• Title, Summary, Keyword: Calmodulin

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Generation and characterization of calmodulin-DHFR sandwich fusion protein

  • Han, Chang Hoon
    • 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.

Carnitine and Calmodulin N-Methylation in Rat Testis; Calmodulin May beInvolved in Carnitine Biosynthesis

  • Oh, Suk-Heung;Cha, Youn-Soo;Sohn, Hee-Sook
    • Preventive Nutrition and Food Science
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    • v.3 no.3
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    • pp.251-255
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    • 1998
  • Rat testis known to contain all of the enzymes required for carnitine biosynthesis also contains high concentration of calmodulin, a protein which may or may not contain trimethyllysine, the major substrate in carnitine biosynthesis. The purpose of this study was to investigate the levels of carnitine and the state of calmodulin N-methylation in rat testes, and to discuss the possibility of the involvement of calmodulin incarnitine biosynthesis. Nonesterified carnitine , acid soluble acyl carnitine, and acid insoluble acyl carnitine of ra tests were 273 nmole, 62nmole, and 4 nmole/g tissue, respectively. Total carnitine level was 339 nmole/g testes tissue. Calmodulin purified from rat tests was assayed for methylation potential using N-methyltransferase from the rat testes. Rat testes calmodulin showed no 3H-methyl incorporation indicating that the calmodulin was trimethylated already by endogenous calmodulin N-methyltransferase. Amino acid composition analysis revealed that the rat testes calmodulin containd one mole of trimethyllysine per mole of calmodulin. These data suggest that testes calmodulin could provide the trimethyllysine needed for the synthesis of carnitine in the rat tests.

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Effects of Various Calmodulins on the Activation of Glutamate Decarboxylase and Nicotinamide Adenine Dinucleotide Kinase Isolated from Tobacco Plants

  • Oh, Suk-Heung;Yun, Song Joong
    • Journal of Applied Biological Chemistry
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    • v.42 no.1
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    • pp.19-24
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    • 1999
  • Plants have been shown to contains $Ca^{2+}$/calmodulin-stimulated GAD and NAD kinase. To test how calmodulin and calmodulin methylation affect the activation of GAD and NAD kinase, GAD and NAD kinase were partially purified from tobacco plants. GAD was also partially purified from E. coli transformed with a plasmid carrying a cloned tobacco GAD gene. We find that GAD from the transformed E. coli showed 60-fold $Ca^{2+}$/calmodulin-dependent activation. However, GAD from tobacco plants was stimulated only about 3.8-fold by the addition of calmodulin in the presence of calcium, suggesting high background activity of the enzyme was possibly due to bound endogenous tobacco calmodulin. There were no significant differences in the tobacco GAD activator properties between calmodulins. A monoclonal antibody against petunia GAD interacted strongly with both GAD from tobacco plants and GAD from cloned gene. NAD kinase from tobacco plants showed a complete $Ca^{2+}$/calmodulin dependency for activity. Unmethylated calmodulins activated GAD in a manner similar to methylated calmodulin. However, the maximum level of NAD kinase activation obtained with unmethylated calmodulins is approximately 4-fold higher than methylated calmodutins. These data suggested that endogenous tobacco calmodulin may interact more tightly with GAD than NAD kinase and that calmodulin methylation affects the activator properties of calmodulins for tobacco NAD kinase but not for GAD.

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Role of Calmodulin in the Generation of Reactive Oxygen Species and Apoptosis Induced by Tamoxifen in HepG2 Human Hepatoma Cells

  • Lee, Yong-Soo
    • The Korean Journal of Physiology and Pharmacology
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    • v.6 no.4
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    • pp.187-191
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    • 2002
  • Tamoxifen, an antiestrogen, has previously been shown to induce apoptosis in HepG2 human hepatoblastoma cells through activation of the pathways independent of estrogen receptors, i.e., intracellular $Ca^{2+}$ increase and generation of reactive oxygen species (ROS). However, the mechanism of tamoxifen to link increased intracellular $Ca^{2+}$ to ROS generation is currently unknown. Thus, in this study we investigated the possible involvement of calmodulin, a $Ca^{2+}$ activated protein, and $Ca^{2+}$/calmodulin-dependent protein kinase II in the above tamoxifen-induced events. Treatment with calmodulin antagonists (calmidazolium and trifluoroperazine) or specific inhibitors of $Ca^{2+}$/calmodulin-dependent protein kinase II (KN-93 and KN-62) inhibited the tamoxifen-induced apoptosis in a dose-dependent manner. In addition, these agents blocked the tamoxifen-induced ROS generation in a concentration-dependent fashion, which was completely suppressed by intracellular $Ca^{2+}$ chelation. These results demonstrate for the first time that, despite of its well-known direct calmodulin-inhibitory activity, tamoxifen may generate ROS and induce apoptosis through indirect activation of calmodulin and $Ca^{2+}$/calmodulin-dependent protein kinase II in HepG2 cells.

Structural Organization of Calmodulin Gene and Expression in Transgenic

  • 최영주
    • Journal of Life Science
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    • v.4 no.2
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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.

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Regulation of γ-Aminobutyric Acid Production in Tobacco Plants by Expressing a Mutant Calmodulin Gene

  • Oh, Suk-Heung;Cha, Youn-Soo
    • Journal of Applied Biological Chemistry
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    • v.43 no.2
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    • pp.69-73
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    • 2000
  • In order to understand the biological role of calmodulin in plants, transgenic plants expressing a mutant calmodulin (VU-4, Iys to ile-115) have been analyzed. We found that tobacco plants expressing VU-4 calmodulin have approximately twofold higher $\gamma$-aminobutyric acid (GABA) levels than the control plants. Cell suspension cultures established from the stem explants of the transgenic tobacco seedlings also have higher levels of GABA than the control cell cultures. Specific activity of glutamate decarboxylase (GAD), which catalyzes the decarboxylation of glutamate to $CO_2$ and GABA, of the transgenic tobacco cell extracts was about twofold higher than the activity of the control cell extracts. Western-blot analysis showed that the GAD is highly expressed in the transgenic tobacco plants. GAD partially purified from tobacco cell extracts showed approximately threefold $Ca^{2+}$/calmodulin-dependent activation. These data suggest that GABA synthesis in the transgenic tobacco plants is elevated, possibly due to higher levels of the calmodulin-dependent GAD enzyme and/or as a result of enhanced activation due to increased levels of the foreign calmodulin.

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Interaction of a Kinesin Superfamily Protein 1A (KIF1A) with Calmodulin

  • Seog, Dae-Hyun
    • Journal of Life Science
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    • v.12 no.2
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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.

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Sanjoinine-A의 Ionophore 활성과 Calmodulin과의 결합에 관한 연구

  • 박만기;박정일;이봉진;예덕천;박명환;한용남;한병훈
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.250-250
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    • 1994
  • 진정성 cyclopeptide인 sanjoinine-A의 진정작용을 분자 수준에서 밝히고자 sanjoinine-A의 ionophore 활성과 칼슘 결합 단백질인 calmodulin과의 결합을 CD와 NMR을 이용하여 연구하였다 sanjoinine-A는 칼슘과 마그네슘에 대하여 ionophore 활성이 있었으며 calmodulin과 두 단계로 결합한다는 것을 알 수 있었다. sanjoinine-A는 칼슘의 유무에 관계없이 calmodulin과 결합하였으며 CD와 NMR 스펙트럼상에서 현저한 변화가 있었다. sanjoinine-A가 calmodulin과 결합시 생체 내에서 활성 상태인 칼슘결합 calmodulin에 비하여 a-helix 구조비율이 감소하였으며 칼슘결합 loop에서 수소결합을 하고있는 amide 수소들의 exchange rate가 현저히 빨라졌다. sanjoinine-A는 calmodulin과 결합하여 칼슘결합 loop 주위의 공간구조를 변형시켜 그 활성을 변화시키는 것으로 보인다.

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Characterization of an Easter Lily Calmodulin cDNA Clone (백합실물에서 하나으 Calmodulin cDNA 클론 연구)

  • Kim, Seong-Ryong;An, Gyu-Heung
    • Journal of Plant Biology
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    • v.39 no.1
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    • pp.9-13
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    • 1996
  • A clone, LCM1, which encodes calmodulin (CaM) was isolated and characterized from monocot lily (Lilium longiflorum Thunb.) plants. The clone is 681 bps and contains the 447 bp coding region, 8 bp leader sequence, 210 bp 3'-untraslated region, and a poly(A) tail. The coding region of 149 amino acids encodes a protein of predicted Mr 17 kD. Comparison of the LCM1 amino acid sequence with other CaMs revealed that the protein is highly conserved among various living organisms. The expression level of calmodulin gene in lily was studied by RNA blot analysis. The LCM1 mRNA was present in all tissues tested. However, a higher level of calmodulin was observed in anther and floral bud. The level of calmodulin mRNA in anther was about 10 times higher than that in anther was about 10 times higher than that in vegetative tissues. The anther preferential expression of CaM in lily is currently investigated in dicot plants.

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Transgenic Tobacco Plants Expressing a Mutant VU-4 Calmodulin Have Altered Nicotinamide Co-Enzyme Levels and Hydrogen Peroxide Levels

  • Oh, Suk-Heung;Park, Yoon-Sick;Yang, Moon-Sik
    • BMB Reports
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    • v.32 no.1
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    • pp.1-5
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    • 1999
  • In order to understand the biological role of calmodulin in plants, transgenic tobacco plants expressing a calmodulin mutant (VU-4 calmodulin, lys to ile-115) gene have been analyzed. SDS-PAGE and Western-blot analyses showed that the foreign calmodulin mutant is stably and highly expressed in the transgenic tobacco plants. The levels of $H_2O_2$were elevated approximately 2-fold in the transgenic plants. Furthermore, the transgenic tobacco plants have more than 6-fold higher levels of NADPH compared to control tobacco plants. The present findings, combined with previous data showing differences in the susceptibility of the transgenic tobacco seeds and normal tobacco seeds to fungal contamination (Oh and Yang, 1996), suggest that the expression of the calmodulin derivative gene in tobacco plants could increase resistance to infection by fungal pathogens.

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