• Korean Journal of Veterinary Research
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• v.48 no.3
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• pp.243-250
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• 2008

A calmodulin-dihydrofolate reductase (DHFR) sandwich fusion protein was generated by insertion of calmodulin into the $\beta$-bulge region of DHFR to observe the effects of structurally constraining the calmodulin structure. The calcium binding properties of the sandwich protein were almost identical to calmodulin. Similar to calmodulin ($10.7 {\mu}M$), the sandwich protein bound four equivalents of calcium, with half saturation ($K_{0.5}$) observed at a [$Ca^{2+}$] of $8{\mu}M$. However, nicotinamide adenine dinucleotide (NAD) kinase activation property of the sandwich protein was lower than that of calmodulin. The sandwich protein activated NAD kinase, but to only half of the level obtained with calmodulin. The K 0.5 for both calmodulin and the sandwich protein were approximately the same (1-2 nM). Methylation analyses of the sandwich protein show that insertion of calmodulin into DHFR results in a large decrease in methylation. The $V_{max}$ observed with the sandwich protein (95 nmole/min/ml) was only 22% of the value observed with calmodulin (436 nmol/min/ml) in the presence of calcium. Addition of trimethoprim to the reaction significantly inhibited the observed methylation rate. Overall, the data suggest that the insertion of calmodulin into the DHFR structure has little effect on calcium binding by the individual lobes of calmodulin, but may constrain the lobes in a manner that results in altered interaction with the calmodulin-dependent proteins, and severely perturbed the methyltransferase recognition site.

• The Korean Journal of Zoology
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• v.33 no.2
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• pp.191-199
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• 1990

Sarcoplasmic reticulum subfractions were isolated from rabbit sarcoplasmic reticulum vesicles using ultracentrifugation in a continuous sucrose gradient (12.5% 50%) after French pressure treatment. And proteins in sarcoplasmic reticulum were detected by SDS-polyacrylamide gel electrophoresis and glycoproteins were identified through the reaction with 1251-concanavalin A.The electrophoresis showed that sarcoplasmic reticulum contained predominantly $Ca^2$+-AThase and calsequestrin along with high affinity calcium binding protein, intrinsic glycoprotein 160 Kd, 94 Kd, 80 Kd, 38 Kd, 34 Kd and 24 Kd proteins. Among these, the protein of about 80 Kd which has been known as one of heat shock proteins was especially enriched in the terminal cistemae of sarcoplasmic reticulum. Meanwhile, autoradiogram of 125 I-concanavalin A bound to the stained gels showed the distribution of glycoproteins which included 160 Kd glycoprotein, 94 Kd glycoprotein, calsequestrin and intrinsic glycoprotein Among these, the protein of about 160 Kd was especially enriched in longitudial sarcoplasmic reticulum and T-tubule, and the protein of about 94 Kd which has been known as one of glucose-regulated proteins was also enriched in T-tubule and sharply reduced in terminal cistemae.

• Journal of the Korean Magnetic Resonance Society
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• v.2 no.2
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• pp.141-151
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• 1998

Human annexin I is a member of annexin family of calcium dependent phospholipid binding proteins, which have been implicated in various physiological roles including phospholipase A2(PLA2) inhibition, membrane fusion and calcium channel activity. In this work, the structure of N-terminally truncated human annexin I ({{{{ DELTA }}-annexin I) and its interactions with Ca2+, ATP and cAMP were studied at atomic level by using nuclear magnetic resonance (NMR) spectroscopy. The effect of Ca2+ binding on the structure of {{{{ DELTA }}-annexin I was investigated. The addition of Ca2+ to {{{{ DELTA }}-annexin I caused some changes in 13C NMR spectra. Carbonyl carbon resonances of some histidines were significantly broadened by Ca2+ binding. However, in the case of methionine, phenylalanine, and tyrosin, small changes could be observed. We found that ATP and cAMP bind {{{{ DELTA }}-annexin I, and the binding ratio of ATP to {{{{ DELTA }}-annexin I is 1. These results are well consistent with the report that cAMP and ATP interact with annexin I, and affect the calcium channels formed by annexin I. Because {{{{ DELTA }}-annexin I is a large protein with 35 kDa molecular weight, site-specific (carbonyl-13C) labeling technique was used to study the interaction sites of {{{{ DELTA }}-annexin I with Ca2+. NMR study was focused on the carbonyl carbon resonances of tyrosine, phenylalanine, methionine and histidine residues of {{{{ DELTA }}-annexin I because the number of these amino acids is small in the amino acid sequence of {{{{ DELTA }}-annexin I.

• Proceedings of the Korean Society of Crop Science Conference
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• 2004.04a
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• pp.12-27
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• 2004

Thanks to spectacular advances in the techniques for identifying proteins separated by two-dimensional electrophoresis and in methods for large-scale analysis of proteome variations, proteomics is becoming an essential methodology in various fields of plant sciences. Plant proteomics would be most useful when combined with other functional genomics tools and approaches. A combination of microarray and proteomics analysis will indicate whether gene regulation is controlled at the level of transcription or translation and protein accumulation. In this review, we described the catalogues of the rice proteome which were constructed in our program, and functional characterization of some of these proteins was discussed. Mass-spectrometry is a most prevalent technique to identify rapidly a large of proteins in proteome analysis. However, the conventional Western blotting/sequencing technique us still used in many laboratories. As a first step to efficiently construct protein data-file in proteome analysis of major cereals, we have analyzed the N-terminal sequences of 100 rice embryo proteins and 70 wheat spike proteins separated by two-dimensional electrophoresis. Edman degradation revealed the N-terminal peptide sequences of only 31 rice proteins and 47 wheat proteins, suggesting that the rest of separated protein spots are N-terminally blocked. To efficiently determine the internal sequence of blocked proteins, we have developed a modified Cleveland peptide mapping method. Using this above method, the internal sequences of all blocked rice proteins (i. e., 69 proteins) were determined. Among these 100 rice proteins, thirty were proteins for which homologous sequence in the rice genome database could be identified. However, the rest of the proteins lacked homologous proteins. This appears to be consistent with the fact that about 30% of total rice cDNA have been deposited in the database. Also, the major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that fumed out to be calreticulin, gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activate in rice, and leginsulin-binding protein in soybean have functions in the signal transduction pathway. Proteomics is well suited not only to determine interaction between pairs of proteins, but also to identify multisubunit complexes. Currently, a protein-protein interaction database for plant proteins (http://genome .c .kanazawa-u.ac.jp/Y2H)could be a very useful tool for the plant research community. Recently, we are separated proteins from grain filling and seed maturation in rice to perform ESI-Q-TOF/MS and MALDI-TOF/MS. This experiment shows a possibility to easily and rapidly identify a number of 2-DE separated proteins of rice by ESI-Q-TOF/MS and MALDI-TOF/MS. Therefore, the Information thus obtained from the plant proteome would be helpful in predicting the function of the unknown proteins and would be useful in the plant molecular breeding. Also, information from our study could provide a venue to plant breeder and molecular biologist to design their research strategies precisely.

• Journal of Plant Biotechnology
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• v.30 no.3
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• pp.281-291
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• 2003

In this review, we described the catalogues of the rice proteome which were constructed in our program, and functional characterization of some of these proteins was discussed. Mass-spectrometry is the most prevalent technique to rapidly identify a large number of proteome analysis. However, the conventional Western blotting/sequencing technique has been used in many laboratories. As a first step to efficiently construct protein cata-file in proteome analysis of major cereals, we have analyzed the N-terminal sequences of 100 rice embryo proteins and 70 wheat spike proteins separated by two-dimensional electrophoresis. Edman degradation revealed the N-terminal peptide sequences of only 31 rice proteins and 47 wheat proteins, suggesting that the rest of separated protein sports are N-terminally blocked. To efficiently determine the internal sequence of blocked proteins, we have developed a modified Cleveland peptide mapping method. Using this above method, the internal sequences of all blocked rice proteins(i, e., 69 proteins) were determined. Among these 100 rice proteins, thirty were proteins for which homologous sequence in the rice genome database could be identified. However, the rest of the proteins lacked homologous proteins. This appears to be consistent with the fact that about 45% of total rice cDNA have been deposited in the EMBL database. Also, the major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that tuned out to be calreticulin, gibberellin-binding protein, which is ribulose-1.5-bisphosphate carboxylase/oxygense active in rice, and leginsulin-binding protein in soybean have functions in the signal transduction pathway. Proteomics is well suited not only to determine interaction between pairs of proteins, but also to identify multisubunit complexes. Currently, a protein-protein interaction database for plant proteins(http://genome.c.kanazawa-u.ac.jp/Y2H)could be a very useful tool for the plant research community. Also, the information thus obtained from the plant proteome would be helpful in predicting the function of the unknown proteins and would be useful be in the plant molecular breeding.

• Journal of Integrative Natural Science
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• v.17 no.1
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• pp.31-41
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• 2024

Calcium ions play an important role in development and intracellular signaling. Dictyostelium discoideum has 14 genes encoding calcium -binding proteins (CBPs), but the function of most CBPs during development has not yet been studied. In this study, we investigated the specific functions of CBP7, one of 14 CBPs, in development using RNA interference cell lines of CBP7, cell lines overexpressing CBP7, cell lines with point mutations in the EF-hand domain, and cell lines expressing fragment proteins. was intended to reveal. CBP7 consists of 169 amino acids and contains 4EF-hand domains. The CBP7-overexpressing cells showed complete loss of developmental process. These cells remained in the single-cell growth stage under development -inducing conditions, while wild-type cells formed aggregations within 6-8h of development and eventually formed fruiting bodies. The experiments using point-mutated CBP7 protein showed that all EF-hand domains of CBP7 were important for CBP7 to function during developmental process. These results suggest that CBP7 plays an important role in developmental processes across all EF-hand domains.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.86-86
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• 1997

Human annexin I is a member of annexin family of calcium dependent phospholipid binding proteins, which have been implicated in various physiological roles including phospholipase A$_2$ (PLA$_2$) inhibition, membrane fusion and calcium channel activity. In this work, the structure of N-terminally truncated human annexin I (Δ-annexin I) and its interactions with Ca$\^$2+/, ATP and cAMP were studied at atomic level by using $^1$H, $\^$15/N, $\^$l3/C NMR (nuclear magnetic resonance) spectroscopy. The effect of Ca$\^$2+/ binding on the structure of Δ-annexin I was investigated, and compared with that of Mg$\^$2+/ binding. The addition of Ca$\^$2+/ to Δ-annexin I caused some changes in the high field and low field regions of $^1$H NMR spectra. Whereas, upon addition of Mg$\^$2+/ to Δ-annexin I, almost no change could be observed. Also we found that the binding ratio of ATP to Δ-annexin I is 1. Because Δ-annexin I is a large protein with 35 kDa molecular weight, site-specific (carbonyl-$\^$l3/C, amide-$\^$15/N) labeling technique was used to determine the interaction sites of Δ-annexin I with Ca$\^$2+/ and ATP. Assignments of all the histidinyl carbonyl carbon resonances have been completed by using Δ-annexin I along with its specific 1,2-subdomain. The carbonyl carbon resonances originating from His52 and His246 of Δ-annexin I were significantly affected by Ca$\^$2+/ binding, and some Tyr and Phe resonances were also affected. The carbonyl carbon resonances originating from His52 is significantly affected by ATP binding, therefore His52 seems to be involved in the ATP binding site of Δ-annexin I.

• Parasites, Hosts and Diseases
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• v.44 no.4 s.140
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• pp.331-341
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• 2006

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to $10{\mu}M$ of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.97-99
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• 2000

Allergy to Brassica pollen has been reported in some countries. We have cloned a cDNA encoding a Brassica pollen allergen, Bra r 1. Bra r 1 belongs to a new family of $Ca^{2+}$-binding proteins, characterized by the presence of two EF-hand calcium-binding domains. Bra r 1 was detected in the tapetum, microspores, pollen coat and pollen tubes, indicating Bra r 1 is involved in pollen pistil interaction and pollen tube growth. We have engineered the hypoallergenic mutants of Bra r 1 for immunotherapy. Here we describe the review of molecular characterization of Bra r 1.

• Journal of the Korean Magnetic Resonance Society
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• v.2 no.2
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• pp.131-140
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• 1998

The calcium-induced structural changes in the skeletal muscle regulatory protein troponin C (NTnC) involve a transition from a ‘closed’to an ‘open’structure with the concomitant exposure of a large hydrophobic interaction site for target proteins. Structural studies have served to define this conformational change and elucidate the mechanism of the linkage between calcium binding and the induced structural changes. There are now several structures of NTnC available from both NMR and X-ray crystallography. Comparison of the calcium bound structures reveals differences in the level of opening. We have considered the concept of a flexible open state of NTnC as a possible explanation for this apparent discrepancy. We also present simulations of the closed-to-open transition which are in agreement with the flexibility concept and with experimental energetics data.