• Title, Summary, Keyword: Calmodulin methyltransferase

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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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Effect of Moutan Cortex Radicis on gene expression profile of differentiated PC12 rat cells oxidative-stressed with hydrogen peroxide (모단피의 PC12 cell 산화억제 효과 및 neuronal 유전자 발현 profile 분석에 대한 연구)

  • Kim Hyun Hee;Rho Sam Woong;Na Youn Gin;Bae Hyun Su;Shin Min Kyu;Kim Chung Suk;Hong Moo Chang
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.17 no.2
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    • pp.529-541
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    • 2003
  • Yukmijihwang-tang has been widely used as an and-aging herbal medicine for hundred years in Asian countries. Numerous studies show that Yukmijihwangtang has anti-oxidative effect both in vivo and in vitro. It has been reported that Moutan Cortex Radicis extract (MCR) was the most effective herb in Yukmijihwang-tang on undifferentiated PC12 cells upon oxidative-stressed with hydrogen peroxide. The purpose of this study is to; 1) evaluate the recovery of neuronal damage by assessing the anti-oxidant effect of MCR on PC12 cells differentiated with nerve growth factor (NGF), 2) identify candidate genes responsible for anti-oxidative effect on differentiated PC12 cells by oligonucleotide chip microarray. PC12 cells, which were differentiated by treating with NGF, were treated without or with hydrogen peroxide in the presence or absence of various concentration of MCR. Cell survival was determined by using MTS assay. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2DCFDA assay The viability of cells treated with MCR was significantly recovered from stressed PC12 cell. In addition, wide rage of concentrations of MCR shows dose-dependent inhibitory effect on ROS production in oxidative-stressed cells. Total RNAs of cells without treatment(Control group), only treated with H₂O₂ (stressed group) and treated with both H₂O₂ and of MCR (MCR group) were isolated, and cDNAs was synthesized using oligoT7(dT) primer. The fragmented cRNAs, synthesized from cDNAs, were applied to Affymetrix GeneChip Rat Neurobiology U34 Array. mRNA of Calcium/calmodulin-dependent protein kinase II delta subunit(CaMKII), neuron glucose transporter (GLUT3) and myelin/oligodendrocyte glycoprotein(MOG) were downregulated in Stressed group comparing to Control group. P2X2-5 receptor (P2X2R-5), P2X2-4 receptor (P2X2R-4), c-fos, 25 kDa synaptosomal attachment protein(SNAP-25a) and GLUT3 were downregulated, whereas A2 adenosine receptor (A2AR), cathechol-O-methyltransferase(COMT), glucose transporter 1 (GLUT1), EST223333, heme oxygenase (HO), VGF, UI-R-CO-ja-a-07-0-Ul.s1 and macrophage migration inhibitory factor (MIF) were upregulated in MCA group comparing to Control group. Expression of Putative potassium channel subunit protein (ACK4), P2X2A-5, P2X2A-4, Interferon-gamma inducing factor isoform alpha precursor (IL-18α), EST199031, P2XR, P2X2 purinoceptor isoform e (P2X2R-e), Precursor interleukin 18 (IL-18) were downregulated, whereas MOO, EST223333, GLUT-1, MIF, Neuronatin alpha, UI-R-C0-ja-a-07-0-Ul.s1, A2. adenosine receptor, COMT, neuron-specific enolase (NSE), HO, VGF, A rat novel protein which is expressed with nerve injury (E12625) were upregulated in MCR group comparing to Stressed group. The results suggest that decreased viability and AOS production of PC12 cell by H₂O₂ may be, at lease, mediated by impaired glucose transporter expression. It is implicated that the MCR treatment protect PC12 cell from oxidative stress via following mechanisms; improving glucose transport into the cell, enhancing expression of anti-oxidative genes and protecting from dopamine cytotoxicity by increment of COMT and MIF expression. The list of differentially expressed genes may implicate further insight on the action and mechanism behind the anti-oxidative effects of herbal extract Moutan Cortex Radicis.