Mechanism for the change of Cytosolic Free Calcium Ion Concentration by Irradiation of Red Light in Oat Cells

Chae, Quae,Han, Bong Deok,Park, Moon Hwan,Lee, Sang Lyul 생화학분자생물학회 1979 BMB Reports Vol.28 No.6

In our previous studies (Chae et al., 1990; Chae et ol., 1993), we found that a phytochrome signal was clearly connected with the change in cytosolic free Ca^(2+) concentrntion ([Ca^(2+)]_i) in oat cells. It was determined that the [Ca^(2+)]_i change occured both by mobilization out of the intracellular Ca^(2+) store and by influx from the medium. The specific aim of this work is to elucidate the processes connecting Ca^(2+) mobilization and influx. The cells treated with thapsigargin (increasing [Ca^(2+)]_i by inhibition of the Ca^(2+)-ATPase in the calcium pool) in the presence of external Ca^(2+) showed the same increasing pattern (sustained increasing shape) of [Ca^(2+)]_i as that measured in animal cells. Red light irradiation after thapsigargin treatment did not increase [Ca^(2+)]_i. These results suggest that thapsigargin also acts specifically in the processes of mobilization and influx of Ca^(2+) in oat cells. When the cells were treated with TEA (K^+ channel blocker), changes in [Ca^(2+)]_i were drastically reduced in comparison with that measured in the absence of TEA. The results suggest that the change in [Ca^(2+)]_i due to red light irradiation is somehow related with K^+ channel opening to change membrane potential. The membrane potential change due to K^+ influx might be the critical factor in opening a voltage-dependent calcium channel for Ca^(2+) influx.

Identification of Phospholipase C Activated by GTPγS in Plasma Membrane of Oat Cell

Chae, Quae,Park, Moon Hwan,Kim, Hyae Kyeong 생화학분자생물학회 1978 BMB Reports Vol.28 No.5

In order to investigate whether phospholipase C (PLC) activity in oat cells is regulated by Gprotein, we have characterized PLC in plasma membranes of oat tissues. To identify the purified plasma membrane, K^+-stimulated, Mg^(2+)-dependent ATPase activity was measured. The activity of ATPase was shown to be proportional to the concentration of membrane protein. To examine the PLC activity regulated by G-protein, we used the inside-out and outside-out plasma membrane mixture isolated from the oat cells. The plasma membrane mixture showed higher PLC activity than the one of the outside-out plasma mem brane. This suggests that PLC activity is located at the cytoplasmic surface of plasma membrane. PLC activity in plasma membrane mixture was dependent on Ca^(2+) with maximum activity at 100 μM Ca^(2+) and it was inhibited by 1 mM EGTA. Using Sep-pak Accell^(TM) Plus QMA chromatography, we found that inositol 1,4,5-trisphosphate (IP₃) was produced in the presence of 10 μM Ca^(2+). The PLC activity in the membrane was enhanced by an activator of G-protein (GTPγS) and not by an inhibitor (GDPβS). This indicates that a G-protein is involved in the activation of PLC in the plasma membrane of oat cells.

Light Effects on the membrane Potential in Oat Cells

Chae, Quae,Kim, Kwan Bae,Park, Moon Hwan 생화학분자생물학회 1978 BMB Reports Vol.28 No.5

One of the reaction pathways in light-invoked signal transduction can be initiated through ion fluxes across the plasma membrane in higher plants. We isolated protoplasts from oat coleoptile and examined the effects of light on the membrane potential using a membrane potential-sensitive fluorescent probe (bisoxonol). Both red and far-red light initially induced a hyperpolarization in oat cells. Red light-induced hyperpolarization was effectively dissipated by 100 mM K^+, but the hyperpolarization induced by far-red light was not depolarized by any of the cations (K^+, Ca^(2+), Li^+, Na^+) tested. The depolarization induced by red light and K^+ was inhibited by 200 mM TEA, which is a K^+ channel blocker. These results suggest that K^+ influx through the inward K^+ channel may be a depolarization path in the phytochrome-mediated signal transduction.

Oligomerization State of the Plasma Membrane Proteolipid Apoprotein Purified from the Bovine Kidney, Probed by the Fluorescence Polarization

Chae, Quae,Nam, Sang-Rye Korean Chemical Society 1988 Bulletin of the Korean Chemical Society Vol.9 No.4

In order to investigate the oligomerization state of the plasma membrane proteolipid apoprotein purified from the bovine kidney, fluorescence polarization experiment was carried out in the two different solvent systems, i.e., water and organic solvent(chloroform-methanol). The molecular volumes of the proteins estimated from the Perrin equation, were to be 45,258$A^3$ and 17,608$A^3$ in water and organic solvent, respectively. These values indicate that a trimerization is possibly occurring in the aqueous environment. As an auxiliary experiment for the calculation of the molecular volume using Perrin equation, fluorescence quenching constants ($K_q$) with the quencher acrylamide and fluorescence lifetimes (${\tau}_F$) of the intrinsic fluorophore tryptophan residue were estimated in the two different solvent systems. $K_q$ in water was 18.21$M^{-1}$ and it was 46.24$M^{-1}$ in organic solvent. Fluorescence lifetimes of tryptophan residue were calculated to be 2.80 nsec. in water and 3.81 nsec. in organic solvent, respectively.

Identification and Characterization of Phytochrome - Regulated Phospholipase D in Oat Cells ( Avena sativa L . )

Chae, Quae,Park, Moon Hwan,Park, Cheon 생화학분자생물학회 1982 BMB Reports Vol.29 No.6

The activation of phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline in plants as well as animals. To determine the presence of PLD in oat cells, we prepared inside-out plasma membrane and cytosolic fractions from oat tissues. PLD activities in both cytosol and plasma membrane were detected by ion chromatography method. The activity of PLD in plasma membrane was dependent upon Ca^(2+) concentration and was heat stable. To investigate whether G-protein couples to PLD, the effects of GTPYS and GDPβS on the PLD activity were measured. PLD activity was dramatically increased 300∼400% in the presence of 50 qM GTPYS but not in the presence of 50 μM GDPβS. These results indicate that G-protein may be involved in regulation of PLD activity. To identify whether PLD is regulated by red light receptor, phytochrome, we irradiated red, far-red, or red/far-red/red light on oat protoplasts. PLD activity has increased 5-fold and 3-fold by treatment with red light and red/far-red/red light, respectively. In contrast, irradiation with far-red light had little or no effect on PLD activity. These results suggest that phytochrome regulates PLD activity through activation of G-protein in oat cells.

Protein Phosphatase and Kinase Involved in the Phytochrome - mediated Signal Transduction in Oat Cell

Quae Chae 생화학분자생물학회 1991 생화학분자생물학회 춘계학술발표논문집 Vol.- No.-

Mechanism for the Change of Cytosolic Free Calcium Ion Concentration by Irradiation of Red Light in Oat Cells

Han, Bong-Deok,Lee, Sang-Lyul,Park, Moon-Hwan,Chae, Quae Korean Society for Biochemistry and Molecular Biol 1995 Journal of biochemistry and molecular biology Vol.28 No.6

In our previous studies (Chae et al., 1990; Chae et a1., 1993), we found that a phytochrome signal was clearly connected with the change in cytosolic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in oat cells. It was determined that the $[Ca^{2+}]_i$ change occured both by mobilization out of the intracellular $Ca^{2+}$ store and by influx from the medium. The specific aim of this work is to elucidate the processes connecting $Ca^{2+}$ mobilization and influx. The cells treated with thapsigargin (increasing $[Ca^{2+}]_i$ by inhibition of the $Ca^{2+}$-ATPase in the calcium pool) in the presence of external $Ca^{2+}$ showed the same increasing pattern (sustained increasing shape) of $[Ca^{2+}]_i$ as that measured in animal cells. Red light irradiation after thapsigargin treatment did not increase $[Ca^{2+}]_i$ These results suggest that thapsigargin also acts specifically in the processes of mobilization and influx of $Ca^{2+}$ in oat cells. When the cells were treated with TEA ($K^+$ channel blocker), changes in $[Ca^{2+}]_i$ were drastically reduced in comparison with that measured in the absence of TEA. The results suggest that the change in $[Ca^{2+}]_i$ due to red light irradiation is somehow related with $K^+$ channel opening to change membrane potential. The membrane potential change due to $K^+$ influx might be the critical factor in opening a voltage-dependent calcium channel for $Ca^{2+}$ influx.

분자 분광학적 방법에 의한 소 심장 Proteolipid 의 구조적 연구

채쾌,경기열 생화학분자생물학회 1991 BMB Reports Vol.19 No.2

UV-VIS difference and fluorescence spectroscopic techniques were employed to probe the structural changes of the membrane proteolipid with the different solvent systems. The purified proteolipid from bovine heart was dissolved in two different solvent systems (water, organic solvent: chloroform-methanol) and various optical spectra with these solvent systems were measured. The number of perturbed tryptophan residues were estimated to be 1.8 in water and 1.2 in organic solvent, respectively. In addition, the higher fluorescence quenching effects on tryptophan residues and/or ANS by acrylamide were observed in water than the one in organic solvent system. The two results strongly support the hydrophobic interaction needed for the oligomerization of the proteolipid in water environment. However, a certain elucidation for the oligomerization state of this protein can not be provided at the moment.

The Effect of Acidic pH on the Spectral Properties of Bacteriorhodopsin

채쾌,Quae Chae Korean Chemical Society 1979 대한화학회지 Vol.23 No.5

Halobacterium halobium으로부터 분리 정제된 퍼플멤브레인을 7.5% 폴리아크릴아미드겔에 혼합시켰다. 이 겔을 사용하여 pH 변화에 따른 흡수 스펙트라와 원편광 이색성스펙트라를 얻었다. pH 7.0에서 보여준 이들 스펙트라의 성질들은 수용액내에 부유하고 있는 퍼플멤브레인으로부터 얻은 것들과 동일하였다. pH 2.7에서는 최대흡광도를 605nm에서 나타내었으며 pH 0.8에서는 565nm에서 보여주었다. 광에 노출되지 않았던 겔의 경우는 광에 노출되었던 겔과는 달리 등흡광점을 보여주었다. pH 2.7과 pH 0.8에서 측정한 원편광 이색성스펙트라는 pH 7.0에서 보여준 bilobed형을 유지하였으며 UV영역에서 분자편광도나 스펙트라의 모양도 pH에 따라 큰 영향을 받지 않았다. pH 2.7에서 생성된 bR605acid 가 퍼플멤브레인의 정상 광화학 순환기 중간 생성물인 $O^{640}$와 유사한 성질을 가진종이 아닐까 추측된다. Purple membrane from Halobacterium halobium was incorporated into 7.5% polyacrylamide gels. Absorption and circular dichroic spectra of purple membrane incorporated with gels were obtained at various pH. The spectra of these gels measured at pH 7.0 were essentially identical with those obtained in the aqueous suspension of purple membrane. Acid titration of the gels showed the transition to a form absorbing at 605nm $(bR_{605}^{acid}$) at pH 2.6, and to a second form at 565nm $(bR_{565}^{acid})$ at pH 0.8. Dark-adapted gels showed an isosbestic point for each transition whereas light-adapted gels did not. Visible CD spectra of $bR_{570}^{LA},\;bR_{305}^{acid}\;and\;bR_{565}^{acid}$ all showed the typical bilobed pattern. CD spectra measured at UV wavelength region were also independent of the variation of pH in terms of molar ellipticity and spectral shape. The protonated species $bR_{605}^{acid}$ may be one of the intermediates formed during the normal photochemical cycle of purple membrane. Most probably, the species $bR_{605}^{acid}$ is considered to be $O^{640}$ in the cycle.

잎담배 성분중 갈색고분자 물질의 분리정제 및 열분해에 관한 연구

채쾌,박지창,Chae, Quae,Park, Ji-Chang 한국연초학회 1980 한국연초학회지 Vol.2 No.1

건조잎담배로부터 단백질로 간주되는 고분자 물질의 gel filtration column chromatography(Sephadex G-75), 투석 그리고 흡착 chromatography의 일종인 brushite column chromatography분리를 시도한 결과, 짙은 갈색의 고분자 물질을 얻었다. Sephadex column에 의한 분리 profile를 보면 분자량이 상이한 두종류의 갈색고분자 물질이 존재함을 확인하였고 brushite column의 분리 profile에 의하면 분자의 전자구조가 다른 두 종류의 고분자가 있음이 나타났다. Burley와 Hicks의 경우 단백질 분해효소에 의한 분해효과를 측정해본 결과, 가장 큰 분해효과를 보인 효소는 chymotrypsin으로 Burley에서 16-30%수준까지 분해현상을 나타내 주었으며 Hicks인 경우 38-57%까지 감소현상을 보여 주었다. Pepsin처리효과는 chymotrypsin처리구와 비슷한 수준을 보였으나 trypsin경우는 매우 낮은 분해현상을 나타냈다. 단백질 분해효소로 처리된 시료의 sephadex column분리 profile을 살펴보면, 분자량이 보다 큰 fraction의 경우 peak가 거의 완전하게 사라짐을 관찰할 수 있으나, 작은 분자량Peak는 약간의 감소현상만을 보여 주었다. 투석후의 갈색 고분자물질을 $300^{\circ}C$에서 연소시킨 경우, 연소전에 관찰할 수 없었던 강한 형광성 물질이 생성되었는데 TLC에 의한 분리후, 이 형광성 물질끈 흡수 스펙트라를 측정해 본 결과, 최대흡수 파장이 265. 275nm(benzene용매)로 나타났으며 스펙트럼 끈 모양이 polynuclear aromatic hydrocarbon(PAH) 계열 화합물의 것과 유사함을 보여 주었다. Gel filtration column chromatography (Sephadex G-75), dialysis an d Brushite column chromatography were carried out to separate the brown pigmented macromolecule from water extracts of the cured leaf tobaccos. The two distinct macromolecules having different molecular weight were separated by the Sephadex column chromatography. Brushite also separated two different species of macromolecules which might have different electronic structures. According to the enzymatic degradation of protein in Burley and Hicks, chymotrypsin showed the best degradation ratio, ie., 16-30% in Burley and 38-57% in Hicks. Similar effect was observed with pepsin. However, very low effect of degradation was revealed with trypsin. The sample treated with the proteolytic enzymes revealed the disappearance of the first peak and the slight decrease of the 2nd peak height in the separation profile of Sephadex. After dialysis, the brown pigmented macromolecule was pyrolyzed at $300^{\circ}C$ and the strongly fluorescent components not identified before pyrolysis were detected with TLC separation. Absorption spectrum of these fluorescent compounds was monitored in benzene and the absorption maxima at 265nm and 275 nm were obtained. Considering absorption maxima and shape of the spectrum, those fluorescent compounds seem to be PAH derivatives.