• 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.

• BMB Reports
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• v.28 no.4
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• pp.306-310
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• 1995

A Mono Q HR 5/5 anion-exchange column with a FPLC system was used to separate exogenously expressed calmodulin from endogenous tobacco calmodulins. Transgenic tobacco calmodulins were purified by fractionation with ammonium sulfate, precipitation with sulfuric acid and hydrophobic chromatography on phenyl-Sepharose CL-4B. The purified calmodulins were chromatographed in the FPLC using the column. This method was selected because of the slight differences in the net charge of foreign and endogenous plant calmodulins due to amino acid sequence differences. By this approach, the exogenously expressed calmodulin was isolated from endogenous tobacco calmodulins. The isolated calmodulin was characterized by amino acid composition analysis as well as methylation analysis.

• Archives of Pharmacal Research
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• v.28 no.2
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• pp.159-163
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• 2005

The effects of sedative peptide alkaloids from Zizyphus species on calmodulin- dependent protein kinase II were investigated. Protein kinase II activity was assayed on the basis of its ability to activate tryptophan 5-monooxygenase as its substrate in the presence of calmodulin. All thirteen alkaloids tested were stronger inhibitors than chlorpromazine ($IC_50,\;98{\mu}M$) on calmodulin-dependent protein kinase II. Among them, the most potent inhibitor was daechuine S27 ($IC_{50},\;2.95{\mu}M$), which was stronger than pimozide ($IC_{50},\;15.0{\mu}M$).

• Journal of Yeungnam Medical Science
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• v.7 no.1
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• pp.39-50
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• 1990

The one event on signalling mechanism is the cleavage by adenyl cyclase of ATP into second messenger, cyclic AMP. The other transfer system of inositol metabolism. it is widely recognized that hydrolysis of the minor membrane lipid phosphoinositide bisphosphate($PIP_2$) initiated by occupation of certain receptors and catalyzed by phospholipase C, lead to toe generation of the two intracellular messengers, inositol triphosphate($IP_3$) and diacylglycerol(DG). $IP_3$ is converted to inositol tetrakisphosphate($IP_4$) by $IP_3$ kinase. In the present study, it is that purification of calmodulin is used by phenyl-Sepharose CL-4B chromatography. it's molecular weigh, 17.000 in SDS-polyacrylamide gel electrophoresis. In order to observe the affinity between calmodulin (CaM)-Affigel 15 and $IP_3$ kinase, and isolated $IP_3$ kinase, was applied in CaM-Affigel with $Ca^{2+}$ equilibirum buffer and EGTA equilibirum buffer. We compared with binding and elution effect of $IP_3$ kinase in several condition of buffer. In affinity of binding. $Ca^{2+}$ equilibrium buffer was in the most proper condition. and elution, CaM/$Ca^{2+}$ buffer(CE1 10.36, CE2 12. 76pM/min/mg of protein) was effected much more than EGTA buffer(E2 1.48, E3 2.43pM/min/mg of protein), but CaM/$Ca^{2+}$ stimulate the activity of $IP_3$ kinase. And then, several detergents such as sodium deoxycholate, tween 20. cholic acid, polyethylene glycol, chaps were applied. The 0.2% chaps buffer(E2 23.19, E3 8.05pM/min/mg of protein) was the most effective in elution of $IP_3$ kinase.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.4
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• pp.234-237
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• 2007

Calmodulin (CaM) is a $Ca^{2+}$-binding protein essential for biological functions mediated through $Ca^{2+}$-dependent mechanism. A cDNA clone for CaM was isolated from a cDNA library of olive flounder Paralichthys olivaceus. The CaM cDNA concists of 782 bp and encodes a polypeptide of 149 amino acids with four $Ca^{2+}$-binding motifs EF-hands (EF-I, EF-II, EF-III, and EF-IV). The deduced amino acid sequence of CaM shows 97-100% amino acid sequence identity to other CaM sequences. Semi-quantitative PCR analysis revealed that the CaM transcription was began during early development and the CaM mRNA is expressed highly in brain and intestine, and moderately in kidney, gill, and eye of healthy olive flounder. Taken together, CaM may be necessary for early olive flounder development and that it may have a part in homeostasis.

• Applied Biological Chemistry
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• v.43 no.4
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• pp.231-235
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• 2000

The changes of calmodulin levels, calmodulin-binding proteins, and $Ca^{2+}$/calmodulin-dependent glutamate decarboxylase during the growth of tobacco suspension cells were investigated. Tobacco cells exhibited a typical growth curve, including an exponential growth phase between 3 and 5 days after inoculation, and an apparent stationary phase occurring after 5 day. Although slight changes were observed from sample to sample, calmodulin protein levels remained similar during the phases of culture growth. Several $Ca^{2+}-dependent$ calmodulin-binding proteins including 56, 46, 36, and 32-kDa proteins were detected in tobacco cell extracts. The 56-kDa protein was identified as glutamate decarboxylase by Western-blot analysis using an anti-GAD monoclonal antibody. The levels of GAD protein and the specific activity of GAD enzyme were highest during the middle exponential phase of the culture growth cycle. These data suggest that $Ca^{2+}$/calmodulin-dependent glutamate decarboxylase is modulated during the growth of tobacco suspension cells.

• Journal of Chest Surgery
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• v.22 no.6
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• pp.901-913
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• 1989

This study was investigated under the postulation that activation of intracellular calcium- calmodulin complex during ischemia-reperfusion leads to myocardial injury. The protective effects of calcium channel blocker, diltiazem and calmodulin inhibitors, trifluoperazine, flunarizine and calmidazolium from ischemic injury in rat hearts were observed by using Langendorff apparatus when the antagonists were infused for 3 min in the beginning of ischemia. Thereby, an increase in resting tension developed during 30-min ischemia was analyzed with regard to [1] the degree of cardiac functional recovery following 60-min reperfusion, [2] changes in biochemical variables evoked during 30-min ischemia. The results obtained were as follows: l. In the ischemic group, the resting tension was increased by 4.1*0.2 g at 30-min ischemia. However, the increase in resting tension was markedly reduced not only by pretreatment with diltiazem [3.3 p M] but also with calmodulin inhibitors, trifluoperazine [3.3 p M], flunarizine [0.5 p M] and calmidazolium [0.5 p M], respectively. 2. Recovery of myocardial contractility, +dF /dt and coronary flow were much reduced when evoked by reperfusion in the ischemic group. These variables were significantly improved either by pretreatment with diltiazem or with calmodulin inhibitors. 3. The resting tension increment evoked during ischemia was significantly inversely correlated with the degree of cardiac function recovered during reperfusion. 4. Following 30-min ischemia, the production of malondialdehyde and release of lysosomal enzyme were much increased in association with a decrease in creatine kinase activity. 5. The increases in malondialdehyde production and release of free lysosomal enzyme were suppressed by pretreatment with calmodulin inhibitors as well as diltiazem. Likewise, the decrease of creatine kinase activities was prevented by these calcium antagonists. With these results, it is indicated that a increase in resting tension observed during ischemia has an inverse relationship to the cardiac function recovered following reperfusion, and further, the later may be significantly dependent on the degree of biochemical alterations occurred during ischemia such as decrease in creatine kinase activity, increased production of malondialdehyde and increased release of free lysosomal enzyme. Thus it is concluded that calmodulin plays a pivotal role in the process of ischemic injury.

• Archives of Pharmacal Research
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• v.24 no.3
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• pp.202-206
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• 2001

The effects of various sedative cyclopeptides and peptide alkaloids from the Zizyphus species on calmodulin-dependent $Ca^{2+}$ -ATPase and phosphodiesterase were Investigated. Calmodulin-induced activation of $Ca^{2+}$-ATPase was strongly inhibited by sanjoinine-A dialdehyde (IC_{50}$, 2.3$\mu\textrm{m}$), -Ah1 (IC_{50}$, 4.0$\mu\textrm{m}$), -A (IC, 4.6$\mu\textrm{m}$), and -G2 (IC_{50}$, 7.2$\mu\textrm{m}$), while calmodulin-induced activation of phosphodiesterase was strongly inhibited by both deachuine- S10 (IC_{50}$, 4.9$\mu\textrm{m}$) and sanjoinine-D (IC_{50}$, 9.0$\mu\textrm{m}$). The inhibitory activity of the various cyclopeptides and peptide alkaloids on $Ca^{2+}$-ATPase was found to correlate well with their Sedative activity.

• BMB Reports
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• v.38 no.4
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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.

• Analytical Science and Technology
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• v.8 no.3
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• pp.221-227
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• 1995

The fiber optic fluorosensor that shows a specific selectivity for calcium ion is studied. This sensor employs protein Calmodulin(CaM) which forms a fluorescent chelate with $Ca^{2+}$. A dialysis membrane is used to entrap a fluorescein isothiocyanate-labeled CaM solution at the common end of a bifurcated fiber optic bundle. The sensing mechanism of this sensor is based on the shifts in the fluorescence spectrum of metal-calmodulin complexes which FCaM forms a chelate with $Ca^{2+}$. Upon binding with $Ca^{2+}$, CaM undergoes a conformational change which induces a change in the fluorescence of FCaM. This change in fluorescence signal which is measured by photomultiflier tube is related to the concentration of $Ca^{2+}$ for calibration curve. Detection limit for $Ca^{2+}$ and the interference effects by $Mg^{2+}$, $Eu^{3+}$ and $La^{3+}$ for this sensor are studied. Response time and life time for this fluorosensor are also investigated.