21t
Biochem. J. (1974) 142, 211-219
Printed in Great Britain
The Development of Ribonuclease and Acid Phosphatase During
Germination of Pisum arvense
By GEOFFREY R. BARKER, CLIFFORD M. BRAY and TREVOR J. WALTER
Department ofBiochemistry, University ofManchester, Manchester M13 9PL, U.K.
(Received 25 January 1974)
1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic,
the time of appearance of the two phases depending on the conditions of growth. 2. Acid
phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development ofribonuclease activity in phase II but not in phase I. 4. '4C-labelled amino acids are
not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of
ribonuclease isolated during phase I is not affected by imbibition of the seed in 80%
deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the
cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase
activity during germination is inhibited by treatment of the seed with cycloheximide
or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in
the presence of '4C-labelled amino acids suggests that the enzyme is radioactively labelled.
9. Germination of peas in the presence of 80% deuterium oxide results in an increase
in the buoyant density of acid phosphatase. 10. The results suggest that increase in
ribonuclease activity during the first 4 days of germination does not result from synthesis
of protein de novo, but that the corresponding increase in acid phosphatase activity does
result from synthesis de novo.
A number of enzymic activities develop during the
early stages of the germination of peas. These include
a-amylase, proteinase, phosphatase (Young &
Varner, 1959), amylopectin 1,6-glucosidase (Shain &
Mayer, 1968) and ribonuclease (Barker & Hollinshead, 1967). The development of enzymic activity
during the germination of seeds may take place either
through activation of an existing protein or by
synthesis de novo. Thus activation of fl-amylase
has been demonstrated in wheat (Rowsell & Goad,
1962) and amylopectin 1,6-glucosidase in peas
(Shain & Mayer, 1968). Varner & Chandra (1964)
found that radioactively labelled amino acids are
incorporated into a-amylase preparations from
germinating barley, but the correctness of the
conclusion that the enzyme is synthesized de novo
depends on the completeness of the purification of
the enzyme. That the enzyme is synthesized de novo
was more clearly demonstrated by the use of heavy
isotopes in conjunction with centrifugation in CsCl
(Filner & Vamer, 1967). The same technique has
been used for studies of proteinase in barley (Jacobsen
& Vamer, 1967), isocitrate lyase in peanuts (Longo,
1968; Gientka-Rychter & Cherry, 1968) and malate
synthetase in peanuts (Longo, 1968). The development of ribonuclease II (EC 3.1.4.23) activity in
germinating peas is of particular interest by virtue
of its biphasic nature, and the present report is
concerned in the first place with this enzyme. Since
experiments indicated the absence of synthesis of
Vol. 142
ribonuclease during phase I of the activity, it was
considered desirable to confirm the validity of the
experimental techniques used by examination of an
enzyme produced by synthesis de novo under similar
conditions of germination. Preliminary experiments
indicated that this is the case with acid phosphatase
(EC 3.1.3.2), and this enzyme has therefore been
studied in detail by using the same techniques as for
ribonuclease.
Experimental
Materials
Cotyledons were removed from seed of Pisum
(var. Minerva Maple) (supplied by Messrs.
Gartons Ltd., Warrington, U.K.), germinated as
previously described (Barker & Hollinshead, 1964)
unless otherwise stated. -4C-labelled protein hydrolysate from Chlorella was purchased from The Radiochemical Centre, Amersham, Bucks., U.K. Deuterium
oxide was purchased from I.C.I. Ltd., Agricultural
Division, Northwich, Cheshire, U.K., and was diluted
with sterile water. CsCl was optical grade, purchased
from Harshaw Chemical Co., Cleveland, Ohio,
U.S.A. Yeast RNA (British Drug Houses Ltd.,
Poole, Dorset, U.K.) was used in the assay for
ribonuclease and was purified as follows. RNA
(2g) was dissolved in approx. 50ml of 50mM-TrisHCI buffer, pH7.0, containing IOmM-MgCI2. The
arvense
212
G. R. BARKER, C. M. BRAY AND T. J. WALTER
solution was shaken with an equal volume of 90%
(v/v) phenol containing 0.1 % (w/v) 8-hydroxyquinoline for 15min at 0°C. The solution was centrifuged
at 0°C at 2000g for 15min and the upper layer was
removed and treated with phenol as described above.
The upper layer was removed, deoxyribonuclease
(Sigma Chemicals Co. Ltd., London S.W.6, U.K.)
was added to a final concentration of 5,ug/ml and the
mixture was kept at 0°C for 15min. Protein was then
removed by using 0.5vol. of phenol solution as
described above. This treatment was repeated until
no protein was visible at the interface after shaking
with phenol. RNA was precipitated from the upper
layer by adding 2vol. of 95 % (v/v) ethanol containing
0.3 M-potassium acetate. The precipitate was collected
by centrifugation, washed with 95% (v/v) ethanol,
ethanol-ether (1 :1, v/v) and ether and dried under
reduced pressure.
Methods
Measurement ofribonuclease activity. Ribonuclease
activity was determined by a modification of the
method of Dickman et al. (1956). Enzyme solution
(1 ml) in 0.1 M-sodium citrate buffer, pH 5.6, containing 0.01 % (w/v) gelatin was incubated with 0.5ml
of 1 % (w/v) RNA in the same buffer for 30min at
37°C, and 0.5ml samples were withdrawn before and
after the incubation and added with vigorous shaking
to 1.5ml of acetic acid-2-methylpropan-2-ol (1:2,
v/v). The mixture was kept at -18°C for 1.5h and
then centrifuged at 2000g for 10min at 4°C. Tubes in
which the supernatant was opalescent were either
discarded or, if possible, re-centrifuged to give a clear
supernatant. Clear supernatant (0.Sml) was diluted
with 5.Oml of water and the extinction was measured
at 260nm. One unit of ribonuclease activity is defined
as the amount of enzyme that causes an increase in
E260 of 1.0 under the above conditions.
Measurement of phosphatase activity. Acid phosphatase was determined by a modification of the
method of Torriani (1960). Enzyme solution (1.6ml)
in 0.2M-sodium acetate buffer, pH 5.6, was incubated
with 0.4ml of 0.04M-p-nitrophenyl phosphate (BDH
Biochemicals Ltd., Poole, Dorset, U.K.) in the same
buffer for 10min at 37°C. The reaction was stopped
by the addition of 2.Oml of 1.0M-Tris-HCl buffer,
pH8.5, containing 0.4M-KH2PO4. Blank readings
were obtained by addition of the Tris-phosphate
solution to the incubation mixture before addition
of the enzyme extract. The extinction of the solution
at 420nm was measured. One unit of phosphatase
activity is defined as the amount of enzyme that
causes an increase in E420 of 1.0 under the above
conditions.
Preparation of tissue extracts. Previous experiments
(Barker & Hollinshead, 1967) had shown that
ribonuclease activity is confined to the supernatant
fraction. For determinations of ribonuclease activity,
cotyledons from five plants were ground with 10ml of
0.1 M-sodium citrate buffer, pH6.6, and a little acidwashed sand. The homogenate was filtered through
muslin and the filtrate was centrifuged at 10OOg for
5min at 4°C. The supematant was decanted, the
volume recorded and samples were removed for
enzyme determination. For measurements of phosphatase activity, cotyledons from ten plants were
ground with 20ml of 0.1 M-Tris-HCl buffer, pH7.4
(measured at 20°C), and a little acid-washed sand.
The homogenate was filtered through muslin and the
filtrate was further centrifuged for 1 h at 20000g.
The supernatant was decanted, the volume recorded
and samples were withdrawn for enzyme measurement.
Measurement of radioactivity. Radioactivity was
determined by using a Packard Tri-Carb model 3001
liquid-scintillation counter. The scintillation solution
consisted of 0.04 % (w/v) 1,4-bis-(4-methyl-5-phenoxazol-2-yl)benzene and 0.4% (w/v) 2,5-diphenyloxazole in xylene containing 30% (w/v) ethanol.
Counting efficiency was estimated by internal
standardization with ['4C]hexadecane to be 38-41 %.
Radioactivity in polyacrylamide-gel slices was
determined by the method ofTishler & Epstein (1968)
except that gel slices were incubated with 0.3ml of
Hyamine hydroxide (Nuclear Enterprises Ltd.,
Edinburgh, U.K.) for 1 h at 45°C before addition
of 10ml of liquid scintillator.
Experiments with deuterium oxide. Peas were
surface-sterilized in a 1 % (w/v) solution of sodium
hypochlorite for 10min and allowed to germinate
under aseptic conditions on absorbent paper
moistened with 30ml of 80% (v/v) 2H20 or with H20
in a 1000ml Erlenmeyer flask. The neck of the flask
was plugged with cotton-wool and the seeds were
allowed to germinate for the required time. Cotyledons of seed germinated for 4 days under these conditions in 80% 2H20 had 85% of the ribonuclease
activity and 65% of the acid phosphatase activity
of seed germinated in H20.
To establish the applicability of centrifugation in
CsCl to ribonuclease and acid phosphatase, 5mg
portions of partially purified iibonuclease and acid
phosphatase were separately dissolved in Sml of
0.05M-sodium acetate buffer, pH5.6. Insoluble
material was removed by centrifugation at 2000g for
5min and 2ml samples of the supernatant were
mixed with either 2ml of water or 2ml of 30% (w/v)
CsCl at room temperature. Samples from each
solution were taken immediately, and also after being
maintained at 20°C for 67h, and enzymic activities
were measured. The acid phosphatase activity of
both control and CsCl-treated samples decreased by
35% compared with the original acid phosphatase
activity of the solution. The ribonuclease activity of
the solutions showed virtually no decrease at all over
1974
213
RIBONUCLEASE AND ACID PHOSPHATASE IN GERMINATING PEAS
the 67h incubation period. Thus both enzymes appear
to be sufficiently stable in CsCl solutions over a 67h
incubation period at 20°C, i.e. under the conditions
of an equilibrium density-gradient-centrifugation run.
In one experiment, samples from the gradient were
assayed for carbohydrate by the anthrone method as
described by Filner & Varner (1967).
Results
Development of ribonuclease activity during germination
Fig. 1 shows the biphasic development of ribonuclease activity in the cotyledons of seed imbibed
in water for 24h and germinated in moist vermiculite.
Imbibition of the seed in a solution containing 5,ug
of cycloheximide/ml and watering with the same
solution during germination does not prevent the
initial rise of enzymic activity but abolishes phase II
(Fig. 1). Retardation of growth by germination at
2-4°C results in slower development of both phases
of ribonuclease activity (Fig. 1).
Purification ofribonuclease
Seeds were imbibed in water containing lOO,Ci of
'4C-labelled protein hydrolysate from Chlorella and
germinated for 4 days as described above. A homogenate was prepared by using cotyledon tissue (30g)
4
as described above. It was filtered through two layers
of muslin and the filtrate was centrifuged at lOOOOg
for 10min at 4°C. The supernatant was decanted and
adjusted to pH4.5 by the addition of 1 M-HCI. After
standing for 1 h the suspension was centrifuged at
lOOOOg for 10min, the supernatant decanted and
adjusted to pH5.5 by the addition of 1 M-NaOH.
Solid (NH4)2SO4 was added to give 40% saturation
and, after being left at room temperature for 15min,
the suspension was centrifuged at lOOOOg for lOmin
and the precipitate was discarded. Solid (NH4)2SO4
was added to the supernatant to give 60% saturation
and the precipitate was collected by centrifugation
as above. This material contained 61 % of the enzymic
activity of the crude extract and had a specific activity
of 0.196 unit/mg, representing approximately sixfold
purification. The precipitate was dissolved in 0.1 Msodium citrate buffer, pH5.6 (20ml), and dialysed
for 24h against water (two changes of 5 litres each).
Any precipitate formed was removed by centrifugation at 2000g for 10min and the supernatant freezedried. A portion (100mg) of this material was dissolved in 2ml of 0.005M-sodium phosphate buffer
(pH6.8) and loaded on to a column (12cmx 1.3 cm)
of hydroxyapatite previously equilibrated with the
same buffer. Initial elution of the column was with
0.005M-sodium phosphate buffer, pH6.8, and the
concentration of this buffer increased in a stepwise
manner to 0.025, 0.05, 0.1, 0.2 and 0.3M at the same
pH. The flow rate was 40ml/h. Fractions (9ml) were
(a)
._
04
-0
'40
0
.4)
3
(b)
16
la
.
Z;
2
3
CU
14
9'
PO 1o
2
I.
c)
-UCd
4*
8
0
c)
Ea
CUC
6
+4
c)
0
._
0
0
40
to
30
20
10
6
8
4
12
Age of cotyledons (days)
Age of cotyledons (days)
Fig. 1. Development of ribonuclease activity in cotyledons ofgerminating peas
2
50
a) Seed imbibed and germinated at 26°C; (b) seed imbibed and germinated at 2-40C; *, imbibed and germinated in H20;
imbibed and germinated in presence of cycloheximide (5jug/ml). The vertical bars represent S.D. (four determinations)
for the values shown.
A,
Vol. 142
2, R. BARKER, C. M. BRAY AND T. J. WALTER
214
0.OSm
0.025m
0.0sm
0.iM
0.2M
0.3 M
C.)
CU
30 nL
o
C.)~ ~ ~ ~ ~ ~ ~ ~ aC
2gCU ~10 n'
o lo
0
1O
20
20
3~ ~ ~ ~ -0
-0
30
10 .0
v
0
Fraction no.
Fig. 2. Chronatography of ribonuclease from pea cotyledons on hydroxyapatite
Proteins precipitated from cotyledon extracts with 60%-saturated (NH4)2SO4 were adsorbed on a column (l2cmx 1.3cm)
of hydroxyapatite and eluted with sodium phosphate buffers (pH6.8) of increasing concentrations as indicated by the
arrows. Fractions (9 ml) were collected and samples taken from each for measurements of E280 (A), radioactivity (@) and
ribonuclease activity (n).
collected and measurements were made of E280,
radioactivity and ribonuclease activity. The results
are shown in Fig. 2. Fractions 2-4 were poQled,
dialysed for 24h against water and freeze-dried. The
enzyme fraction eluted from the column had a specific
activity of 4.04 units/mg, corresponding to a 14-fold
purification relative to the crude extract. A portion
(12mg) of this material was dissolved in 2ml of
O.OlM-Tris-HCl buffer, pH7.8 (measured at 20°C),
and loaded on to a column (l0cmx 1.3cm) of
DE-32 DEAE-cellulose (Reeve Angel and Co. Ltd.,
London EC4V 6AY, U.K.) previously equilibrated
with the same buffer. Initial elution of the protein was
with 0.01 M-Tris-HCl buffer, pH 7.8, and subsequent
stepwise elution with NaCl concentrations of 0.025,
with a current of 4mA/gel tube. Gels, stained with
Amido Black, showed the presence of seven bands.
Measurement of ribonuclease activity in slices of
unstained gels indicated a peak of ribonuclease
activity in the same region as three of the stained
bands (Fig. 4). The peak of activity coincided with
a minimum in radioactivity. It was therefore not
possible to ascertain which of the bands contained
enzyme activity and to determine with certainty
whether the enzyme was radioactively labelled, in
spite ofthe fact that enzymic activity and radioactivity
appeared to coincide during elution fromn hydroxyapatite and DEAE-cellulose.
0.05, 0.075, 0.1, 0.2 and 0.3M in 0.01 M-Tris-HCl
buffer, pH7.8. The flow rate was 40ml/h. Fractions
(7.5 ml) were collected and measurements were made
of E280, radioactivity and ribonuclease activity. The
results are shown in Fig. 3. Fractions containing
ribonuclease activity were pooled, dialysed for 24h
against water and freeze-dried. Ribonuclease eluted
in these fractions had a specific activity of 13.5 units/
mg, corresponding to a 46-fold purification relative
to the crude extract. The yield of ribonuclease was
47%.
The freeze-dried material obtained from the DEAEcellulose column was dissolved in 0.25ml of 20%
(w/v) sucrose and subjected to clectrophoresis on
polyacrylamide gel at pH 8.9 by the mnethod of Davis
(1964). Electrophoresis was continued for 50min
Attempted density labelling ofribonuclease
Seeds were germinated in 80 % (v/v) 2H20. After 4
days, cotyledons were removed (20g) and homogeniized in 50ml of 0.1 M-sodium citrate buffer, pH 5.6,
at 2-4°C. Ribonuclease was precipitated with 60%saturated (NH4)2SO4, the precipitate was redissolved
in 2ml of 0.05M-sodium acetate buffer, pH 5.6, and
0.1 ml samples were layered over 4.5 ml of CsCl
solution (density 1.358 g/cm3) and centrifuged at
49000rev./min for 67h at 250C in the SW65 rotor in
Beckman L2-65B centrifuge. Similar samples were
centrifuged at the same time from peas germinated
in H20. Fractions were collected from the centrifuge
tubes and assayed for ribonuclease activity. The
results are shown in Figs. 5(a) and 5(b). There is no
1974
215
RIBONUCLEASE AND ACID PHOSPHATASE IN GERMINATING PEAS
C
Go
N
20
*
c.
;
10
'r3
Zd
I'
Fraction no.
Fig. 3. Chromatography ofribonuclease from pea cotyledons on DE-32 DEAE- cellulose
Protein having ribonuclease activity eluted from hydroxyapatite was dissolved in 0.01 M-Tris-HCl buffer, pH7.8, absorbed
on a column (10cm x 1.3 cm) of DE-32 DEAE-cellulose and eluted with the same buffer containing increasing concentrations
of NaCl as indicated by the arrows. Fractions (7.5ml) were collected and samples taken from each for measurements of E280
(A), radioactivity (@) and ribonuclease activity (m).
>6
Development of acid phosphatase activity during
+-A
:E
2
9 -e
9:5
cd
-
(a)
---
4)
'90
rA4.,
00,
0
04
-mRv
I,
I
.
.,-I2
2001-
(b)
l
l
I
I
+
l
+
444444
.00
5
1oo
-g .0;a
g
,
(b)
0
2
I
II
I
4
Distance into smnall-pore gel (cm)
Fig. 4. Electrophoresis of ribonuclease from pea cotyledons
on polyacrylamide gel
Fractions having ribonuclease activity eluted from DE-32
DEAE-cellulose were freeze-dried, dissolved in 0.25ml
of 20% (w/v) sucrose and submitted to electrophoresis on
polyacrylamide gels at pH8.9. Gels were sliced (2mm) and
examined for ribonuclease activity (a) and radioactivity (b).
Arrows indicate the locations of stained bands in the gel.
germination
Fig. 6 shows the development of acid phosphatase
activity in the cotyledons of seeds imbibed and
germinated as described above. The acid phosphatase
activity in the 20000g-supernatant fraction of the
cell homogenate showed a 100% increase between
the second and fourth days of germination, remnaining
fairly steady before and after this period. The acid
phosphatase activity in the 20000g pellet showed a
slight decrease over the same period of germination.
Imbibition and germination of seed in a solution
containing 5 ug of cycloheximide/ml as previously
described abolished almost completely the development of acid phosphatase activity in the germinating
cotyledons (Fig. 6).
Dry pea seeds (50g) were vacuum-infiltrated for
30min with either water or a solution containing
lOOpg of actinomycin D/ml to facilitate removal of
the testa from the pea seeds. The seeds, minus the
testa, were then allowed to imbibe normally in water
or actinomycin D solution for a further 24h and
germinated in moist vermiculite. The vermiculite was
moistened daily with water. Fig. 6 shows the inhibition
of development of acid phosphatase activity by
actinomycin D.
nuclease from seeds germinated in H20 and 2H20,
Incorporation of '4C-labelled amino acids into acid
phosphatase
and consequently no evidence of synthesis of the
enzyme de novo during the first 4 days of germination.
Dry seed (53g) was surface-sterilized for 10min
in 1% (w/v) sodium hypochlorite and imbibed in
significant difference between the densities of ribo-
Vol. 142
G. R. BARKER, C. M. BRAY AND T. J. WALTER
216
0
'0
0.075
'.5
0.05
1.4
1.3
0.025
1.2
Z-
30 r
CU
a
0
S0 20
C)
o
._a
a
..
U,
CU
C)
Cd
1.5
I0o
c
04
cn
. 0. 05
1.4
0
04
1.3
._
c)
1.2
0
10
20
30
Fraction no.
40
50
Fig. 5. Equilibrium density-gradient centrifugation of
ribonuclease from cotyledons of peas germinated in H20
and 2H20
Seeds were imbibed in H20 (a) or 80% 2H20 (b) and grown
as described in the text. The cotyledons were homogenized
and ribonuclease was precipitated with 60%-saturated
(NH4)2SO4. The precipitate was redissolved in 0.05Msodium acetate buffer (pH 5.6) and layered on a solution
of CsCl (density 1.358 g/cm3). Solutions were centrifuged at
49000rev./min for 67h at 25°C. Samples from the gradient
were collected and assayed for ribonuclease activity (n).
The density of the fractions (o) was calculated from refractive-index measurements.
80ml of water containing JOO,uCi of 14C-labelled
protein hydrolysate from Chlorella for 17h. The seeds
were then placed in moist vermiculite and watered
daily. After 4 days, cotyledons were removed and a
tissue extract was prepared as described above. Solid
(NH4)2SO4 was added to give 40% saturation and,
after being left at room temperature for 15min, the
suspension was centrifuged at 20000g for 10min and
the precipitate was discarded. Solid (NH4)2SO4 was
added to the supernatant to give 70% saturation
and the precipitate was collected by centrifugation as
above. This material contained 52 % of the enzymic
activity of the crude extract. It had a specific activity
of 0.27 unit/mg, representing approximately twofold
purification. The precipitate was dissolved in water,
dialysed for 24h against two changes of water
(5 litres each), the small precipitate was removed by
0
5
10
Age of cotyledons (days)
Fig. 6. Development of acidphosphatase activity in cotyledons of germinating peas
Seeds were imbibed and germinated, as described in the
text, in water (a), cycloheximide (5,cg/ml) (A) or actinomycin D (lOO,pg/ml) (m). The vertical bars represent
S.D. (four determinations) for the values shown.
centrifugation at 20000g for 10min and the supernatant was freeze-dried. A portion (100mg) of this
material was dissolved in 3ml of 0.01M-Tris-HCI
buffer, pH7.0, (measured at 20°C), and introduced
into a column (25cmx1.6cm) of DEAE-cellulose
(DE-32 cellulose) previously equilibrated at 2-40C
with the same buffer. The column was eluted at 2-40C
with a linear gradient of 0-0.3M-NaCl containing
0.01M-Tris-HCl buffer, pH7.0. The flow rate was
16.5ml/h. Fractions (5ml) were collected and E280,
phosphatase activity and radioactivity were measured.
The results are shown in Fig. 7. Fractions 14-22 were
pooled, dialysed for 24h against water and freezedried. Of the enzymic activity introduced on to the
column 70% was recovered. The product had a
specific activity of 7.6 units/mg, corresponding to a
25-fold purification relative to the crude extract.
A portion (0.2mg) of the freeze-dried material
obtained
dissolved
subjected
at pH4.3
from the DEAE-cellulose column was
in 0.05ml of 20% (w/v) sucrose and
to electrophoresis on polyacrylamide gel
by the method of Reisfeld et al. (1962).
Electrophoresis was continued for 1 h with a current
of 4mA/gel tube. Gels stained with Amido Black
1974
217
RIBONUCLEASE AND ACID PHOSPHATASE IN GERMINATING PEAS
*_
.
._
-i5
o
O5
it
l
0
0~
,
Fraction no.
Fig. 7. Chromatography of acidphosphatase from pea cotyledons on DE-32 DEAE-cellulose
Protein precipitated by 70%-saturated (NH4)2SO4 was dissolved in 0.01 M-Tris-HCl buffer (pH7.0) and chromatographed
on a column (25cmx 1.6cm) of DEAE-cellulose. Elution was by a linear gradient of 0-0.3M-NaCl containing 0.01 M-TrisHCl buffer (pH7.0). The flow rate was 16.5ml/h. Fractions (5ml) were collected and samples taken from each for measurements of E280 (A), radioactivity (@) and acid phosphatase activity (o).
29
0.50
o.
results suggest that the development of acid phosphatase activity in the first 4 days of germination
results from synthesis of protein de novo.
_ (a)
0.25
C)
*;
0
-
.,-4
400
io
(b)
,
200
._
I
2
Distance into small-pore gel (cm)
Fig. 8. Electrophoresis of acid phosphatase from
pea
cotyledons on polyacrylamide gel
Fractions having acid phosphatase activity eluted from
DEAE-cellulose were combined, dialysed against water
and freeze-dried. Freeze-dried material (0.2mg) was
dissolved in 0.05ml of 20% (w/v) sucrose and submitted
to electrophoresis on polyacrylamide gels at pH4.3.
Gels were sliced (1 mm) and examined for acid phosphatase
activity (a) and radioactivity (b). Arrows indicate the
locations of stained bands in the gel.
showed the presence of five bands. Measurement of
phosphatase activity in slices of unstained gels
indicated that activity was located in one band only.
Fig. 8 shows the distribution of radioactivity and acid
phosphatase activity in 1mm slices of the gel. The
Vol. 142
Density labelling of acid phosphatase
Seeds were germinated in 80 % (v/v) 2H20. At this
concentration the effect of 2H20 on the developmeilt
of acid phosphatase activity in germinating pea
cotyledons appeared to be to diminish the rate of
appearance of acid phosphatase activity without
affecting the final enzyme activity in the cotyledons
during the germination period studied. A similar
effect of 2H20 on the development of isocitrate lyase
activity in germinating peanut cotyledons has also
been noted (Gientka-Rychter & Cherry, 1968).
After 7 days (for peas germinated in 2H20) or
4 and 7 days (for peas germinated in H20), cotyledons
were removed (15g) and homogenized in 50ml of
0.2M-sodium acetate buffer, pH5.6, at 2-40C. Acid
phosphatase was precipitated by (NH4)2S04 at 70%
saturation, as described above, and the precipitate
redissolved in lOml of 0.2M-sodium acetate buffer,
pH5.6, at 2-4°C. Samples (0.1 ml) were layered over
4.5ml of CsCl solution (starting density 1.358 g/cm3)
and centrifuged at 49000rev./min for 67h at 25°C in
the SW65 rotor in a Beckman L2-65B preparative
ultracentrifuge. After centrifugation the gradients
were fractionated into 5-drop fractions (approx.
O.1ml) and the fractions assayed either for acid
phosphatase activity or used in the determination of
refractive index with a Zeiss-Abbe refractometer.
The density of the fractions could be found from a
standard curve relating refractive index (nv) to
G. R. BARKER, C. M. BRAY AND T. J. WALTER
218
4 days appears not to involve enzyme synthesis de novo
because it is unaffected by cycloheximide.
p=1.360
The above conclusions regarding acid phosphatase
and the first phase of ribonuclease activity receive
0.02
02
support from experiments involving radioactive and
1.3
density labelling. During the purification of acid
phosphatase from seeds germinated in the presence of
.0
1.2 J O~ ~ ~ ~.
S .
O ~~~~~~ ,
@
2
'4C-labelled amino acids, peaks of radioactivity and
ce 0
enzymic activity were coincident in polyacrylamide
0.2 _
p=1.387
(b)
gels. No analogous unambiguous correlation of
peaks of radioactivity and enzymic activity was
.5~
observed during the purification of ribonuclease
from seed after 4 days of germination. The absence of
0. oI
- 1.4
change in the buoyant density of ribonuclease isolated
from seed germinated in 80% 2H20 provides no
evidence for enzyme synthesis de novo. A shift of
and 21.3
1.62% in the buoyant density of acid phosphatase
~~~~~~~~~~~~~1.2from
0
seeds germinated in 80% 2H20 compared with
0
7
d ip=(.360
that from seeds germinated in H20 is consistent with
- 1.5
(c)
synthesis of the enzyme protein de novo. Alternative
interpretations are excluded by the facts first that
0.2
- 1.4
carbohydrate material differed in position in the
density gradient from that of acid phosphatase
(Fig. 9a) and secondly that partial purification of the
-1.2
enzyme in H20 medium eliminates possible effects
of deuterium-hydrogen exchange. Moreover, the
40
50
30
0
10
20
buoyant densities of acid phosphatase isolated after
Fraction no.
germination for 4 days and 7 days were identical,
indicating that the observed increase in buoyant
9.
Fig. Equilibrium density-gradient centrifugation of acid
phosphatase from cotyledons of peas germinated in H20
density was not due to the longer time required for
and 21H20
germination in 2H20. These results clearly demonstrate
synthesis of acid phosphatase de novo during
Seeds were imbibed and grown for 4 days in H20 (a) or for
germination of the pea seed. The occurrence of some
7 days in 2H20 (b) or 1120 (c). The cotyledons were
concomitant activation of a pre-existing protein is
homogenized and acid phosphatase was precipitated
unlikely, since this would result in broadening the
with70fa -saturated (NH4)2S04. The precipitate was
redissolved in 0.2m-sodium acetate buffer (pH5.6),
peak of activity of acid phosphatase isolated from
layered on to a solution of CsCl and centrifuged as
seed germinated in 2H20, and this was not observed.
described in the text. Samples from the gradient were
assayed for acid phosphatase activity (an) and for carboGrateful acknowledgement is made to the Science
hydrate (Filner & Varner, 1967), (E650 El). The density of
Research Council for the award of studentships to
the fraction (0) was calculated from measurements of
T. J. W. and C. M. B., to the Agricultural Research Council
refractive index.
for financial support and to Mrs. I. Dimond for excellent
0.04
(a)
1.5
1.4
technical assistance.
density (p25). The results shown in Fig. 9 confirm the
above conclusion that acid phosphatase is synthesized
de novo during germination.
Discussion
The presence of cycloheximide in the medium used
for imbibition prevented the development of acid
phosphatase activity during the first 6 days of
germination and ribonuclease activity from the sixth
day onwards. These activities are therefore believed
to arise by enzyme synthesis de novo. In contrast, the
development of ribonuclease activity during the first
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