of primers, a specific gene product, 580 bp in size, was produced. Upon Southern hybridization of the PCR products with Schistosomn mnnsoni tropomyosin (SMTM) CDNA, only one signal appeared at the band of 580 bp product. This 580 bp product was considered to encode C. sinensis tropomyosin (CSTM) and cloned in pGEM-3Zf(-) for DNA sequencing. CSTM cDNA was 575 bp containing one open reading frame of 191 predicted amino acids, which revealed 86.3% homology with SMTM and 51.1% with rrichostronsylur coeubnlormis tropomyosin. CSTM cDNA obtained will serve as a probe in the studies of molecular cloning of CSTM.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.227-234
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• 1998

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (${\times}2,000$). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various celluar functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.

• Korean Journal of Food Science and Technology
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• v.9 no.4
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• pp.295-305
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• 1977

The procedures for the Preparation of regulatory proteins of myofibrill were developed and postmortem changes in the regulatory proteins of myofibrill were investigated. Both the physiological property and molecular shape of ${\alpha}-actinin$ from pre-rigor muscle did not differ from those of ${\alpha}-actinin$ from post-rigor muscle. On the other hand, although tropomyosin of myofibril changed negligibly during the post-mortem storage of muscle, troponin of myofibril changed remarkably.

• 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.

• The Korean Journal of Pharmacology
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• v.8 no.2
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• pp.55-61
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• 1972

Superprecipitation of actomyosin has been considered to be an in vitro model of the muscle contraction. The superprecipitation and ATPase activity (which supplies the energy for contraction) are influenced by several factors which are the large amount of changes in ionic strength, Mg and ATP concentrations. But those behaviors are found to be promptly influenced by the change in a small range of calcium concentration which can be controlled by the cellular function of muscle physiologically only in the presence of the modullatory proteins, tropomyosin and troponin. In order to elucidate the precise roles of calcium in the muscle contraction and relaxation, the effects of calcium on the actin- myosin interaction was observed in the presence of tropomyosin and troponin using the superprecipitation system. The results are summarized as follows: 1. EGTA (glycol ether diaminetetraacetic acid)prolonged the initiation of the superprecipitation of natural actomyosin. 2. Superprecipitation curve was declined by adding EGTA at the time when tile curve reached the half- maximum. The degree of declining was proportional to the amount of EGTA added. Especially, upon adding 0.25 mM EGTA the curve was lowered to the level before the protein superprecipitated. But addition of EGTA did not affect the curve after attaining the maximum. 3. Superprecipitation of Perry myosin B was not affected by EGTA added both before and during the course of the reaction. 4. Tropomyosin did not change the response of Perry myosin B to EGTA added at any time of the reaction. 5. Troponin also did not change the response of Perry myosin B to EGTA. 6. Both tropomyosin and troponin together rendered the Perry myosin B to obtain the same response as natural actomyosin to EGTA. 7. It was concluded that actin-myosin interaction was influenced by the minute change of calcium concentration only in the presence of both tropomyosin and troponin. We could reproduce the contraction and relaxation of the muscle in vitro under the presence of ATP by changing the calcium concentration.

• 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 ${\alpha}-tropomyosin$.

• Korean Journal of Food Science and Technology
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• v.46 no.5
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• pp.538-543
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• 2014

A sandwich enzyme-linked immunosorbent assay method (sELISA) for detecting the presence of shrimp in processed foods was developed using rabbit polyclonal antibodies against tropomyosin produced by black tiger prawns (shrimp). Based on the standard curve derived using this method, the detection range of shrimp was determined to be $1-100{\mu}g/mL$. The cross-reactivity of these antibodies toward black tiger prawns, fleshy prawns, cocktail prawns, lobster, and blue crab was 100, 73, 155, 18, and 0%, respectively. When shrimp was heated for 10 min, the mean assay recovery of tropomyosin was 121-221% at $70-100^{\circ}C$ and 7.8% at $121^{\circ}C$. When shrimp was added to cream soup, weaning food, sausage, fish paste, and sauce, the mean assay recovery was 397, 639, 168, 234, and 0%, respectively. In sample tests involving 14 commercial items, the coincidence ratio of assay results and reference was 79%.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.3
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• pp.189-198
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• 2006

Objective: We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse. We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry. Methods: NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London). Results: Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and ${\alpha}$-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin. Conclusions: This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.