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EDTA가 백서 생체모 MARKER ENZYME의 활성에 미치는 영향에 관하여
Effects of EDTA on the Activities of Marker Enzymes for Membranes from Rat Liver.

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Authors
이용길; 최한웅; 정홍근; 이기영
Issue Date
1979-03
Publisher
서울대학교 의과대학
Citation
Seoul J Med, Vol.20 No.1, pp. 44-54
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.
ISSN
0582-6802
Language
Korean
URI
http://hdl.handle.net/10371/7203
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Medicine (의학과)The Seoul Journal of MedicineThe Seoul Journal of Medicine Vol. 20 No.1 (1979)
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