S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Medicine (의학과) The Seoul Journal of Medicine The Seoul Journal of Medicine Vol. 21 No.4 (1980)
흰쥐 간세포 원형질막 5'-Nucleotidase의 특성에 관한 연구
Properties of 5'-Nucleotidase from Rat Liver Cell Plasma Membrane
- 김광현; 백만기; 정홍근; 김승원
- Issue Date
- 서울대학교 의과대학
- Seoul J Med, Vol.21 No.4, pp. 335-347
- 5'-Nucleotidase bas been shown to be localized in
the plasma membranes of various cell types. It is
widely used as a marker enzyme for plasma membrane
in subcellular fractionation. It has been reported as well that 5' -nucleotidase has broad specificities for
Attempts have been made for such a reason to resolve
this enzyme into isoenzymes, but in most cases
evidences for isoenzymes were not obtained.
5'-Nucleotidase has been reported recently to Increase
in its activity upon aging the plasma membrane
fraction in vitro at low temperature. A mechanism
involved for the increase has been put foward in
effect that a regulatory substance is bound to the
enzyme, forming a latent 5f-nucleotidase.
The present investigation was carried out to prepare
membrane-free 5'- nucleotidase from rat liver plasma
membrane and to study the kinetic properties of
the enzyme. Attempts also were made to resolve 5'nucleotidase
into isoenzymes and to confirm and characterize
the proposed regulatory substance.
The results obtained are summarized as follows:
I. Membrane-free 5' -nucl eotidase obtained by sonification
from rat liver plasma membrane was resolved
into two isoenzyme bands by disk electrophoresis on
the polyacrylamide gel. The slow-moving isoenzyme
was major component whereas the fast-moving Isoenzyme,
a minor component.
2. Membrane-free 5f -nuclcotidase compnzmg the
major isoenzyme was purified about 12-fold from rat
liver homogenate by differential centrifugation and
gel filtration on Sephadex G-200 column equilibrated
with 20mM sodium deoxycholate-20mM Tris, pH 9.2.
3. Activation of membrane·free 5f-nucleotidase was
brought about to the same extent(5%) by the much
lower concentration of MnH (0. 01-0. ImM) and by
much higher concentration of MgH(IOmM).
4. The enzyme was strongly inhibited by EDTA
and recovered completely by the addition of about 5
time as much MgH in molar concentration as EDTA.
5. The enzyme was strongly inhibited by ADP
and to a much less extent by GDP and GTP. Purine
nucleosides did not affect the enzyme activity.
6. The pH optimum and KM value of the enzyme
were 8. a and O. 15mM respectively.
7. Phospholipid was detected in the membrane-free
5f-nucleotidase and the enzyme was activated by widely
used detergents such as sodium dodecyl sulfate
(SDS) , sodium deoxycholate(SDC) and Triton x-roo. Vma x of the enzyme was increased about 2-fold by
addition of 5mg of SDC and 0.2 I' moles of MnH to
2ml of the enzyme assay system, but KM value of the
enzyme was not altered. And the slope of the decrease
in enzyme activity with time due to thermal denaturation
at 30°C was greater in the SDC and
Mnt t-containing assay system than in the control
Based on the above results, it was discussed that
the phospholipid bound to 5' -nucleotidase could be
regulatory substance which could be released by the
detergents, most effectively by SDC combined with
, and by incubation itself, causing the enzyme
to increase in activity.