서지주요정보
Biochemical role of pig heart mitochondrial creatine kinase = 돼지 심장 미토콘드리아에 존재하는 크레아틴 키아제의 생화학적 역할
서명 / 저자 Biochemical role of pig heart mitochondrial creatine kinase = 돼지 심장 미토콘드리아에 존재하는 크레아틴 키아제의 생화학적 역할 / Il-Han Kim.
저자명 Kim, Il-Han ; 김일한
발행사항 [서울 : 한국과학기술원, 1987].
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4104084

소장위치/청구기호

학술문화관(문화관) 보존서고

DBE 8701

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In order to elucidatate the biochemical role of heart mitochondrial creatine kinase in the energy metabolism of heart muscle cell, we studied the mechanism on the coupling between the production of phosphocreatine by respiring heart mitochondria and oxidative phosphorylation. We also studied the effect of the concentration ratio of phosphocreatine to creatine on the rate of coupled respiration as well as that of uncoupled respiration in order to examine the possible regulatory role of the creatine kinase in the energy metabolism. The followings are the main results. 1. Effect of the concentration ratio of phosphocreatine to creatine on the rate of respiration: In the absence of added creatine and phosphocreatine, pig heart mitochondria respiring in the presence of glutamate and malate exhibited an approximately 5-fold increase in the initial State 4 respiratory rate upon the addition of ADP, followed by return to the state of the Post-ADP State 4 rate. When the concentration of creatine was increased, the rate of the State 3 as well as the post-ADP State 4 increased further. The increased rates of the State 4 and 4 respiration due to the addition of creatine were diminished in the presence of phosphocreatin. The half maximal stimulation of post-ADP State 4 respiration was obtained with 8 mM creatine, which is comparable to the value of apparent Km of purified pig heart mitochondrial creatine kinase for creatine(8.5 mM). The State 3 respiratory rate decreased by increasing the concentration ratio of phosphocreatine to creatine with the total concentration of creatine plus phosphocreatine fixed at 20 mM. The maximum state 3 respiratory rate in the presence of excess ADP was further stimulated about 2-fold by the addition of 20 mM creatine. Also, the response of the uncouped respiratory rate in the presence of FCCP to the creatine and phosphocreatine was similar to that of the State 3 rate. These results indicate that the concentration ratio of phosphocreatine to creatine in heart muscle acts as a signal modulating the rate of oxidative phosphorylation. 2. The formation of phosphocreatine by respiring mitochondria in the presence of exogenous ADP: As the concentration of ADP was increased, the rate of phosphocreatine formation by respiring heart mitochondria increased. The value of apparent Km of the phosphocreatine-forming mitochondria for ADP was estimated to be 0.0185 mM. This value was much lower than that of Km for ATP (0.31 mM) which was determined from the reaction of the soluble form of mitochondrial creatine kinase in the presence of 1mM of exogenous ATP. The ATP formed by the respiring heart mitochondria was also measured simultaneously in the presence of ADP. The concentration of the ATP formed remained constant during the phosphocreatine formation at a constant rate. The rate of the accumulation of the oxidative-phosphorylated ATP in the medium surrounding mitochondria was continuously monitored as a function of ADP concentration with the firefly luciferase-coupled assay. Creatine did not affect the rate of accumulation of the ATP in the medium, indicating that the phosphocreatine-forming mitochondria in the presence of ADP did not used the ATP in the medium as a substrate. These results suggest that the heart mitochondrial creatine kinase bound to the inner membrane functionally and tightly coupled to the oxidative phosphorylating system with respect to the ATP within the mitochondria. 3. The formation of phosphocreatine by the respiring mitochondria in the absence of exogenous ADP and ATP: The isolated pig heart mitochondria was found to form phosphocreatine continuously at a rate of 12.5±1.8 nmol per min per mg of the mitochondrial protein in the respiration medium without externally added adenine nucleotides. Its formation rate and respiratory rate in the absence of added adenine nucleotides showed a concentration dependency with respect to both creatine and phosphate, and the apparent Km values of the phosphocreatine-forming heart mitochondria were estimated to be 7.3 and 0.12 mM for creatine and phosphate, respectively. The Km value of this system for creatine was comparable to the value of the soluble form of mitochondrial creatine kinase for the same substrate in the presence of excess ATP, indicating that a catalytic amount of endogenous ATP is very effectively utilized as a substrate for mitochondrial creatine kinase bound to the respiring mitochondria. The synthesis of phosphocreatine by the respiring heart mitochondria was completely inhibited by antimycin, FCCP, atractyloside, and 2,4-dinitrophenol. However, oligomycin had no effect on the rates of phosphocreatine formation as well as respiration in the absence of added ADP. Pig heart mitochondria, which did not show a creatine kinase activity, continuously produced phosphocreatine at a rate of 1.5±0.14 nmol per min per mg of the mitochondrial protein when the soluble form of heart mitochondrial creatine kinase was added. In contrast to the case of heart mitochondria, the formation of phosphocreatine by the respiring liver mitochondrial system was completely blocked by oligomycin. These results indicate that during the continuous formation of phosphocreatine by the respiring heart mitochondria, the adenine nucleotides is not released into the medium. 4. Kinetic properties of the soluble form of mitochondrial creatine kinas: The mitochondrial creatine kinase was detached from the inner membrane of the mitochondria, and its kinetic properties were compared with those of cytoplasmic isoenzyme. The optimum pHs for the forward as well as the reverse reaction of the mitochondrial isoenzyme were slightly lower than the respective pH of the cytoplasmic one. The affinities of mitochondrial isoenzyme toward its substrates were stronger than those of the cytoplasmic one toward its respective substrates. The over-all equilibrium constants at pH 7.4 were estimated to be $1.48×10^{-2}$ and $7.20×10^{-3}$ for the mitochond and the cytoplasmic isoenzymes, respectively. The characteristic pattern of the initial velocity and the product inhibition for the forward reaction of the mitochondrial isoenzyme at pH 7.4 appeared to be the same as that of the cytoplasmic one. These results show that mitochondrial creatine kinase is kinetically disadvantaged for the forward reaction (i.e., phosphocreatine-forming reaction). Based on these results, we proposed that the mitochondrial creatine kinase, adenine nucleotide translocase and coupling ATPase all interact as a "multifunctional enzyme complex" producing phosphocreatine, and that a catalytic amount of bound adenine nucleotides function as a coenzyme for the reaction of phosphocreatine formation. This hypothesis implies that the concentration ratio of phosphocreatine to creatine is a crucial factor regulating the rate of the oxidative phosphorylation in the heart muscle cell.

1. Creatine 의 농도를 증가시킴에 따라 state-4 및 state-3 respiration 속도가 점차적으로 증진되었으며, creatine에 의해 증진된 state-3 및 state-4 respiration 속도는 phosphocreatine 에 의해다시 감소됨을 알수 있었다. Creatine 8 mM 에 의해 state-4 respiration속도는 최대 증진 속도의 반에 달했으며, 이 농도 값은 추출한 미토콘드리아 creatine kinase의 creatine에 대한 Km값과 유사함을 알수 있었다. 또한 phosphocreatine 과 creatine의 농도 합을 20 mM 로 고정시킨 다음 phosphocreatine 의 농도비를 증가 시킴에 따라 state-3 respiration rate가 점차 감소됨을 관찰할 수 있었다. Creatine 이 존재할때 state-3 respiration 속도가 존재하지 않을때에 비해 모든 ADP 농도에서 높았으며, FCCP 에 의해 촉진된 respiration 속도도 creatine과 phosphocreatine에 의해서 현저하게 영향을 받음을 알수 있었다. 2. ADP 농도를 증가시킴에 따라 미토콘드리아에 의한 phosphocreatine의 생성 속도는 포화 경향을 보이면서 증진되었다. 이 조건에서 phosphocreatine 의 생성 속도를 측정하여 ADP에 대한 Km 값을 구해본 결과 18.5μM 임을 알았고, 이 Km 같은 미토콘드리아의 oxidative phosphorylation계와 연계되지 않은 (추출한) 미토콘드리아 creatine kinase 의 ATP에 대한 Km 값인 210μM보다 훨씬 낮음을 알수 있었다. 이외에도 ADP 존재하에 oxidative phosphorylation 에 의한 ATP 생성에 대한 creatine kinase 의 활성의 영향을 살펴본 결과 이 조건에서 phosphocreatine 은 반응시간에 정비례 하게 생성 되었으나, 생성된 ATP 는 반응 시간에 무관함을 보였다. 또한 oxidative phosphorylation 에 의해 미토콘드리아 외부 respiration 용액내에 이 ATP 가 축적되는 속도도 미토콘드리아 creatine kinase 의 phosphocreatine 생성과 무관함을 알 수 있었다. 3. 외부에서 ADP 및 ATP 를 respiration 용액내에 가하지 않은 반응조건 에서도 돼지 심장 미토콘드리아 creation kinase 는 creatine 을 phosphocreatine으로 계속적으로 일정한 속도 12.5±1.8 nmol per min per mg)를 유지하면서 생성하였고, 이 system 내에서 phosphocreatine 의 생성에 대한 creatine 및 phosphate의 km값은 각각 7.3 mM 로 0.12 mM이었다. 이 system 의 creatine 의 Km 값은 돼지 심장 미토콘드리아에서 추출한 creatine kinase를 1.0 mM ATP 존재하에 creatine 농도를 변화시켜 얻은 creatine의 Km 값과 유사하였다. Respiring heart mitochondria 에 의한 phosphocreatine 생성은 antimycin, atractylosiide 및 2,4-dinitrophenol 에 의해 완전히 억제 되었으나 oligomycin 에 의해서는 전혀 영향을 받지 않았다. Creatine kinase 의 활성을 전혀보이지 않은 간 미토콘드리아에 충분량의 creatine kinase (20 units) 를 가해 줄 경우 심장 미토콘드리아에 비해 아주 적지만 일정한 속도 (1.5±0.14 nmol per min per mg) 로 phosphocreatine이 생성됨을 관찰할 수 있었고, 이 경우에는 심장 미토콘드리아의 경우와 대조적으로 oligomycin에 의해서도 phosphocreatine 생성은 완전히 억제됨을 알수 있었다. 4. 돼지 심장 미토콘드리아에서 추출하여 얻어낸 creatine kinase(미토콘드리아 creatine kinase)의 반응 특성을 cytoplasmic creatine kinase와 비교 고찰하였다. 정반응 및 역반응에 있어서 미토콘드리아 creatine kinase의 최적 pH는 cytoplasmic creatine kinase의 것보다 모두 0.5unit 정도 낮았으며, 더욱이 pH 7에서 8 사이에서는 정반응과 역반응의 최대 속도비는 미토콘드리아 creatine kinase의 것이 2배 정도 큼을 알수 있었다. 도한 미토콘드리아 creatine kinase의 기질에 대한 친화도는 정반응 및 역반응에 있어서 모두 cytoplasmic creatine kinase의 것보다 모든 pH 범위에서 강했으며, pH 7.4에서 두 isoenzyme의 평형상수 (keq)는 미토콘드리아 creatine kinase의 경우 $1.48×10^{-2}$, cytoplasmic creatine kinase의 경우 $7.20×10^{-3}$로 미토콘드리아 creatine kinase 반응이 정반응 쪽으로 cytoplasmic creatine kinase의 경우보다 2배 정도 유리하게 일어남을 알수 있었다. 또한 product inhibition pattern 및 initial velocity pattern을 토대로 두 효소의 반응기작을 추윽해 본 결과 두 반응기작은 모두 rapid equilibrium random 형을 따름을 알수 있었다. 5. 이상의 일련의 실험 결과를 토대로, 미토콘드리아 creatine kinase와 oxidative phosphorylation system은 기능적으로 밀접하게 연계되어서 phosphocreatine을 계속적으로 생성할 수 있는 일종의 multifunctional enzyme complex 로 작용된다는 관점 아래 돼지 심장 미토콘드리아 creatine kinase의 심장 세포 에너지 대사내 에서의 생화학적 역학을 제시해 보았다.

서지기타정보

서지기타정보
청구기호 {DBE 8701
형태사항 xii, 98 p. : 삽도 ; 26 cm
언어 영어
일반주기 저자명의 한글표기 : 김일한
지도교수의 한글표기 : 김형만
학위논문 학위논문(박사) - 한국과학기술원 : 생물공학과,
서지주기 Reference : p. 90-95
주제 Enzyme kinetics.
Phosphocreatine.
크레아틴 키나아제. --과학기술용어시소러스
미토콘드리아. --과학기술용어시소러스
효소 반응 속도론. --과학기술용어시소러스
크레아틴인산. --과학기술용어시소러스
Creatine kinase.
Mitochondria.
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