• BMB Reports
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• 제40권5호
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• Journal of Animal Science and Technology
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• 제61권2호
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• pp.87-97
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• 2019

Phytate induced excessive mineral excretion through poultry litter leads to poor performance and environmental pollution. Exogenous microbial phytase supplementation to poultry diets reduce the environmental excretion of nutrient and improve bird's performance. However, excessive dietary sodium (Na) level may hinder the phytase-mediated phytate hydrolysis and negate the beneficial effects of phytase. Therefore, this experiment was conducted to investigate the effects of different concentration dietary Na on phytase activity and subsequent impact on broiler performance, bone mineralisation and nutrient utilisation. In this study, six experimental diets, consisting of three different levels of Na (1.5, 2.5, or 3.5 g/kg) and two levels of microbial phytase (0 or 500 U/kg) were formulated by using $3{\times}2$ factorial design. The six experimental diets were offered to 360 day-old Ross 306 male chicks for 35 days, where, each experimental diet consisted of 6 replicates groups with 10 birds. Along with growth performance, nutrient utilization, intestinal enzyme activity, dry matter (DM) content of litter and mineral status in bone were analysed. Dietary Na and phytase had no effect on bode weight gain and feed intake. Birds on the low Na diet showed higher (p < 0.05) feed conversion ratio (FCR) than the mid-Na diets. High dietary Na adversely affected (p < 0.001) excreta DM content. Phytase supplementation to the high-Na diet increased (p < 0.01) the litter ammonia content. High dietary Na with phytase supplementation improved ($Na{\times}phytase$, p < 0.05) the AME value and ileal digestibility of Ca and Mg. The total tract retention of Ca, P, and Mg was reduced with high Na diet, which was counteracted by phytase supplementation ($Na{\times}phytase$, p < 0.001). The diets containing mid-level of Na improved (p < 0.001) the function of Na-K-ATPase and Mg-ATPase in the jejunum. The overall results indicate that high dietary Na did not affect phytase activity but influenced the nutrient utilization of birds, which was not reflected in bird overall performance.

• Journal of Photoscience
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• 제4권2호
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• pp.35-40
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• 1997

The sensory transduction processes of blue light in guard cells have been suggested the involvement of Ca$^{2+}$/calmodulin-dependent myosin light chain kinase (MLCK) or MLCK-like proteins. The source of Ca$^{2+}$ required for the signal transduction process was investigated in guard cell protoplasts (GCPs). The GCPs showed the typical H$^+$ pumping activity by blue light (200 $\mu$mol m$^{-2}$ s$^{-1}$) and fusicoccin (10 $\mu$M) under background red light (600 $\mu$mol m$^{-2}$ s$^{-1}$). The blue light-dependent H$^+$ pumping was not significantly affected by the externally changed Ca$^{2+}$ concentrations. The addition of 1 mM Ca$^{2+}$ in the bathing medium ratherly inhibited the H$^+$ pumping. In contrast, the blue light-dependent H$^+$ pumping was inhibited by caffeine and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibitor of C$^{2+}$-ATPase in endoplasmic reticulum (ER) without inhibiting the H $^+$ pump. The inhibition by caffeine and BHQ was fully reversible. The extent of inhibition by caffeine and BHQ was larger when they were added together than when added separately. The results suggest that Ca$^{2+}$ required for the blue light-dependent H$^+$ pumping may be released from the intracellular Ca$^{2+}$ stores, probably ER in guard cells.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.67-67
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• 2001

Our previous studies showed that the relaxation defect of diabetic heart was due to the changes in the expressional levels of SR $Ca^{2＋}$-ATPase and PLB. In the diabetic heart contractile abnormalities were also observed, and one of the mechanisms for these changes could include alterations in the expression and/or activity levels of various $Ca^{2＋}$ regulatory proteins involving cardiac contraction.(omitted)

• 한국식품과학회지
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• 제6권4호
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• pp.199-208
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• 1974

근육의 수축 및 사후강직은 myosin 과 actin 그리고 ATP 와의 상호작용에 의한 것임은 널리 알려진 정설(定說)이다. 최근 myosin 과 actin 및 ATP 의 상호작용이 근조절단백질의 지배를 받고 Ca ion 이 관여하고 있다는 것이 알려졌다. 그런데 이들 조절단백질은 수용성단백질로써의 성질을 가지고 있음에도 불구하고 염용성단백질 구분에 들어 있다. 본 연구의 목적은 염용성단백질 구분에 들어 있는 이들 조절단백질의 새로운 분리정제방법을 연구하는 데 있었다. 이를 위하여 본 연구에서는 새로운 정제방법의 flow sheet 를 작성하였다. 이 제안된 새로운 정제방법은 근원섬유중의 조절단백질의 함량을 정량적으로 추적할 수 있는 장점을 가지고 있다는데 그 특색이 있다.

• The Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.529-535
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• 1997

The present study was aimed at investigating whether the vascular calcium regulation is altered in hypertension. Two-kidney, one clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt hypertension were made in rats, and their thoracic aortae were taken 4 weeks later. The isometric contractile response and calcium uptake of the endothelium-denuded aortic preparations were determined. Caffeine ($0.1{\sim}35\;mmol/L$) induced a greater contraction in 2K1C and DOCA-salt hypertension than in normotensive control. When the vascular calcium store was functionally-depleted by a repeated exposure to caffeine, it took longer to reload the store and to resume the initial contraction force in response to caffeine in both 2K1C and DOCA-salt hypertension. The vascular $^{45}Ca$ uptake following the functional depletion of the cellular store was also greater in both models of hypertension than in control. Ryanodine, calcium channel activator of the sarcoplasmic reticulum, attenuated the restoration of caffeine-induced vascular contraction, which was not affected by either 2K1C or DOCA-salt hypertension. Nifedipine, an L-type $Ca^{2+}$ channel blocker, attenuated the restoration of caffeine-induced contraction, which was not affected by DOCA-salt hypertension, but was more pronounced in 2K1C hypertension. Nifedipine also diminished the vascular $^{45}Ca$ uptake, which was not affected by DOCA-salt hypertension, but was more pronounced in 2K1C hypertension. Ouabain, a $Na^+,\;K^+-ATPase$ inhibitor, increased the caffeine-induced contraction by a similar magnitude in control and 2K1C hypertension, which was, however, markedly attenuated in DOCA-salt hypertension. Ouabain enhanced the vascular $^{45}Ca$ uptake, the degree of which was not affected by 2K1C hypertension, but was markedly attenuated in DOCA-salt hypertension compared with that in control. Cyclopiazonic acid, a selective inhibitor of $Ca^{2+}-ATPase$ of the sarcoplasmic reticulum, attenuated the restoration of caffeine-induced contraction, which was not affected by 2K1C hypertension, but was more marked in DOCA-salt hypertension. These results suggest that the increased vascular calcium storage may be attributed to an enhanced calcium influx in 2K1C hypertension, and to an impaired $Na^+-K^+$ pump activity of the cell membrane and subsequently increased calcium pump activity of the cellular store in DOCA-salt hypertension.

• 한국식품과학회지
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• 제30권5호
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• pp.1120-1127
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• 1998

고등어 surimi를 효율적으로 생산하기 위한 연구의 일환으로 감압수세장치를 설계 제작하고 이 장치를 이용하여 각각 상압과, 560 및 660 mmHg의 감압 하에서 각각 1, 3, 5 및 7회 알칼리 수세하고 압력 및 수세횟수에 따른 고등어 surimi의 품질 특성을 검토하였다. 설계 제작한 감압수세장치는 연속수세가 가능하였으며 양질의 surimi를 효율적으로 제조할 수 있었다. 전체의 수세조건을 통하여 surimi의 수분함량은 $72.0{\sim}72.9%$, 조지방 $4.8{\sim}5.7%$ (수세하지 않은 것, 7.0%), pH $6.9{\sim}7.0$ (수세하지 않은 것, 6.0), VBN $6.9{\sim}7.0\;mg/100\;g$ 및 가압드립 $6.7{\sim}8.3%$이었으며, 단백질 추출성은 560 mmHg, 5회 수세 시 염용성, 수용성 및 기질 단백질추출성이 각각 3,694, 6,036 및 1,424 mg/100 g으로써 각각 가장 높았다. $Mg^{2+}-$ 및 $Ca^{2+}-ATPase$ 활성은 560 mmHg, 5회 수세 시 각각 0.25 및 $0.17\;{\mu}mol\;Pi/min/mg$ actomyosin으로써 가장 높았다. Setting gel의 TGase 활성은 560 mmHg, 5회 수세 시 3.932 nmol/mg이었으며 gel 강도는 setting gel 및 cooked gel에서 각각 420 g cm (상압, 320 g cm) 및 485 g cm (상압, 412 g cm)로써 각각 가장 높았다. 전반적으로 보아 고등어 surimi 제조를 위한 가장 적합한 수세조건은 760 mmHg, 660 mmHg 및 560 mmHg 압력 중 560 mmHg의 감압 하에서 5회 반복 수세하는 것이었다.

• 대한물리치료과학회지
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• 제11권1호
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• pp.20-27
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• 2004

It is widely accepted that smooth muscle contraction is triggered by intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) released from intracellular $Ca^{2+}$ stores such as sarcoplasmic reticulum (SR) and from the extracellular space, The increased $[Ca^{2+}]_i$ can phosphorylate the 20-kDa myosin light chain ($MLC_{20}$) by activating MLC kinase (MLCK), and this initiates smooth muscle contraction. In addition to the $[Ca^{2+}]_i$-MLCK-tension pathway, a number of intracellular signal molecules, including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), and Rho-associated coiled coil-forming protein kinase (ROCK), play important roles in the regulation of smooth muscle contraction. However, the mechanisms regulating contraction of caldesmon (CaD), actin-binding protein, are not entirely elucidated in the presence of $Ca^{2+}$. It is known that CaD tightly interacts with actin and inhibits actomyosin ATPase activity. Therefore, the purpose of the present study was to investigate the roles of $Ca^{2+}$-dependent CaD in smooth muscle contraction. Endothelin-1 (ET-1), G-protein coupled receptor agonist and vasoconstrictor, increased both vascular smooth contraction and phosphorylation of CaD in the presence of $Ca^{2+}$. These results suggest that ET-1 induces contraction and phosphorylation of CaD in rat aortic smooth muscle, which may he mediated by the increase of $[Ca^{2+}]_i$.

• BMB Reports
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• 제29권3호
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• 한국가금학회지
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• 제14권2호
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• pp.137-143
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• 1987

육계육의 가승근육과 다리근육을 4$^{\circ}C$ 및 -2$0^{\circ}C$에 저장하면서 근원섬유단백질과 actomyosin을 추출하고 그 추출성, 생물활성 및 용해도를 비교한 결과는 다음과 같다. 1. Myofibril의 추출성은 가슴 및 다리근육 모두 냉장기간이 길어지면서 점차 높아지고. 동결저장기간이 경과되면서 다소 낮아졌다. Actomyosin의 추출성은 가슴 및 다리근육 모두 냉장 중에 큰 변화를 보이지 않았으며, 동결저장의 경우 저장기간이 경과하면서 점차 감소하였다. 2. Myofibril의 $Ca^{2+}$-ATPase 활성은 가슴 및 다리근육 모두 냉장 7일까지 큰 변화는 없었으며, 동결저장의 경우 2주째 가장 농은 활성을 보였고 그 이후는 감소하였다. Actomyosin의 $Ca^{2+}$-ATPase 활성은 가슴 및 다리근육 모두 냉장 중에 큰 변화를 보이지 않았으며, 동결저장의 경우 저장기간이 경과하면서 점차 감소하였다. 3 근원섬유단백질은 염농도에 따라 신선육의 경우 가슴 및 다리근육 모두 0.20M KCl, 냉장육은 가슴 및 다리근육이 각각 0.25M KCl. 0.30M KCl, 그리고 동결저장육은 가슴 및 다리근육 모두 0.30M KCl 에서 용해되었다. Actomyosin은 염농도에 따라 신선육의 경우 가슴 및 다리근육 모두 0.20M KCl에서 용go되기 시작하였고, 가슴근육은 냉장 1일에 0.25M KCl. 냉장 3. 5, 7일에 0.30M KCl, 다리근육은 냉장기간에 관계없이 모두 0.25M KCl에서 용해되기 시작하였다. 또한, 동결저장 중 가승근육은 0.30M KCl, 다리근육은 0.25M KCl에서 용해되기 시작하여 모두 0.40M KCl에서 완료되었다.