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.1 (1980)
흰쥐 간 원형질막 5'-Nucleotidase의 시험관내 활성증가 기전
Mechanism for Increase in 5'-Nucleotidase Activity of Rat Liver Plasma Membrane npon Aging in vitro
- Issue Date
- 서울대학교 의과대학
- Seoul J Med, Vol.21 No.1, pp. 1-13
- 5'-Nucleotidase is one of the marker enzymes for plasma
membrane in subcellular fractionation. In
recent years it ia sbown that the enzyme is not only
important in its diagnostic value for hepatic meta'
static carcillOma. but also responsible for its regulation
of blood Sow through hydrolysis of AMP to adenosine.
a powerful vasodilator.
It was reported as well that its activity of plasma
membrane is increased upon aging the membrane.
While most enzymes bound to membranes decrease in
their activities upon aging the membranes in vitro, 5'nucleotidase
activity is increased to the contrary under
the same condition. The present investigation was
carried out to elucidate the possible mechanism involved
in the phenomenon.
Plasma membrane was purified from rat liver homogenate
by sucrose density gradient and was suspended
in isotonic sucrose solution. part of which
was sonified and ultracentrifuged to obtain membrane-
free supernatant, and both samples were used
as enzyme sources. The behaviors of 5'-nucleotidase
were observed in the control system consisting of
substrate and buffer. and in the activation system
consisting of substrate, buffer, sodium deoxycholate,
The results obtained are summarized as follows.
1. From the results showing the increased activities
of 5' -nucleotidase of both the plasma membrane
suspension and the membrane-free supernatant upon
aging the samples, it is concluded that it is independent
of the membrane structure but is due to
the property of enzyme molecule itself. The 5'nucleotidase
activities of both the suspension and the
supernatant, measured in the activation system were
approximately 100% and 70% respectively higher
than those measured in the control system.
After incubation of the suspension at 45°C for 1
hour. the 5' -nucleotidase activity was increased by
16% in the control system, while it showed no
variation in the activation system.
2. The increase in 5'-nucletidase activity caused
with deoxycholate in the absence of Mn* in the
assay system was a very slow reaction but the reaction
reached immediately to a maximum in the
presence of Mn*.
3. Apparent Km values for 5' -riucleotidase measured with the same amount of suspension In the
control system and in the activation system were
the same, O. 12mM but Vmax for the reaction was
about 2-fold greater in the activation system than
that measured in the control system.
4. The increase in 5'-nucleotidase activities of the
supension and the supernatant was directly dependent
on the concentration of deoxycholate in the
presence of Mn* in the assay system, while their
rates of increase upon aging the enzyme preparation
were inversely dependent on the concentration of
5. The above results may well be explained reasonably
by assuming the presence of a regulatory
substance. R being associated with the catalytic
enzyme molecule, C of 5'-nucleotidase; C, an active
form of the enzyme, whereas C.R, a latent form of
From the foregoing results it can be inferred that
C could be a thermally stable protein, whereas R
may be a thermally unstable substance; the latter
being slowly denatured upon aging and released from
C' R, which leads eventually to the increase in active
form. It can also be inferred that R is released from
C·R by deoxycholate in the activation system for
which the following equdtion could be suggested,
C·R+D=C+R·D(D, the molecule of