1084
BIOCHEMICAL SOCIETY TRANSACTIONS
induction of both systems is subjected to the catabolic repression by glucose (Kloppel &
Hofer, 19746).
A peculiar feature of the repression of pentitol induction by glucose is its persistence
even after glucose disappearance from the medium. Since the systems can be induced in
glucose-grown cells, but not after subsequent glucose catabolism, therefore there must be
a substance in the culture medium that suspends the effect of glucose. Indeed, if a
stationary-phase yeast suspension is incubated with glucose in the culture medium, the
cells are inducible after disappearance of the glucose. By eliminating the individual
components of the culture medium (for its composition see Hofer & Dahle, 1972) it can
be shown that it is the Mg2+that counteracts the effects of glucose. Fig. 1 demonstrates
that omission of Mg2+ from the culture medium leads to the known persistence of the
repression, whereas the addition to the yeast suspension of Mgz+ alone allows a full
induction of the catabolic pathway after disappearance of the glucose.
Analyses of hot-water extracts of R.gracilis cells show a decrease in the cellular content
of soluble magnesium in the presence of glucose (from 10.2 to 7.3m~-Mg’+).However, a
simple decrease in intracellular Mg2+concentration cannot account for the inability of
the cells to induce, since 1 mM-EDTA effects a similar decrease in intracellular Mgz+
concentration without having a measurable effect on the process of induction. Further, if
the preincubation with glucose takes place in the absence of MgZ+,subsequent incubation
with ribitol and Mgz+ does not reverse the persistent repression by glucose. There is
an indirect indication suggesting that Mgz+ may not be extruded from the cells but is
likely to be bound to the cell material during glucose catabolism. The catabolic
repression cannot be avoided by addition of cyclic AMP ( 5 m ~to
) the yeast suspension.
There are two modes of glucose action in the repression of protein induction by
pentitols. (1) The catabolic effect, which persists even after glucose disappearance from
the suspension. It is combined with the decrease of the soluble magnesium and can be
counteracted by externally added Mgz+. (2) The membrane effect, which is effective
only as long as glucose is present in the medium, as indicated by the comparison of curves
5 and 6 in Fig. 1. It consists in the inhibition of the inductor entry at the membrane carrier
level, as has already been described for the D-xylose catabolizing system in R. gracilis
(Hofer & Dahle, 1972).
Hofer, M. & Dahle, P.(1972) Eur. J . Biochem. 29, 326-332
Kloppel, R. & Hofer, M. (1974~)Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1
Orig. Reihe A 228,211-217
Kloppel, R. & Hofer, M. (19746) Proc. In?. Symp. Yeasts 4th, Part 1, pp. 291-292
Lewis, D. H. & Smith, D. C. (1967) New Phytol. 66, 185-204
The Effect of D-Galactosamine and Uridine on the Uracil Nucleotide
Contents of Isolated Rat Hepatocytes
FRIEDRICH HOFMANN and KARL DECKER
Biochemisches Znstitut an der ~ e d i z i n i ~ c Fakultat
~en
der Lrniuersitiit Freiburg, 0-7800
Freiburg, Hermann-Herder Strasse 7, West Germany
D-Galactosamine decreases the hepatic contents of UTP and of UDP-glucose to less
than 10% of normal. Administration of uridine, however, leads to a rapid increase of
the UTP and the UDP-glucose contents. This has been shown in vim (Decker & Keppler,
1972), in the isolated perfused rat liver (Keppler et al., 1969) and in hepatoma cells
(Keppler & Smith, 1974). To facilitate quantitative studies on metabolic pathways
involving uracil nucleotides it was decided to work with isolated rat liver cells. The
following experiments were performed to compare the response of these hepatocytes
with that of the intact organ.
1975
c
1.97
2.07
2.82
3.05
0
20
50
80
8.48
11.41
15.63
18.31
Lactate
dehydrogenase
0.13
0.18
0.36
0.34
Glutamate
dehydrogenase
4.96
4.91
5.94
8.23
Glutamatepyruvate
transaminase
+Galactosamine
0.03
0.02
0.01
Controls
0.23
0.23
0.28
0.30
0
20
50
80
0.34
0.54
0.76
+Uridine
UTP (pmol/g wet wt.)
Time
(mid
I
0.24
0.24
0.25
0.26
0.04
0.03
0.03
+Galactosamine
0.26
0.31
0.42
+Uridine
UDP-glucose (pmol/g wet wt.)
Controls
r
Each value is the mean of at least three independent determinations.
.
r
Activity in medium (% of that in homogenate)
Table 2. A&ustment of UTP, UDP-glucose and [Z UMP] by 4m~-~-galactosarnine
and 3mwuridine
Nucleotide
pyrophosphatase
Time
(min)
with that of a total homogenate.
6.3
8.1
9.9
11.3
1.31
1.41
1.20
1.13
Controls
1.69
1.92
2.29
+Galactosamine
1.75
2.06
2.42
+Uridine
1
Iditol
dehydrogenase
[Z UMPI @mol/g wet wt.)
2.31
3.70
4.09
4.39
Glutamateoxaloacetate
transaminase
L, Table 1. Leakage of enzymes into the incubation medium
Each value is the mean of at least five independent determinations. The data represent the percentage of the activity found in the medium compared
o_
1086
BIOCHEMICAL SOCIETY TRANSACTIONS
Isolated rat hepatocytes were prepared by the method of Berry & Friend (1969), with
the following modifications. The livers were perfused in situ for about 8min with an
oxygenated, Cazf-free Krebs-Henseleit (1 932) buffer, pH 7.55, supplemented
with 5.5m~-glucose and O S ~ M - E G T A *The
.
perfusion was performed at room
temperature (20"C),the flow rate being 50ml/min. This was followed by a 3 min perfusion
with the same medium without EGTA. The livers were then transferred to the recirculating system containing the medium without EGTA, but supplemented with 2.5 %
defatted high-purity albumin and an amino acid mixture. Then 5min later, CaCI, (final
concn. 3 m ~ and
) collagenase (final concn. 0.5%) were added and the perfusion was
continued for 35min at 37°C. After filtration and centrifugation, 96% of the resulting
cells excluded Trypan Blue given as a 0.4% solution.
The functional integrity of the hepatocytes was monitored by determination of the
contentsof some nucleotidesand by the activity of intracellular enzymes in the incubation
medium (Table 1).
The intracellular ATP, CTP, GTP, UTP and UDP-glucose remained in the range of
concentration found in uiuo and in the isolated perfused rat liver. The leakage of lactate
dehydrogenase, glutamate dehydrogenase, glutamate-oxaloacetate transaminase,
glutamate-pyruvate transaminase, iditol dehydrogenase and nucleotide pyrophosphatase to the incubation medium was slightly increased as compared with the isolated liver
perfused with the same medium. The wet weight of the cells was determined as a routine
by an assay of the nucleotide pyrophosphatase activity in a cell suspension: 1g wet wt.
of liver was found to correspond to 21 .Ot0.7 units of this enzyme. The yield of isolated
cells was about 60% of the liver weight.
Addition of D-galactosamine resulted in a dose- and time-dependent decrease in the
UDP-glucose and the UTP contents. D-Galactosamine 4 ( m ~in
) the incubation medium
decreased the contents to 10% of the normal values (0.24k0.03 and 0.23kO.O6pmol/g
wet wt. respectively) within 30min (Table 2). The GTP and the ATP contents remained
nearly constant, whereas intracellular CTP increased. The stimulation of the synthesis
de nouo of uracil nucleotides led to an increase of the [EUMP] (sum of the acidsoluble uracil nucleotides) of the liver cells (Table 2). This has been shown to be due to
the relief of the UTP-mediated feedback inhibition of cytosolic carbamoyl phosphate
synthetase (Pausch et al., 1975). D-Ghcosamine added to the incubation medium in the
same concentration as D-galactosamine did not elicit a comparable change in the UTP
content.
Administration of uridine led, in a time- and dose-dependent manner, to a higher
content of uracil nucleotides (Table 2). Uridine (3 mM) doubled the UDP-glucose content
within 200min, whereas the UTP content increased fivefold. Uridine however, was able
to reverse the effect of D-galactosamine administration. Hepatocytes treated with 4mMD-galactosamine for lOmin recovered their UTP and UDP-glucose contents within 60
min after addition of 3mM-uridine to the cell suspension.
It is concluded that the metabolic integrity of the hepatocytes, and the close similarity
of their response to both D-galactosamine and uridine make these cells suitable for
studies of UTP- and UDP-sugar-dependent processes.
* Abbreviation: EGTA, ethanedioxybis(ethy1amine)tetra-acetic acid.
This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn-Bad
Godesberg, West Germany
Berry, M. N. & Friend, D. S. (1969) J. Cell. Biol43,506-520
Decker, K. & Keppler, D. (1972)Prog. Liver Dis. 4,183-199
Keppler, D. & Smith, D. F. (1974) Cancer Res. 34,705-711
Keppler, D. Frohlich, I., Reutter, W., Wieland, 0. &Decker, K. (1969) FEBS Lett. 4,278-280
Krebs, H. A. & Henseleit, K. (1932) Hoppe-Seyler's Z. Physiol. Chern. 210, 33-66
Pausch, J., Wilkening, J., Nowack, J. &Decker, K. (1975) Eur. J . Biochem. 53,349-356
1975