The Korean Journal of Zoology (한국동물학회지)
- Volume 28 Issue 1
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- Pages.31-43
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- 1985
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- 0440-2510(pISSN)
Purification and Characterization of ($Ca^{2+}$ +$Mg^{2+}$ )-ATPase of Sarcoplasmic Reticulum from Rat Skeletal Muscle
쥐 근소포체의 ($Ca^{2+}$ +$Mg^{2+}$ )-ATPase의 분리정제와 그 효소특성에 관하여
- Lee, Jong-Soon (Dept. of Zoology, College of Natural Sciences, Seoul National University) ;
- Ha, Doo-Bong (Dept. of Zoology, College of Natural Sciences, Seoul National University) ;
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Chung, Chin-Ha
(Dept. of Zoology, College of Natural Sciences, Seoul National University)
- Published : 1985.01.01
Abstract
The $(Ca^{2+}+Mg^{2+})$-ATPase has been purified homogeneously from sarcoplasmic reticulum of rat skeletal muscle by sucrose density gradient centrifugation. The purified enzyme has a molecular weight of 115,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dedecyl sulfate, and therefore has the same size of the enzyme in rabbit and chick skeletal muscle. $Ca^{2+}, Mg^{2+}, Fe^{2+}, Co^{2+}, and Mn^{2+}$ at 50 $\\muM$ show stimulatory effect on the ATP-ase, while $Zn^{2+}, Cu^{2+}, and Hg^{2+}$ inhibit it at the same concentration. The ATPase activity is insensitive to antimalarial drugs such as quinine and quinacrine, but is sensitive to inhibition by p-hydroxymecurie benzoate and phenylmethylsulfonylfluoride. The enzyme has optimum pH of 6 to 7 and Km value for ATP is estimated to be 98 $\\muM$. Thus, a number of biochemical properties of this enzyme appear to be different from those of the enzyme that have been isolated from rabbit skeletal muscle. The $(Ca^{2+}+Mg^{2+})$-ATPase appears to be selectively degraded in microsomal fraction. The activity of metalloendoprotease is evident in the microsomal preparation when assayed by radioactively labeled protein substrate, such as $^{3}H-casein and $^{125}I$-insulin. However, it is presently unclear whether the metalloendoprotease is responsible for the degradation of the $(Ca^{2+}+Mg^{2+})$-ATPase.
$(Ca^{2+}+Mg^{2+})$-ATPase를 쥐의 근소포체로부터 sucrose density gradient centrifugation의 방법을 사용하여 분리 정제하였다. 정제된 효소를 폴리아크릴 아마이드 젤에서 전기영동한 결과, 토끼와 닭의 경우에서와 같이 분자량 115,000인 단일 단백질 띠로 나타났다. 정제된 이 효소의 활설도는 50 $\\muM$의 $Mg^{2+}, Ca^{2+}, Co^{2+}, Fe^{2+}, Min^{2+}$에 의해서는 증가되었고, 같은 농도의 $Zn^{2+}, Cu^{2+}, Hg^{2+}$에 의해서는 감소되었다. Quinine와 quinacrine 같은 antimalarial drug는 이 효소의 활성도에 큰 영향을 주지 않았으나, p-hydroxymercuric benzoate와 phenylmethylsulfonylfluoride는 이 효소의 활성을 억제하였다. 이 효소는 pH 6과 7 사이에서 가장 높은 활성을 나타내었고, ATP를 기질로 사용하였을 때 Km 값은 98 $\\muM$이었다. $(Ca^{2+}+Mg^{2+})$-ATPase는 microsomal fraction에서 선택적으로 분해되었다. $^{3}H-casein$ 이나 ^{125}I-insulin같은 방사성 동위원소로 표지된 기질을 사용하여 단백질 분해에 대한 활성도를 조사해 본 결과, microsomal preparation에 metalloendoprotease가 존재하였다. 그러나 아직까지는 그 효소가 $(Ca^{2+}+Mg^{2+})$-ATPase를 분해하는지는 확실하지 않다.
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