• Analytical Science and Technology
• /
• v.24 no.6
• /
• pp.527-532
• /
• 2011

IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is a well-known $Ca^{2+}$-independent calmodulin (CaM) binding protein, is involved in various cellular functions such as cell proliferation and cell migration. IQGAP1 has four repeated IQ motifs, which are crucial for CaM binding. It has been shown that all four IQ motifs of IQGAP1 can bind to $Ca^{2+}$/CaM, while the third and fourth IQ motifs of IQGAP1 can bind to apoCaM. However, it has not been clear whether the CaM binding of IQ motifs of IQGAP1 was mediated directly or indirectly. In this study, we examined whether the binding between CaM and each IQ motif of IQGAP1 was direct in vitro. As a result, we found that IQ1 motif has a weak $Ca^{2+}$-dependent CaM binding. In contrast, IQ3 has a $Ca^{2+}$-dependent CaM binding. All other motifs have no significant CaM binding. We also found that IQ(2.7-3) and IQ(3.5-4.4) have CaM binding capacity. This finding indicates that IQ motifs of IQGAP1 plays a dynamic role via different motif interactions with $Ca^{2+}$/CaM or proCaM.

• Journal of Applied Biological Chemistry
• /
• v.43 no.1
• /
• pp.5-11
• /
• 2000

Three cDNA clones encoding rice calmodulin (CaM) isoforms (OsCaM-1, OsCaM-2, and OsCaM-3) were isolated from a rice cDNA library constructed from suspension-cultured rice cells treated with fungal elicitor. The coding regions of OsCaM-1 and O.sCaM-2 were 89% homologous at DNA Ievel, whereas the 5' and 3' untranslated regions were highly divergent. The polypeptides encoded by OsCaM-1 and OsCaM-2 was identical except two conservative substitution at position 8 and 75. The coding region of OsCaM-3 was consist of a typical conserved CaM domain and an additional C-terminal extension. The amino acid sequence of conserved CaM domain of OsCaM-3 shared only 86% identity with that OsCaM-1. The OsCaM-3 cDNA is belongs to a novel group of calmodulin gene due to its C-terminal extension of 38 amino acids, a large number of which are positively charged. The extension also contains a C-terminal CaaX-box prenylation site (CVlL). Genomic Southern analysis revealed at least six copies of CaM or CaM-related genes, suggesting that calmodulin may be represented by a small multigene family in the rice geneme. Expression of OsCaM gene was examined through Northern blot analysis. Transcript level of OsCaM-3 was increased by treatment with a fungal elicitor, whereas the OsCaM-1 and OsCaM-2 genes did not respond to the fungal elicitor. The expression of OsCaM-3 gene was remarkable inhibited in the rice cells treated with cyclosporine A, calcinurin inhibitor.

• Molecules and Cells
• /
• v.19 no.2
• /
• pp.268-278
• /
• 2005

We investigated the effect of cytosolic and extracellular $Ca^{2+}$ on $Ca^{2+}$ signals in pancreatic acinar cells by measuring $Ca^{2+}$ concentration in the cytosol($[Ca^{2+}]_c$) and in the lumen of the ER($[Ca^{2+}]_{Lu}$). To control buffers and dye in the cytosol, a patch-clamp microelectrode was employed. Acetylcholine released $Ca^{2+}$ mainly from the basolateral ER-rich part of the cell. The rate of $Ca^{2+}$ release from the ER was highly sensitive to the buffering of $[Ca^{2+}]_c$ whereas ER $Ca^{2+}$ refilling was enhanced by supplying free $Ca^{2+}$ to the cytosol with $[Ca^{2+}]_c$ clamped at resting levels with a patch pipette containing 10 mM BAPTA and 2 mM $Ca^{2+}$. Elevation of extracellular $Ca^{2+}$ to 10 mM from 1 mM raised resting $[Ca^{2+}]_c$ slightly and often generated $[Ca^{2+}]_c$ oscillations in single or clustered cells. Although pancreatic acinar cells are reported to have extracellular $Ca^{2+}$-sensing receptors linked to phospholipase C that mobilize $Ca^{2+}$ from the ER, exposure of cells to 10 mM $Ca^{2+}$ did not decrease $[Ca^{2+}]_{Lu}$ but rather raised it. From these findings we conclude that 1) ER $Ca^{2+}$ release is strictly regulated by feedback inhibition of $[Ca^{2+}]_c$, 2) ER $Ca^{2+}$ refilling is determined by the rate of $Ca^{2+}$ influx and occurs mainly in the tiny subplasmalemmal spaces, 3) extracellular $Ca^{2+}$-induced $[Ca^{2+}]_c$ oscillations appear to be triggered not by activation of extracellular $Ca^{2+}$-sensing receptors but by the ER sensitised by elevated $[Ca^{2+}]_c$ and $[Ca^{2+}]_{Lu}$.

• Horticultural Science & Technology
• /
• v.32 no.2
• /
• pp.178-183
• /
• 2014

This study aimed to evaluate the fruit quality characteristics and incidence of flesh browning in response to air storage at $0{\pm}1^{\circ}C$ a fter controlled atmosphere s torage (CA condition: $O_2$ $2.5{\pm}0.5$%, $CO_2$ $1.5{\pm}0.5$%) at $0{\pm}1^{\circ}C$ in 'Fuji' apples (Malus domestica Borkh.). The storage system was performed as followed: air storage for one month, CA storage 4 months + air storage 3 months ( CA 4M + A ir 3M), CA storage 5 m onths + air storage 2 months ( CA 5M + Air 2M) a nd C A storage 6 m onths + air storage 1months (CA 6M + Air 1M), while the control fruits were stored at CA storage for 8 months right after harvest. The incidence of flesh browning ranged from 17.1% to 30.2% during CA storage but not detected under the treatments of CA 4M + Air 3M and CA 5M + Air 2M. The respiration rate was not affected by storage treatments for 6M while the respiration rate was lower in the treatments of CA 4M + Air 3M and CA 5M + Air 2M than the other storage treatments after 7 months. Ethylene production and internal ethylene concentration were lowest in rapid CA storage and increased with a decreasing CA storage duration. Therefore, the results indicate that CA 5M + Air 2M storage treatment should be recommended to maintain the fruit quality and reduce the risk development of flesh browning rather than typical CA storage in 'Fuji' apples.

• YAKHAK HOEJI
• /
• v.41 no.6
• /
• pp.773-781
• /
• 1997

The activity of $Ca^{2+}$calmodulin (CaM)-dependent protein kinase Ia (CaM kinase Ia) is shown to be regulated through direct phosphorylation by CaM kinase I kinase (CaMK IK). In the present study, three distinct CaMKIK peaks were separated from Q-Sepharose colunm chromatography of pig brain homogenate using a Waters 650 Protein Purification System. The purified CaMKIK from the major peak potently and rapidly enhanced CaM kinase Ia activity, reaching a maximal stimulation within 2min at the concentrations of 12-15nM. The activated state of CaM kinase Ia is characterized by a markedly enhanced $V_{max}4 as well as significantly decreased $K_m\;and\;K_a$ values toward peptide substrate and CaM, respectively. These observations suggest the activation process of CaM kinase Ia. The phosphorylation of CaM kinase Ia by CaMKIK may induce its conformational change responsible for the alterations in the kinetic properties, which ultimately leads to the rapid enzyme activation.

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.2
• /
• pp.101-110
• /
• 2004

Voltage-sensitive release mechanism was pharmacologically dissected from the $Ca^{2+}-induced\;Ca^{2+}\;release$ in the SR $Ca^{2+}$ release in the rat ventricular myocytes patch-clamped in a whole-cell mode. SR $Ca^{2+}$ release process was monitored by using forward-mode $Na^+-Ca^{2+}$ exchange after restriction of the interactions between $Ca^{2+}$ from SR and $Na^+-Ca^{2+}$ exchange within micro-domains with heavy cytosolic $Ca^{2+}$ buffering with 10 mM BAPTA. During stimulation every 10 s with a pulse roughly mimicking action potential, the initial outward current gradually turned into a huge inward current of $-12.9{\pm}0.5\;pA/pF$. From the inward current, two different inward $I_{NCX}s$ were identified. One was $10\;{\mu}M$ ryanodine-sensitive, constituting $14.2{\pm}2.3%$. It was completely blocked by $CdCl_2$ (0.1 mM and 0.5 mM) and by $Na^+-depletion$. The other was identified by 5 mM $NiCl_2$ after suppression of $I_{CaL}$ and ryanodine receptor, constituting $14.8{\pm}1.6%$. This latter was blocked by either 10 mM caffeine-induced SR $Ca^{2+}-depletion$ or 1 mM tetracaine. IV-relationships illustrated that the latter was activated until the peak in $30{\sim}35\;mV$ lower voltages than the former. Overall, it was concluded that the SR $Ca^{2+}$ release process in the rat ventricular myocytes is mediated by the voltage-sensitive release mechanism in addition to the $Ca^{2+}-induced-Ca^{2+}\;release$.

• Korean Journal of Plant Tissue Culture
• /
• v.21 no.6
• /
• pp.363-367
• /
• 1994

In an effort to investigate the role of calmodulin (CaM) as a modulating molecule in the signal transduction system in plant cells, we established methods for introduction of purified CaM into cultured soybean cells. CaM was purified from bovine testis, and was labelled with fluorescein isothiocyanate (FITC). Suspension -cultured cells were healed with saponin (0.1 mg/mL) to permeabilize the plasma membrane and coincubated with FITC-CaM complex. Saponin pretreatment was found to increase the fluorescence in the suspension cultured cells, indicating that the FITC-CaM complex could be incorporated into the cytoplasm. Optimal conditions for introducing FITC-CaM complex into protoplasts by electroporation were established with various electric pulses. With increasing field strength, the fluorescence in the protoplase was increased, while the viability of the protoplase decreased. FITC-CaM complex was successfully introduced into the protoplasts by electroporation and the amount of FITC-CaM complex in the protoplase was estimated.

• The Korean Journal of Physiology
• /
• v.20 no.1
• /
• pp.31-41
• /
• 1986

The sufficient myoplasmic $Ca^{++}$ to react with the contractile proteins is necessary to induce contraction of a cardiac muscle. These $Ca^{++}$ for the production of muscle contraction are supplied from the three recognized $Ca^{++}$ sources; internal $Ca^{++}$ release via the sarcoplasmic reticulum(SR), $Ca^{++}$ influx through a gated Ca-channel in the membrane as a Isi, and $Ca^{++}$ transport by the mechanism of Na/ca exchange. However, it is still controversial which $Ca^{++}$ sources act as a main contributor for myoplasmic $Ca^{++}$, Therefore, this study was undertaken in order to examine the $Ca^{++}$ sources for the contraction of frog ventricle. There is evidence that the SR is sparse in frog ventricular fibers, and that T-tubules are absent. Isolated ventricular strips of frog, Rana nigromaculata, were used in this experiment. Isometric tension was recorded by force transducer, and membrane potentials of ventricular muscles were measured through the intracellular glass microelectrodes, which were filled with 3M KCI and had resistance of $30{\pm}50M{\Omega}$. All experiments were performed at room temperature in a tris·buffered Ringer solution which was aerated with 100% $O_2$. Isotonic high K, low Na solution was used to induce K-contracture, K-contracture appeared at the concentration of 20 to 30mM-KCI and was potentiated in parallel with the increase in KCI concentration. The contracture had two components: an initial rapid phasic and a subsequent slow tonic contractile responses. Membrane Potentials measured at normal Ringer solution(2.5mM KCI) was -90 to -100 mV, and decreased linearly as the KCI concentration increased; -55mV at 20mM.KCI, -45mV at 30 mM.KCI, -30 mY at 50 mM.KCI, and -12 mV at 100 mM.KCI. K-contracture was evoked firstly at the membrane potential of -45 mV. The contracture was potentiated by the increase of bathing extracellular $Ca^{++}$ concentration. However, in the absence of $Ca^{++}$ the contracture was almost not induced by 50 mM.KCI solution. Caffeine(20mM) in normal Ringer solution, which is known to release $Ca^{++}$ from SR without substantial effects on the $Ca^{++}$ fluxes across the surface membrane, did not affect membrane potential and also not initiate contracture, but the caffeine in 20 mM-KCI Ringer solution produced a contracture. Above results suggest that the main $Ca^{++}$ source for the K·contracture of frog ventricle is $Ca^{++}$ influx through the voltage-dependent Ca-channel, and that in the K-contracture at the concentration of 100 mM-KCI, the mechanism of Na/ca exchange also partly contributs, in addition to the $Ca^{++}$ influx.

• Korean Journal of Soil Science and Fertilizer
• /
• v.32 no.4
• /
• pp.333-343
• /
• 1999

To investigate the relationship between the translocation and distribution the monovalent K and Na and the divalent Sr and Ca, the natrophile and calcitrophic plant sugar beet (Beta vulgaris L.) was used. Strontium uptake and distribution are concentration and growth stage dependent. The highest Ca content occurred in the treatment of 4 : 1 mM ratio of Ca to Sr, while the highest Sr content in old leaves in the presence of 1 mM Ca and 4 mM Sr. The addition of low concentration of Sr stimulates Ca-uptake. Reversely. Sr-uptake is highest in the presence of 1 mM Ca. This result may be an antagonistic effect between Ca and Sr. The ratios of Mg to Ca and Sr are satisfactorily presented by the regression analysis. The sum of Sr and Ca contents are most significant linear to the ratio of Mg to one, showing a negative correlation. This result implies that the absorption of Mg and Ca or Sr is antagonistic. In the presence of only 5 mM Sr, K and Na-uptakes increases, while Sr in the presence of Ca does not affect the change in the K and Na assimilation and their ratios. The ratios of K to Na is also not changed. A little addition of Sr could more effectively retain the chlorophyll loss while only in the presence of Sr, the chlorophyll levels are considerably reduced.

• Korean Journal of Animal Reproduction
• /
• v.26 no.1
• /
• pp.33-39
• /
• 2002

This study was conducted to investigate the effect of $Ca^{2+}$ concentration in fusion medium on the fusion, nuclear morphology and the development of bovine somatic cell nuclear transfer embryos. Bovine skin cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion medium containing with 0.05 to 1.0 mM Cacl$_2$. Nuclear transfer embryos were activated with a combination of A23187 and cycloheximide. Nuclear transfer embryos were fixed at 3 h after fusion or cultured for 7 ~8 days. Fusion rate was significantly (P＜0.01) increased by increasing the $Ca^{2+}$ concentrations in the fusion medium from 0.05 mM (56.6%) to 0.5 mM (50.1%) and 1.0 mM (84.3%). More than 80% of reconstituted embryos underwent premature chromosome condensation (PCC) with 0.05, 0.1 mM CaCl$_2$, whereas 54.5% and 59.3% of embryos formed pronucleus (PN) directly without PCC in the 0.5 and 1.0 mM CaCl$_2$, groups. Blastocyst formation rates were significantly (P＜0.05) different between 0.1 mM and 1.0 mM CaCl$_2$groups. From the present result, it is suggested that the elevated $Ca^{2+}$ concentrations in fusion medium can enhance the fusion and blastocyst formation rates of bovine nuclear transfer embryos.bryos.