Biochem. J. (1963) 86, 130
130
The Metabolism of Isolated Rat-Liver Nucleoli and other Subnuclear
Fractions
THE ACTIVE SITE OF AMINO ACID INCORPORATION IN THE NUCLEUS
BY K. R. REES, G. F. ROWLAND AND J. S. VARCOE
Department of Chemical Pathology, University CoUege Hospital Medical School, University Street,
London,
W.C.
1
(Received 28 June 1962)
It was shown by Rees & Rowland (1961) that
rat-liver nuclei isolated in 025 M-sucrose incorporated amino acids into protein, and nucleotides into
RNA. Although the incorporation was inhibited to
varying degrees by anoxia and by several inhibitors
of oxidative phosphorylation, it was not affected by
detergents, by freezing and thawing the nuclei, or
by disruption of the nuclei by ultrasonic vibration
(Rees, Ross & Rowland, 1961). We have now studied
isolated nucleoli and other subnuclear fractions
in order to investigate the active site of synthesis
of proteins and nucleic acids within the nucleus.
In addition the enzymic and chemical composition
of the various fractions has been examined.
Part of this work has been published in a preliminary form (Rowland, Rees & Varcoe, 1962).
MATERIALS AND METHODS
Animals. Male albinorats,weighing200-250g., were used.
Reagent8. These were as described by Rees & Rowland
(1961). In addition [1-34C]valine and [1-14C]leucine were
obtained from The Radiochemical Centre, Amersham,
Bucks.
Nuclear and8ubnuclearpreparation&. Nuclei were isolated
from rat liver in 0*25M-sucrose as described by Rees &
Rowland (1961).
Scheme 1 shows the method used for isolating nucleoli
and other subnuclear fractions. Batches of isolated nuclei,
suspended in 15-20 ml. of 0 25m-sucrose, were subjected to
ultrasonic vibration (20 keyc./sec.) at 20 in a MSE ultrasonic disintegrator (60w) with a titanium probe of 1 cm.
diam. until all nuclei were disrupted (usually 7 min.). The
resulting suspension was centrifuged for 30 sec. at 2500g to
remove probe debris and coagulated protein. The nucleoli
Disrupted nuclei
Centrifuged at 412 x 104g,^ min.
I~~~~~
I
Sediment
Supernatant
Recentrifuged twice
in water (see text)
Centrifuged at 2*5 x 10g5,. min.
Nucleoli
(fraction A)
I
Sediment
(fraction B)
Supernatant
Centrifuged at 1-2 x 106g,V min.
I
Sediment
(fraction C)
Supernatant
Centrifuged at 6-3 x 106g,v min.
Sediment
(fraction D)
I
Supernatant
Centrifuged at 3 15 x 107g,y* min.
4
Sediment
Supernatant
(fraction F)
(fraction E)
for
1.
Procedure
subnuclear
fractions
of
rat-liver nuclei. Full details are given
Scheme
isolating
in the Materials and Methods sectioxi.
Vol. 86
METABOLISM OF SUBNUCLEAR FRACTIONS
131
Table 1. Percentage recovery of nitrogen and chemical composition of fractions obtained
from rat-liver-cell nuclei
The fractionation procedure and the analytical methods are given in the Materials and Methods section. The
results are the means of six similar experiments, with the ranges given in parentheses.
Percentage
Chemical composition (,ug./mg. of protein N)
recovery of
Fraction
nuclear N
RNA P
DNA P
Phospholipid P
Intact nuclei
100
36-7 (25-8-52 3)
113 (77-3-124)
39-5 (28.5-450)
A
5-5 (4.5-7.3)
65-5 (60 0-73.5)
131 (113-142)
37-8 (35-2-41-8)
(nucleoli)
B
24-2 (16-0-38-6)
45-8 (32 9-58 0)
186 (170-221)
31-8 (17-5-45-7)
C
54-1 (46-9-61-5)
106-5 (88-8-129)
39-0 (30.2-46.8)
(23
35-9
5-42-0)
D
8-1 (7-0-13-8)
34-4 (21-9-43-8)
36-0 (13-3-51.6)
61-3 (53 5-68 8)
E
3*0 (2-1-4-7)
14-9 (9-5-18-8)
17-3 (94-25.0)
91-4 (84-2-102-5)
F
4-7 (2-3-7.0)
7-8 (1-6-11-8)
13-8 (9.5-19.3)
6-3 (3.9-7 8)
(fraction A)
were
then isolated by centrifuging the
super-
natant at 2100g for 20 min. (3000 rev./min. in a MSE
Major refrigerated centrifuge at 20). The nucleolar sediment was washed, twice, by resuspending it in glass-distilled
water, and centrifuging at 2100g for 15 and 10 min., discarding the supernatant each time. Fraction A was resuspended in water by gentle homogenization in a vertical-
action hand-operated homogenizer (Rees & Rowland,
1961). Fractions B-F were isolated from the original supernatant remaining after the nucleoli had been removed.
This supernatant was centrifuged at 10 OOOg for 25 min.
(10 000 rev./min. in a MSE Angle 13 refrigerated centrifuge
at 20) to yield a sediment (fraction B) and supernatant.
The supernatant was then centrifuged at 105 OOOg for
12 min. (40 000 rev./min. in a Spinco model L ultracentrifuge) to sediment fraction C. Fraction D was sedimented
by centrifuging the supernatant at 105 OOOg for 1 hr. The
supernatant was recentrifuged at 105 OOOg for 5 hr. to
yield a sediment (fraction E) and a supernatant (fraction
F). Fractions B-E were resuspended in cold 0-25 M-sucrose.
Analytical method8. DNA, RNA, phospholipid and
nitrogen were determined by the methods used by Rees &
Rowland (1961), modified as follows for application to the
small amounts of material obtained in some of the subnuclear fractions. A suspension of material was precipitated
with 10% (w/v) trichloroacetic acid and washed twice
with 5 % (w/v) trichloroacetic acid, and the precipitate was
extracted for the determination of phospholipid as described by Wheeldon & Collins (1957) except that acetone
was used once and chloroform-ethanol (2:1, v/v) three
times. The residue was extracted with 5 % trichloroacetic
acid at 900 and the extract divided into two for the determination of DNA by the diphenylamine method of Dische
(1955) and of RNA by the orcinol method of Mejbaum
(1939). The residual protein was analysed for nitrogen by
the micro-Kjeldahl method.
Cytochrome oxidase, succinoxidase, dihydronicotinamide-adenine dinucleotide-cytochrome c reductase and
dihydronicotinamide-adenine dinucleotide-neotetrazolium
reductase were determined as described by Rees & Rowland
(1961), and lactate dehydrogenase was determined by the
method of Kornberg (1955).
The incorporation of radioactivity in vitro was measured
as described by Rees & Rowland (1961), the incubation
mixture being: 50 jumoles of sodium phosphate buffer,
pH 7-4; 50 umoles of NaCl; 1 ,uc of "4C-labelled substrate;
1-5 ml. of subnuclear fraction suspended in 0-25M-sucrose;
water to 2-5 ml. The temperature of incubation was 380.
Histochemical methods. Reactions with acid haematin
after various types of fixation to show the presence of
phospholipid associated with heterochromatin were as
described by La Cour, Chayen & Gahan (1958).
The methyl green-pyronin reaction was as described by
Darlington & La Cour (1960).
RESULTS
Chemical composition. From the analytical
results in Table 1, the nucleoli may be seen to
represent about 6 % of the nuclear nitrogen,
whereas nearly 80 % is recovered in fractions B
and C. Also shown in Table 1 are the ratios of
RNA phosphorus to protein nitrogen, DNA
phosphorus to protein nitrogen, and phospholipid
phosphorus to protein nitrogen for each fraction.
Of the fractions, the nucleoli have the highest
RNA:protein ratio, fraction B the highest DNA:
protein ratio, and fractions D and E, which are
low in nucleic acids, have very high phospholipid:
protein ratios. Fraction F, the supernatant, is
mainly protein and is very low in nucleic acids and
phospholipids.
Enzyme studies. As in previous papers (Rees &
Rowland, 1961; Rees, Ross & Rowland, 1962) the
activities of various enzymes in the nuclear preparations have been determined and an attempt
has been made to localize the site of the enzymes
within the nucleus. Succinoxidase determinations
were carried out as an indicator of mitochondrial
contamination of the intact nuclei. In many preparations no succinoxidase activity was detected,
but, where present, mitochondrial contamination
was calculated to be less than 2 %. Succinoxidase
activity was not detected in any of the subnuclear
fractions even when these were isolated from
nuclei which showed slight activity.
Table 2 shows the activities of various enzymes
in intact nuclei, disrupted nuclei, nucleoli and sub9-2
K. R. REES, G. F. ROWLAND AND J. S. VARCOE
132
nuclear fractions. Apart from cytochrome oxidase,
where there is a definite increase, ultrasonic disruption does little to alter the enzyme activities.
The nucleoli have clearly very little enzyme
activity and the low values may be due to slight
contamination with material from the other
fractions. The results also show that fractions D
and E are generally the most active fractions with
respect to the enzymes studied, fraction F also
being rich in cytochrome oxidase and lactate dehydrogenase.
Synthetic reactions. Intact nuclei, nucleoli and
the other fractions were each incubated with
various 14C-labelled amino acids or orotic acid for
various times in the simple medium as used by
Rees & Rowland (1961). The results of three
typical experiments are shown in Table 3 rather
than a mean of all experiments, since the level of
incorporation varied from preparation to preparation. However, the pattern of ability to incorpor-
1963
ate by the various fractions was always the same.
The nucleoli (fraction A) and also fractions D and
E are many times more active in incorporating
amino acids and orotic acid than the intact nuclei,
whereas the other fractions are generally somewhat less active than the original nuclear preparation. Another feature is that fraction E is always
more active than the nucleoli in a given experiment.
On the basis of these experiments it appears that
there are two main 'sites' (fraction A and fractions
D plus E) for the incorporation of both amino acids
and nucleotides. Chemical analysis indicates that
fractions D and E are not just disrupted nucleoli,
but the question arises whether the two 'sites' are
structurally associated in the intact nucleus. To
investigate this possibility batches of nuclei were
divided in two. One half of the batch was subjected to ultrasonic vibration for the minimum
time to give full nuclear breakage (Ij-2 min.) and
Table 2. Activity of enzymes in isolated rat-liver nuclei, ultrasonic extracts of nuclei, nucleoli
and subnuclear fractions
The fractionation procedure and the enzyme-assay systems are given in the Materials and Methods section. The
results are the means of at least four experiments, with the ranges given in parentheses. In all cases the necessary
blanks have been subtracted and enzyme activity shown to be linearly related to enzyme concentration.
Fraction
Intact nuclei
Disrupted nuclei
A
Cytochrome oxidase
(Id. of 02/hr./mg. of N)
350 (284-472)
650 (506-800)
0
(nucleoli)
B
C
D
E
F
423 (277-570)
280 (251-323)
1090 (1020-1200)
2260 (1900-2700)
1490 (840-1870)
NADH-neotetrazolium reductase
(mg. of formazan
of cytochrome c reduced/hr./mg. of N) 1produced/hr./mg. of N)
6-1 (4.8-7.7)
21 (18-27)
5-8 (4.6-7.9)
28 (24-35)
0-3 (0 1-0-4)
3 (1-4)
NADH-cytochrome c
reductase (,umoles
15
19
32
15
8
(12-19)
(17-20)
(26-40)
(11-19)
(6-10)
2-0 (1-2-2.9)
2-4 (2.0-2.8)
4-6 (3.6-6.2)
3.9 (2.3-5.8)
0-5 (0-3-0-8)
Lactate dehydrogenase (units/mg.
of protein)
0-29 (0-21-0-37)
0-30 (0.24-0.35)
0-11 (0-09-0-15)
0-25 (0.17-0.36)
0-60 (0.48-0-87)
0-57 (0.43-0J74)
Table 3. Incorporation in vitro of 14C-labelled amino acids into protein and of [6-14C]orotic acid
into ribonucleic acid by nuclei, nucleoli and subnuclear fractions of rat liver
The system used was as described in the Materials and Methods section. The results are those of three representative experiments of a group of nine similar experiments.
Radioactivity in nuclei, nucleoli and sub-nuclear fractions
(counts/min./cm.2 at infinite thickness)
Inuibation
ti'lime
Substrate
Expt.
E
D
F
C
B
Nuclei A (nucleoli)
no.
,hr.)
2820
54
780
92
99
1
1240
104
1
[2-14C]Glycine
8000
99
2050
185
165
2
3350
134
1600
7
323
37
27
464
1
12
[1-14C]Valine
710
10
3650
63
1290
34
2
20
915
58
286
115
139
1
510
166
[6-_4C]Orotic acid
467
115
2500
207
316
1310
2
208
565
122
227
273
81
1
152
316
2
[2-_4C]Glycine
1590
136
438
593
150
501
2
213
111
95
398
56
71
127
1
135
[6-14C]Orotic acid
170
1082
37
186
142
2
266
334
2077
177
570
950
443
310
1237
3
3
[2-14C]Glycine
644
1971
127
405
205
717
389
3
[1-14C]Leucine
VMETABOLISM OF SUBNUCLEAR FRACTIONS
Vol. 86
the remainder for 10 min. Nucleoli and subnuclear
fractions were then isolated from each batch of
disrupted nuclei and their ability to incorporate
[1-14C]leucine was studied. Table 4 shows that
increasing the time that nuclei are subjected to
ultrasonics results in a decrease in the incorporation by the nucleoli, and in an increase in that by
fractions D and E. Preliminary results of chemical
analysis suggest that a longer period of disruption
results in a decrease in the lipid content of the
nucleolar fraction and in a corresponding rise in
fractions D and E. These results support the contention that the two synthetic sites are closely
related structures within the nucleus.
Further information on the localization of these
fractions was obtained by histochemical techniques. Confirmation that fraction A corresponds
to the nucleolus was obtained by staining with
Table 4. Effect of different ultrasonic-disruption
times on the ability of subnuclear fractions of ratliver nuclei to incorporate [1-14C]leucine into protein
The system used was as described in the Materials and
Methods section. Incubation was for 3 hr. The results are
those of a representative experiment of a group of three
similar experiments.
Radioactivity in subnuclear
fraction (counts/min./cm.2
at infinite thickness)
Isolated from
Isolated from
nuclei disrupted nuclei disrupted
for 1 min.
for 10 min.
974
384
228
192
225
204
1432
2000
1556
1946
162
258
Fraction
A (nucleoli)
B
C
D
E
F
133
methyl green-pyronin (Table 5). When intact
nuclei are treated with this stain the nucleoli stain
pink and the remainder of the nucleus stains green.
The structures visible under the microscope in
fraction A also stain pink. La Cour et al. (1958)
have shown that phospholipid of the heterochromatin stains black with acid haematin when
tissues are fixed with Lewitzsky's fluid [1 % (w/v)
chromium trioxide-o 0 % (v/v) formalin (1:1, v/v)],
but not when fixation was carried out in Baker's
solution [1 % (w/v) calcium chloride in 4 % (v/v)
formalin which has been kept over marble chips].
These reactions, when applied to fractions A and E
(Table 5), suggest that fraction E contains large
quantities of heterochromatic phospholipid and
that the nucleoli also contain a small amount of
this lipid. These results indicate that fractions D
and E represent heterochromatin which in the
intact nucleus is associated with the nucleolus and
which has become separated during disruption of
the nuclei.
The experiments described above were all based
on separation of subnuclear material before the
incorporation of labelled substrates. Table 6 shows
the results of fractionation of the nuclei after the
incorporation of [1-14C]leucine in vitro. A very
high specific activity was obtained in the protein
of the nucleoli in comparison with that of the
other fractions, D and E being particularly low.
DISCUSSION
The site of protein synthesis within the nucleus
was once a subject of interest (Caspersson, 1947),
but has subsequently been overshadowed by the
interest in cytoplasmic protein synthesis. Recent
techniques permitting the isolation of metabolic-
Table 5. Staining reactions of subnuclear fractions A and E from rat-liver nuclei
The fractionation procedure and the staining techniques are given in the Materials and Methods section.
Staining reaction
r
Stain
Acid haematin
&
Fixation of smear before staining
A~~~
Reaction
Phospholipids
stain dark brown
to blue-black
Colour reaction of
subnuclear fraction
t-
Fixative
None
Baker's soln. (1 %
CaCl2 in 4%
formaldehyde)
Lewitsky-s fluid
[I % chromium
trioxide-1O %
formalin (1: 1,
Reaction
Extracts phospholipid associated with heterochromatin
Retains phospholipid associated with heterochromatin
A (nucleoli)
Light brown
None
E
Brown-black
Pale yellow
Brown-black
Intense black
Pink (as in
intact nuclei)
Slightly pink
v/v)]
Methyl greenpyronin
RNA stains pink
(due to pyronin)
DNA stains green
(due to methyl
green)
None
134
K. R. REES, G. F. ROWLAND AND J. S. VARCOE
Table 6. Fractionation of rat-liver nuclei after
incorporation of [1-14C]leucine in vitro
The system used was as described in the Materials and
Methods section. The reaction was stopped after 3 hr. by
cooling to 00, and the nuclei were washed several times with
cold 0-25M-sucrose containing unlabelled leucine, subjected to ultrasonics for 7 min.; the fractions were then
isolated as described in the Materials and Methods section.
Results are those of a representative experiment of a group
of three similar experiments.
Radioactivity of protein
(counts/min./cm.2 at
infinite thickness)
Fraction
70
Nuclei
1225
A (nucleoli)
400
B
121
C
44
D
15
E
188
F
ally active preparations of nuclei (Allfrey, Mirsky
& Osawa, 1957; Rees & Rowland, 1961) open up the
possibility of reinvestigating this problem.
The only structure visible in the resting nucleus
is the nucleolus, and so the method for fractionation
was begun with the isolation of this subnuclear
component from disrupted nuclei. The isolation of
nucleoli from disrupted rat-liver nuclei has been
described by Monty, Litt, Kay & Dounce (1956).
The procedure, however, involved a sedimentation
step of over 12 hr. and was carried out with 1 %
(w/v) gum arabic. Such a lengthy procedure is
unnecessary since a satisfactory isolation may be
achieved by differential centrifuging. The subsequent isolation of nuclear material from the
nucleoli-free supematant was based on a series of
arbitrary centrifugings in which the centrifugal
force is increased fivefold each time. Although this
fractionation was arbitrary the results of chemical
analysis indicate that a separation of four distinct
nuclear constituents, including the nucleoli, was
achieved. Evidence that fraction A is truly
nucleolar comes from microscopic examination of
the fresh material, from the staining reaction and
from the chemical composition. Chemical analysis
of nucleoli isolated by Monty et al. (1956) showed a
similar picture to that reported in this paper. Of
particular interest are their RNA: DNA ratios,
which indicate a definite amount of DNA associated with the nucleolus. This is confirmed by the
analyses reported in this paper. Fractions B and C,
which comprise almost 80 % of nucleus, are considered on the basis of chemical composition to
represent the chromosomal material.
The high lipid: protein ratios found in fractions D
and E suggest that they are a single nuclear component and may represent the heterochromatin
(La Cour et al. 1958). This leaves the supernatant
1963
(fraction F) which is low in nucleic acids and lipids
and which is considered to be the nuclear sap.
All the subnuclear fractions incorporated amino
acids into protein to some extent, but it is clear
that two main components of high activity have
been isolated from the nucleus. Since an arbitrary
fractionation scheme was adopted it is considered
that the activity that persists in the intermediate
fractions is due to contamination by material from
the active components. Caspersson (1947) suggested that a region of chromatin associated with
the nucleolus (heterochromatin) secreted substances of a protein nature, and Sirlin (1958),
using radioautography, has produced evidence that
14C-labelled amino acids are incorporated by
nucleoli and their associated chromatin. La Cour
et al. (1958) have shown the presence of a lipid
material in chromosomes and in particular in the
heterochromatin. Since fractions D and E are rich
in phospholipid and show a high synthetic ability
it seemed likely that these fractions contained
heterochromatin. Conceivably these fractions
could have become separated from the nucleoli
during the ultrasonic disruption of the nuclei. For
this reason an attempt was made to reduce the
disruption time to the point where nucleoli and
heterochromatin were still structurally associated.
This was not completely achieved, although with a
reduced disruption time the activity of the nucleoli
was higher and that of fractions D and E lower. In
addition there appeared to be a transfer of lipid
material from the nucleolus to fractions D and E
with a prolonged disruption time.
Fractions D and E possess similar staining properties to the lipid material surrounding the
nucleoli in intact nuclei. It is concluded from this
evidence that fractions D and E are probably the
heterochromatin long recognized by the cytologist,
and that in the liver-cell nucleus it is structurally
associated with the nucleolus.
The fractionation of nuclei after incubation at
370 with [1-14C]leucine does not follow the same
pattern as that obtained with freshly isolated
nuclei. This is borne out by the finding that the
quantities of nuclear material recovered in the
fractions differ markedly depending on whether
preincubated or fresh nuclei are used. This may be
the explanation for the high labelling in the nucleolus and the low activity in fractions D and E,
and, if correct, it must be assumed that the heterochromatin has not been separated from the
nucleolus during the disruption of the nuclei in
this type of experiment.
The question remains whether there are two
different sites of incorporation of amino acids in
the nucleus, the nucleolus and heterochromatin, or
whether the nucleolar activity can be explained in
terms of residual heterochromatin not separated
Vol. 86
METABOLISM OF SUBNUCLEAR FRACTIONS
during nuclear disruption. This latter view is
supported by the results of a prolonged period of
disintegration in which nucleolar activity was
considerably decreased. In addition, the staining
reactions of nucleoli with acid haematin indicate
the presence of some heterochromatic phospholipids
even after such a prolonged disruption time. It is
concluded therefore that, although the site of
incorporation of amino acids in the nucleus is in the
region of the nucleolus, it is probably in the
associated heterochromatin and not the nucleolus
itself. Whether this state of affairs is true for the
incorporation of orotic acid into RNA is not yet
known, but, since the pattern of incorporation of
orotic acid in the. subnuclear fractions follows that
of incorporation of amino acids, it may well prove
to be localized in the heterochromatin region also.
The possibility of a close structural association
between the sites for nuclear protein synthesis and
RNA synthesis is of interest when considering
whether nuclear protein synthesis depends on the
presence of ribonucleoprotein particles as are
present in microsomes. Such microsomal particles
or ribosomes are characterized by their RNA and
protein composition, their sedimentability and
their ability to incorporate amino acids into protein
when fortified with 'pH 5 enzymes' from cell sap,
ATP, GTP, Mg2+ ions and an ATP-generating
system. Various groups of workers have described
nuclear ribonucleoprotein particles but it is
difficult to compare their results since different
methods of fractionation have been used.
Frenster, Allfrey & Mirsky (1960) described the
isolation of a range of ribonucleoprotein particles
from thymus-cell nuclei with certain similarities to
cytoplasmic ribosomes. The greatest incorporation
into both protein and RNA occurred in ribonucleoprotein particles that had a very low RNA: protein
ratio, a situation resembling that described in this
paper more closely than that in microsomes.
Szafranski, Wehr & Golaszewski (1961) obtained
three fractions from guinea-pig-liver nuclei of
which one was described as consisting of ribonucleoprotein particles. Although this fraction
incorporated amino acids in a system without an
added energy source, the supernatant fraction
(similar to fractions D and E described in the
present paper) was three times as active as their
so-called ribonucleoprotein particles.
Rendi (1960) isolated subnuclear fractions from
rat-liver by using deoxycholate and Lubrol to
disrupt the nuclei. However, when the nuclei were
fractionated after the incorporation of amino acids,
the most active fraction was not that consisting of
the so-called ribonucleoprotein particles but was
one without RNA or DNA. This fraction, moreover, incorporated amino acids without the addition of an external energy source.
135
It would appear, therefore, that the active
fractions described in the present work do not
correspond to the nuclear ribonucleoprotein
particles described by other workers since they are
very low in RNA. It appears, however, that similar
subnuclear material, low in nucleic acids, has been
isolated by these other workers and that this
material is often more active after incorporation
than the ribonucleoprotein particles. Unfortunately, none of the other workers have analysed
their subnuclear fractions for phospholipid, which
we have found to be a major constituent of the
active fractions D and E.
The presence in nuclei of certain enzymes (Rees
& Rowland, 1961) suggested that an electrontransport chain similar to the respiratory chain in
mitochondria may exist in rat-liver nuclei and may
play a role in the production of energy for synthetic reactions. Since the nucleoli and heterochromatin incorporate actively when isolated from
the rest of the nuclear material, they should be
rich in such enzymes if these enzymes are involved
in energy-yielding reactions necessary for the incorporation. The limited number of enzymes studied
in this respect all appear to be concentrated in
fractions D and E and all are very low in the
nucleoli. Although it appears that the site for
incorporation is localized in the heterochromatic
material it is difficult to understand why the
nucleoli that retain enough of this material to
incorporate actively do not also retain high concentrations of the enzymes. It must be concluded that
there is insufficient evidence at present to confirm
that an oxidative mechanism is responsible for the
energy production needed for the incorporation of
amino acids into protein in the nucleus.
SUMMARY
1. Rat-liver nuclei isolated in 0-25 m-sucrose
were disrupted by ultrasonic vibration and subjected to differential centrifuging to isolate nucleoli. The remainder of the disrupted nuclear
material was arbitrarily fractionated by centrifuging into four sedimentable fractions and a
supernatant.
2. Determinations of RNA, DNA, phospholipid
and protein, together with staining reactions of the
subnuclear fractions, suggest that a separation of
nucleoli, chromosomal material, a lipid-rich
material and nuclear sap was achieved.
3. Although all the subnuclear fractions will
incorporate 14C-labelled amino acids into protein
and [6-14C]orotic acid into RNA without an additional external energy source, the nucleoli and the
lipid-rich material have by far the greatest
activity.
4. There is high specific activity of certain
136
K. R. REES, G. F. ROWLAND AND J. S. VARCOE
oxidative enzyme systems in the lipid-rich fractions but they appear to be absent from nucleoli.
5. It is concluded that the lipid-rich material
may be the heterochromatin associated with the
nucleoli and that this is the active site for the
incorporation of amino acids by rat-liver nuclei.
We thank Professor C. Rimington, F.R.S., and Dr J.
Chayen for helpful discussions, and Dr P. S. Gahan for his
help with the histochemistry. The work was supported by
a grant from the British Empire Cancer Campaign.
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Darlington, C. D. & La Cour, L. F. (1960). The Handling of
ChroMnWome8, p. 143. London: Allen and Unwin Ltd.
Dische, Z. (1955). In The Nucleic Acids, vol. 1, p. 285.
Ed. by Chargaff, E. & Davidson, J. N. New York:
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Physicochemical Studies on Cytochrome b2
SEDIMENTATION, DIFFUSION AND ELECTROPHORESIS OF THE CRYSTALLINE
DEOXYRIBONUCLEOPROTEIN
BY J. McD. ARMSTRONG,* J. H. COATESt AND R. K. MORTON
Department of Agricultural Chemi8try, Waite Agricultural Research Institute, University
of Adelaide, South Australia
(Received 4 May 1962)
Cytochrome b2 is the L( + )-lactate-cytochrome c this paper. The molecular weight of the enzyme in
oxidoreductase [L( + )-lactate dehydrogenase] of solution has been determined by a number of
baker's yeast (Bach, Dixon & Zerfas, 1946; methods.
Appleby & Morton, 1954; Boeri, Cutolo, Luzzati &
MATERIALS AND METHODS
Tosi, 1955). The enzyme was obtained as a crystalline, apparently homogeneous, deoxyribonucleoGeneral
protein containing equimolecular amounts of riboCytochrome b2 (Type I). This was prepared from dried
flavin phosphate and of protohaem (Appleby &
yeast essentially as described by Appleby & Morton
Morton, 1954, 1959a, b, 1960). Both of these pros- baker's
(1959a). Solutions were stored under nitrogen at -15°.
thetic groups are reduced in the presence of lactate The
enzyme was recrystallized before use, and experiments
(Appleby & Morton, 1954; Hasegawa & Ogura, with any one sample were carried out within 3 days of
1961).
recrystallization. The enzyme was dissolved in buffer
Further physicochemical studies of the crystal- composed of (final concentrations) 03M-sodium lactate,
line deoxyribonucleoprotein (now known as Type I 0-05M-tetrasodium pyrophosphate and 0.1 mM-EDTA
cytochrome b2; Morton, 1961a) are described in (disodium salt), adjusted to pH 6*8 with hydrochloric acid.
Present address: Department of Physical Biochemistry, Australian National University, Canberra, Australia.
t Present address: Department of Physical and Inorganic Chemistry, University ofAdelaide, South Australia.
*
This buffer (I 0*63) was used to obtain the high ionic
strength necessary to give adequate concentrations of
cytochrome b2 (see Appleby & Morton, 1959a) and because
it was found that the enzymic activity was retained for
long periods in pyrophosphate buffer.
