• 대한수의학회지
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• 제48권3호
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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.

• 생명과학회지
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• 제4권2호
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• pp.50-59
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• 1994

신호전달과정의 연구는 calcium이 messenger로서 작용한다고 밝혀진 후로 식물에서 $Ca^{++}$ -messenger system에 대한 생화학적 및 분자생물학적 분야에서의 연구는 급속하게 발전하게 되었다. 식물세포에서 calcium 이온들의 많은 작용은 EF hand family로서 알려진 calcium binding protein에 의해서 조절된다. Calmodulin (CaM)은 highy conserve 되어 있으며, 4개의 calcium binding domain을 가진 ubiquitous한 단백질이다. 본 연구는 calmodulin 유전자의 발현에 미치는 calcium, EGTA, calcium ionophore 및 calmodulin antagonist의 영향과 또한 외부신호(light, wounding), chemical 및 auxin 등의 영향을 reporter화 유전자의 분석에 의해서 CaM유전자의 발현기작을 규명하고자 하였고, 또한 calmodulin 유전자의 organ-specific 발현 및 calmodulin의 새로운 생리적인 기능도 연구하고자 하였다.

• Archives of Pharmacal Research
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• 제16권4호
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• pp.289-294
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• 1993

A binding protein of radio-labeled sanjoinine-A (fangufoline) in rat brain cytoplasm was investigated, using an equilibrium dialysis technique. The labeled agent was bound to the cytosol fraction with two distinctly different types of sets in calclum ion-dependent manner. The bound protein was identified as calmodulin by dgel filtration of the sanjoinine-A bound cytosol fraction on a Sephadex G-75 column. Calmodulin was bound to sanjoinine-A bound at two sets of binding sites in the calculated as two at high affinity sites $(Kd=1.1\;mu{M)}$ and four at low affinity sites $(Kd=3.1\;\mu{M)}$.

• Journal of Life Science
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• 제12권2호
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• pp.43-46
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• 2002

Kinesin Superfamily Protein 1A (KIF1A) is an anterograde monomeric motor transporting a subset of synaptic vesicle precursors and plays an important role in neuronal function and survival. Here, f have used the yeast two-hybrid system to identify the proteins that interacts with the tail region of KIF1A. Calmodulin was found to interact specifically with the tail region of KIF1A. Calmodulin regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. KIF1A interacts with calmodulin in the yeast two-hybrid assay, which is proved by immunoprecipitation with calmodulin in brain fraction. These results indicate that KIF1A is associated with calmodulin, suggesting that calmodulin may be a key role in the regulation of anterograde transport of synaptic 1 vesicle precursors.

• Applied Biological Chemistry
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• 제43권4호
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• pp.231-235
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• 2000

담배 배양 세포의 성장과정 중의 칼모듈린 농도변화 및 칼모듈린 결합 단백질의 종류에 대하여 조사하고 이들 단백질들 중 글루탐산 탈탄산효소를 immunodetection과 활성측정으로 확인하였다. 담배세포는 유도기(초기 $1{\sim}2$일간), 대수증식기($3{\sim}5$일), 정지기 등의 전형적인 성장 패턴을 보였다. 칼모듈린의 농도는 비록 대수증식기에 약간 감소하는 경향을 보이다 정지기에 이르면서 유도기의 수준을 회복하는 것으로 나타났지만 전체적으로는 성장단계에 관계없이 유사한 수준을 유지하는 것으로 나타났다. 주요 칼슘-의존형 칼모듈린 결합단백질은 56, 46, 36, 32-kDa의 4종류인 것으로 조사되었고, 모노클로날 항체를 이용하여 immunodetection을 실시해 본 결과 56-kDa 단백질이 담배 글루탐산 탈탄산효소로 확인되었다. 56-kDa의 글루탐산 탈탄산효소는 대수증식기에 수확한 세포에서 가장 많이 검출되었고, 이와같은 패턴은 효소활성 측정에서도 확인되었다. 이러한 결과들은 담배세포의 성장과정 중에 칼슘/칼모듈린-의존형 글루탐산 탈탄산효소 농도가 조절되고 있음을 제안해 주는 것이다.

• Molecules and Cells
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• 제24권2호
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• pp.276-282
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• 2007

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.

• Journal of Microbiology
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• 제37권1호
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• pp.21-26
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• 1999

Two novel calcium-binding proteins, named CAB-I and CAB-II, have been isolated from Streptomyces coelicolor. Purification of the calcium-binding proteins involved heat treatment, fractionation with ammonium sulfate, acid treatment, anion exchange and hydrophobic interaction column chromatography, FPLC gel filtration, and preparative isoelectric focusing. A chelex competitive assay and 45Ca autoradiography verified the calcium-binding ability of the proteins. The major band CAB-II has an apparent molecular weight of 26,000 determined by SDS-polyacrylamide gel electrophoresis and 340,000 determined by gel filtration. The isoelectric point of this molecule showed the acidic nature of the molecule. N-terminal amino acid sequence analysis shows homology to rat Ca2+/calmodulin-dependent protein kinase-II (CAB-II) and yeast phosphoprotein phosphatase (CAB-I).

• BMB Reports
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• 제38권4호
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• pp.432-439
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• 2005

$Ca^{2+}$/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of $Ca^{2+}$-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic $Ca^{2+}$-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stress induced expression of OsCam1 gene requires the $[Ca^{2+}]_{cyt}$ elevation that is known to occur in response to these stimuli.

• Journal of Yeungnam Medical Science
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• 제7권1호
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• pp.39-50
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• 1990

세포막의 정보전달기전중 phosphoinositide system은 정보가 전달될때 phospholipase C 효소의 작용으로 phosphatidyl inositol bisphosphate로부터 inositol triphosphate($IP_3$)와 diacylglycerol이 생성되며 $IP_3$는 다시 $IP_3$kinase에 의해 inositol tetrakisphosphate($IP_4$)로 되어 이차전령 물로서 작용한다. 본 연구는 $IP_3$kinase효소가 $Ca^{2+}$와 calmodulin에 의해 활성화되는 성질을 이용하여 calmodulin을 정제하고 $IP_3$kinase효소와의 친화도를 비교 관찰하였다. Calmodulin정제는 phenyl-Sepharose resin을 이용하여 column chromatography를 시행하여 정제확인하였으며 분자량이 17,000임을 SDS-polyacrylamide gel 전기영동으로 확인하였다. 정제된 calmodulin을 affigel column에 결합시킨 gel에 소의 뇌로부터 분리한 $IP_3$kinase효소가 담긴 시료를 calmodulin-affigel column에 적용하여 결합 및 유출정도를 비교하였으며 $Ca^{2+}$이 든 buffer에서 친화도가 가장 컸으며 유출은 EGTA용액에서 일부 유출되었으며 calmodulin/$Ca^{2+}$이 든 buffer에선 강한 유출정도를 관찰하였다. 그러나 calmodulin/$Ca^{2+}$는 $IP_3$kinase효소의 활성을 증가시키며 calmodulin이 단백질이어서 정제면에서 효소와의 분리가 쉽지않아 여러 다른 detergent를 적용하였으나 0.2% chaps buffer에서 집중된 유출을 관찰하였다.

• Plant Resources
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• 제7권3호
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• pp.174-180
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• 2004

Calmodulin, a $Ca^{2+}$-binding protein, has no enzyme activity. It combines with $Ca^{2+}$ and makes variable proteins to an active form. Calmodulin 2 is a ubiquitous protein in plants. To investigate the defense mechanism against various stresses, a clone encoding a calmodulin 2 protein was isolated from a cDNA library prepared from taproot mRNAs of Codonopsis lanceolata. The cDNA, designated CICAM2, is 719 nucleotides long and has an open reading frame of 450 bp with a deduced amino acid sequence of 149 residues. The deduced amino acid sequence of CICAM2 showed a high similarity with calmodulins of P. x hybrida (P27163) 97%, N. tabacum (BAB61908) 97%, S. tuberosum (AAA74405) 96%, Z. mays (CAA74307) 92%, C. richardii (AF510075) 93%, M. truncatula (AAM81203) 91%, and G. max (P62163) 91%. The transcriptional expression of the CICAM2 gene, was gradually increased by the CaCl$_2$ treatment. Whereas its expression And it was gradually decreased in the cold stress treatment.ent.