EDTA가 백서 생체모 MARKER ENZYME의 활성에 미치는 영향에 관하여

Abstract

For the examination of effects of EDTA on the activities of membrane marker enzymes, plasma membrane. mitochondria and microsomal membrane were partially purified from rat liver. O. 1M EDTA solntion buffered with 5mM Tris (pH 7.4) was added to these su bcellular particle suspensions to be 1mM or added directly to the enzyme assay system to survey the effects of EDTA on the enzyme activities under the varying conditions. The results obtained are as follows: 1. 5'-Nucleotidase and p-hydroxvbutyric dehydrogenase were enriched io.s-reu in the plasma membrane fraction and 8. Lfold in the mitochondrial fraction, respectively through differential centrifuga· tion followed by discontinous sucrose density gradient. The activities of (MgH+Na++K')-ATPase and glucose-fiephosphatase were slightly elevated by the presence of 1mM EDTA in the enzyme preparations. 2. According to kinetic study, the apparent Km for 5' -nucleotidase was 0.38mM and 5'-nucleotidase was inhibited competitively by EDTA. The enzyme activity was suppressed up to around 80% in the presence of O. 1M EDTA, but no further inhibition was observed beyond this concentration, and thus the rest of 20% is supposedly caused by other nonspecific phosphatases, 3. A modified method was attempted to establish determination of 5' -nucleotidase, taking advantages of selective inhibition of this enzyme by EDTA. The activity of phosphatases other than this enzyme is measured in the presence of O. 1M EDTA and the enzyme assay is once repeated but in the absence of EDTA, thus the difference of above two assayed activities would be the proper activity of 5' -nucleotidase, 4. Despite p-hydroxbutyric dehydrogenase (P-HBD) was actually inhibited by the presence of O.OlmM EDTA in the enzyme assay system, this mitochondrial enzyme activity, however, increased considerably when it was measured with the mitochondrial preparation with 1mM EDTA which was diluted to O. 01mM in the enzyme assay system. This apparent contradiction can be explained on the following assumption. The mitochondrial membrane structure is impaired by EDTA through the chelation of Cat" bound to membrance, rendering them vulnerable to the hypotonicity of enzyme assay medium. which results in the ruptured structures of mitochondrial membrane, offering much enhanced surface area with exposed enzyme. The resulting increased enzvme activity could well cover its inhibition. This presumption was proved by appropriate experimental design using intact mitochondria and fragmented one by sonication. 5. The ratio of intact mitochondria to ruptured Doe in mitochondrial preparation could be easily measured by the increase in p-HBD activity in the presence of 1mM EDTA, as compared with that of the sample without EDTA.