Archives of

Microbiology

Arch. Microbiol. 117, 2 7 - 34 (1978)

9 by Springer-Verlag 1978

Amino Acid Pool and Protein Turnover

during Differentiation (Spherulation) of Physarumpolycephalum
GERTRUD WENDELBERGER-SCHIEWEG and ALOYS H1JTTERMANN
Forstbotanisches Institut der Universitfit G6ttingen,
Biisgenweg 2, D-3400 G6ttingen-Weende, Federal Republic of Germany

Abstract. The composition of the amino acid pool

during spherulation was determined. It changes in size
and in composition, the concentration of each amino
acid behaving individually. The first response to the
onset of spherulation either by starvation or osmotic
shock (0.5 M mannitol) always is a decrease of the
pool's size, which during further starvation expands for
a short period and then decreases again. During development induces by mannitol in the presence of
external amino acids, the pool size increases continuously after the initial depletion.
As shown by radioactive labeling, amino acids
were actively released from the plasmodium into a
medium containing amino acids, but retained by the
microplasmodia in an amino acid-free medium. The
kinetics of the uptake of radioactive amino acids from
the medium is biphasic, indicating the existence of
multiple pools. Even after a labeling period of 8 h
the amino acid pool is not yet in equilibrium with the
medium. The possibility of a compartimentation of
the pool was confirmed by density labeling of two
different enzymes.
Whereas the turnover of total protein is only very
low during growth, it is rather high in spherulating
microplasmodia. At least 70 ~ of the originally existing protein is degraded during this development, while,
simultaneously, at least 50 ~ of the protein present
after 24 h starvation is newly synthesized during that
period.
Key words: Amino acid pool - Protein turnover Differentiation - Physarumpolycephalum - Spherulation- Pool compartimentation- Density labelingde novo enzyme synthesis.

by a variety of metabolic changes (Goodman and

Rusch, 1969; McCormick et al., 1970a, b; Mohberg
and Rusch, 1971; Htittermann, 1973; Goodman and
Beck, 1974; Zaar and Kleinig, 1975).
Especially conspicuous is a significant loss of protein of about 55 ~ within 32 h starvation (Sauer et al.,
1970). Some individual proteins, however, are synthesized de novo during that differentiation (Hfittermann etal., 1971, 1975; Hfittermann, 1972; Hoffmann and Hfittermann, 1975).
At present no detailed data are available on the
changes of the amino acid pool and the turnover of
total protein during starvation. Such an investigation
of possible changes is important for correct interpretation of any labeling experiment which should
prove whether the spherulation is initiated by differential gene expression (Sussman, 1966) or by metabolic
changes (Wright, 1973). In addition, it will give insights into this area of metabolism which apparently
is rather important for the differentiation of Physarum
polycephalum to spherules.
MATERIALS AND METHODS

Chemicals
U-14C-protein hydrolysate of Chlorella was purchased from Amersham Buchler, Braunschweig, West Germany. Unisolve was obtained from Koch-Light Laboratories Ltd., Colnbrook Bucks.,
England. The deuterated amino acids came from Sharp and Dohme,
Mfinchen, West Germany. The biochemicals were obtained from
Boehringer-Mannheim, Mannheim, West Germany. All other
chemicals were bought from E. Merck, Darmstadt, West Germany.
fl-Glucuronidase from beef liver was a product of Serva, Heidelberg, West Germany.

Cultures
spherulation of Physarumpolycephalum induced by
starvation in nonnutrient salts medium is accompanied

In this study the strain M3C IV of Physarumpolycephalum was

used. This organism was grown in a semidefined medium containing
yeast extract, tryptone, and heroin, as reported by Daniel and Baldwin (1964). For the labeling experiments tryptone was omitted and

0302-8933/78/0117/0027/$ 01.60

28
supplemented with either light, radioactive or heavy amino acid
mixture described below. The induction of spherulation by transferring the microplasmodia to a nonnutrient salts medium was
carried out according to Daniel and Baldwin (1964), the induction
of spherulation in a synthetic medium (16 amino acids, glucose,
salts vitamins, and hemin) supplemented with 0.5 M mannitol
according to Chet and Rusch (1969). Our transfer schedules and
special culture conditions were described earlier (Htittermann, 1972).

Isotopes
Labeling was done with a mixture of amino acids (10 mg amino
acids/ml medium). The composition of the mixture was the same
for both, density and radioactive labeling, containing per litre
medium: 529 mg lysine-HC1, 155 mg histidine-HC1-H20, 470 mg
arginine, 1028 mg aspartic acid, 500 mg threonine, 476 mg serine,
1058 mg glutamic acid, 632 mg proline, 604 mg glycine, 839 mg
alanine, 520 mg cysteine-HCi-H20, 763 mg valine, 193 mg methionine, 472 mg isoleucine, 1105 mg leucine, 118 mg tyrosine, and
598 mg phenylalanine. For radioactive labeling 10 mg amino acids
mixture was supplemented by 0.625 gCi U-14C-protein hydrolysate.
For density labeling experiments this mixture contained amino
acids with deuterium.

Labeling Experiments
Cultures growing in the log-phase were harvested and washed
three times with nonnutrient salts medium. The microplasmodia
were then transferred either to growth medium (without tryptone)
or to nonnutrient salts medium. Each culture then was labeled by
the addition of the corresponding amino acids mixture (10 mg/ml).
The cultures were harvested by centrifugation for 1 rain at 500 x g,
and the supernatant (-= medium) was counted for radioactivity.
The microplasmodia were washed four times with salts medium
and then extracted.

Extraction Procedures

Arch. Microbiol., Vol. 117 (1978)

tillation analyzer-which corrected efficiencies so that d/min were
obtained.
The amount of amino acids were determined according to Stegmann (1960).
Proteinwas measured by the Lowry-method (Lowry et al., 1951).
The amino acid composition was determined in 0.2 N sodium citrate
buffer, pH 2.2, in a Beckman Unichrome amino acid analyzer. The
determinations were kindly done by Prof. Dr. Hilschmann, MaxPlanck-Institut for experimentelle Medizin, G6ttingen, Germany.

Analysis of Density Labeling Experiments

in Rubidium Chloride
Gradients were made according to the method of Hu et al. (1962).
The extract of Physarumpolycephalum plus the appropriate amount
of 0.1 M tris-acetate-buffer, pH 7.1 was brought to a final concentration of 35 ~ (w/w) RbC1 solution by adding solid RbC1. As a
density marker/~-glucuronidase from beef liver was used.
5 ml of this solution were centrifuged for 65 h in a SW 50.1
rotor at 250000 x g (48000 rpm) at 4 ~C in a L2-65B ultracentrifuge
(Spinco). After the run, about 65 fractions of 5 drops (0.08 ml)
each were collected, the gradients being dripped from the bottom.
The refractive index of every tenth fraction was determined at 25 ~C
using an Abb6-refractometer (Zeiss), the other fractions were
assayed immediately for enzymic activities. The densities of the
fractions were calculated from the refractive indices using the
following formula (Wendelberger-Schieweg, 1977):
Q25 = 9.3282

n 25

--

11.4558.

Determination of Enzymic Activities

The activity of Glutamatedehydrogenase was assayed according to
Hfittermannet al. (1971) of glucose-6-phosphate dehydrogenase
according to Hfittermann et al. (1970). The activity of/Lglucurouidase was measured according to Bergmeyer et al. (1974), with
4-nitrophenyl-/Lglucuronopyranoside in acetate-buffer, pH 4.5, as
substrate.

For analysing the density labeling experiments extracts were made

according to Htittermann and Gebauer (1973).

For determining the composition of the amino acidspool of the

microplasmodia as a whole about 1 g (wet weight) microplasmodia
were disrupted by sonication (Branson sonifier B-12, intensity 10)
in 20 ml 80 ~ ethanol for 1 min. The homogenates were filled up
with 80 ~ ethanol to 100 ml and refluxed for 20 min. Cell fragments
and precipitated proteins were pelleted by centrifugation at 50 000 x g
for 20 rain at 4~ C, and reextracted in 80 ~ ethanol. The combined
supernatants were evaporated at 50~ and the residues dissolved
in 10 ml 0.2 N sodium citrate buffer, pH 2.2.
For determining the specific radioactivity the washed microplasmodia were resuspended in 10 ml distilled water and sonicated.
An equal volume of trichloroacetic acid in acetone (10~o w/v) was
added to the homogenates and the mixture left for at least 3 h at
0~C. After centrifugation at 50000 x g for 15 rain the supernatant
was used to determine the specific radioactivity of the amino acids.
The pellet was washed three times with the trichloroacetic acid
solution and then hydrolyzed in 10 ml 0.4 N NaOH.
Determination of Radioactivity,
Amount of Amino Acids and Protein,
and of Composition of Amino Acids
For determiningradioactivity0.1 ml solution was mixed with 10 ml
Unisolve. The radioactivity was counted in a Philips liquid scin-

RESULTS

1. Size and Composition

of the Amino Acid Pool during Spherulation
F o r t h e e x a m i n a t i o n o f c h a n g e s in size a n d c o m p o sition of the amino acid pool during spherulation
m i c r o p l a s m o d i a w e r e h a r v e s t e d a f t e r 6, 12, 18, a n d
24 h a f t e r t h e t r a n s f e r to n o n n u t r i e n t salts m e d i u m ,
o r a f t e r 4, 8, 12, a n d 16 h d i f f e r e n t i a t i o n in t h e a m i n o
acid containing mannitol medium (Chet and Rusch,
1969) a n d e x t r a c t e d f o r a m i n o acids. T h e e x t r a c t i o n
m e t h o d u s e d c a u s e d n o loss o f a m i n o acids. T h i s was
c h e c k e d b y c o m p a r i n g s a m p l e s o f 50 m l e t h a n o l
h o m o g e n a t e s w h i c h w e r e s u p p l e m e n t e d w i t h 0.5 o r
1.0 m g l e u c i n e w i t h s o l u t i o n s o f 0.5 o r 1.0 m g l e u c i n e
in 50 m l e t h a n o l w i t h a n d w i t h o u t e x t r a c t i o n .
The changes of the composition of the amino acid
p o o l d u r i n g s p h e r u l a t i o n in t h e n o n n u t r i e n t salts
m e d i u m are g i v e n in T a b l e 1, t h o s e d u r i n g s p h e r u l a t i o n
in t h e m a n n i t o l m e d i u m in T a b l e 2.

G. Wendelberger-Schieweg and A. Hfittermann: Amino Acid Pool and Protein in

Table 1. Composition of the amino
acid pool of Physarumpotycephalum
during spherulation caused by transfer
to nonnutrient salts medium

Physarumpolycephalum

Amino acid
Time of spherulation (h)
(nmole/g wet weight)
0
6
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Cysteine

143 ,+ 29
543 51
551 ,+ 52
161 21
2364 54
63 14
601 24
609 51
20 2
200 8
250 13
69 19
41 5
70 15
92 6
39 3
not detectable

47
461
243
275
398
53
155
34
7
16
86
27
12
45
92 +
33

5
46
15
27
36
6
48
3
3
7
11
3
1
2
37
4

12

29

18

54 8
248 ,+ 28
417 15
255 15
798 _+ 117
76 4- 7
235 ,+ 4
67 4
18 5
26 3
105 18
27 2
5 I
72 19
96 21
68 22

24

31 8
~167 5
328 27
291 1
1273 82
119 __+ 11
293 26
200 53
25 3
65 3
210 3
30 3
25 3
99 12
218 _+ 3
102 6

34
188
197
252
944
53
200
46
21
34
65
20
23
99
39
48

4
+ 11
13
8
4
3
9
1
2
1
+ 0
_+ 2
2
18
1
28

The indicated values are averages and standard deviations of four independent determinations
Total sum

Table 2. Composition of the amino

acid pool of Physarumpolycephalum
during spherulation caused by
medium with 0.5 M mannitol

5816 367
100 ~

1984 263
34 ".,

Amino acid
Time of spherulation (h)
(mnole/g wet weight)
0
4
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanhe
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Cysteine

143 _+ 29
543 51
551 _+ 52
161 21
2364 54
63 16
601 24
609 51
20 2
200 8
250 13
69 19
41 ,+ 5
70 15
92 6
39 3
not detectable

86
509
578
425
157
133
547
127
I9
55
92
19
17
97
275
70

28
62
2
33
17
7
42
8
2
36
33
2
2
5
1
5

2567 289
44 ~',/,

8
163
1061
980
405
214
129
611
159
23
95
167
29
36
98
284
65
70

3476 250
60 ~/,,

12

0
66
13
34
34
2
54
5
2
2
6
2
I
6
51
7
4

109
1531
950
370
467
102
621
699
25
168
364
43
63
102
207
89
77

2263 107
39

16
I
217
_+ 9
24
_+ 80
9
_+ 54
80
3
24
53
12
18
15
29
,+ 11
9

226
1689
926
708
2463
105
1580
230
51
406
552
134
116
138
348
128
89

,+ 2
170
_+ 9
+ 69
24
_ 8
149
zz 23
~ 5
39
53
+ 12
1l
2 13
_~ 34
2 12
,+ 8

The indicated values are averages and standard deviations of four independent determinations
Total sum

5816 _+ 367
i00~

D u r i n g d i f f e r e n t i a t i o n b y s t a r v a t i o n ( T a b l e 1) t h e
amino acid pool became much. smaller. After 6 h
h u n g e r o n l y a t h i r d o f t h e o r i g i n a l a m o u n t w a s left,
t h e n t h e p o o l filled u p a g a i n a n d d e c r e a s e d a f t e r w a r d s
t o 39 ~ o f t h e size p r e s e n t d u r i n g g r o w t h . N o a m i n o
acid had a constant-concentration throughout spherulation. Only the concentration of methionine, an essen-

3206 285
56~

4589 289
79 Z

5996 648
i03~

9889 641
170 ~/o

t i m a m i n o a c i d f o r Physarumpolycephalum ( D a n i e l
et al., 1963), r e m a i n e d r e l a t i v e l y u n c h a n g e d . E s p e cially striking was the reduction of the amount of the
amino acids with a branched C-chain (valine, leucine,
and isoleucine). On the other hand, there was a slight
increase of the concentrations of the polar amino acids
glutamic acid, histidine, and arginine. The amount of

30

lysine being constant for 12 h then doubled and decreased afterwards. The quantity of threonine continuously became lower to 30 ~ of the original concentration. About half the amino acid pool consisted
of proline. The concentration of proline in growing
microplasmodia was six times higher than in 6 h
starved plasmodia; after 24 h there was about half
the original concentration left.
Although in the mannitol medium (Table 2) external amino acids were present in the medium, the
pool first became smaller too, and increased only
afterwards to 170 ~ of its original size. The decrease
of the proline concentration during the first hours was
even stronger than the one observed in the salts medium. As was the case in the nonnutrient salts medium,
the amount of the amino acids with a branched C-chain
decreased strikingly, whereas there was an increase
in the basic amino acids histidine, lysine, and arginine.
Already after 4 h there was three times more glutamic
acid in the pool than during growth, although this
amino acid is not present in this medium. Cysteine,
not being detectable in the pool of growing microplasmodia, was taken up out of the medium, and
appeared accordingly in the pool.

2. Uptake of Radioactive Amino Acids into the Pool

and into the Protein during Growth and Spherulation
To estimate the uptake of amino acids out of the
medium into the amino acid pool and their incorporation into protein, microplasmodia growing in the
log-phase were transferred to growth medium containing a mixture of radioactive amino acids (10 mg
nonradioactive amino acids/ml medium plus 0.625 gCi
14C-protein hydrolysate from Chlorella) instead of
tryptone, or to nonnutrient salts medium. The growing
microplasmodia were labeled for 2, 4, 6, and 8 h. The
starving microplasmodia were transferred after 4, 6,
8, and 10 h (16, 18, 20, and 22 h respectively) of starvation to salts medium supplemented with 10 mg
radioactive amino acid mixture/ml of medium and
harvested after 12 (24 h resp.) induction of differentiation, thus being labeled for 2, 4, 6, and 8 h.
Even after 8 h labeling the amino acid pool was
not in equilibrium with the medium (Fig. I b). In
growing microptasmodia the pool had then about
50 ~, in starving microplasmodia about 30 ~ of the
specific radioactivity of the medium. The incorporation
of radioactive amino acids into protein (Fig. 1 c) was
according to the behaviour of the amino acid pool.
In the medium (Fig. 1 a) the specific radioactivity
of the amino acids decreased considerably, the decrease being more striking for the growth medium
than for the salts medium. This result means that
either the radioactive amino acids were taken up

Arch. Microbiol., Vol. 117 (1978)

dpm/mg
200000-

150000"

120 000-

dpm/mg

.J"
50000-

I
25 000-

|
0

dpm/mg

/.<:r
"~,

0
4
8
Fig. 1 A - C . Uptake of radioactive amino acids into the pool of
growing and starving microplasmodia of Physarumpolycephalum.
(A) Specific radioactivity of amino acids in the medium during
the labeling period. (B) Specific radioactivity of the amino acid pool.
(C) Specific radioactivity in the total protein, e - - i
growing
plasmodia; a
a starving plasmodia harvested after 12 h of
starvation; 9
m starving plasmodia harvested after 24 h of
starvation

preferentially, or that a considerable quantity of amino

acids was released by the microplasmodium into the
medium.
To test for a release of amino acids, microplasmodia
were prelabeled for 36 h. They were then transferred
to growth medium with the nonradioactive amino acid
mixture instead of tryptone or to salts medium with
and without the amino acid mixture. The addition
of 10 mg amino acids/ml medium does not interfere
with the spherulation process (Ht~ttermann et al., 1971).
Two cultures of each type were harvested immediately,
the others at the times indicated in Figures 2 and 3.
The results for growing microplasmodia are shown
in Figure 2. During growth, amino acids were released
into the medium. Within 4 h the specific radioactivity
in the medium increased considerably (Fig. 2a). The
same was true for spherulation in the salts medium
with a supplement of amino acids (without figure).

Physarumpolyeephalum

G. Wendelberger-Schieweg and A. Hiittermann: Amino Acid Pool and Protein in

dpm/mg

31

mg

1000-

200
mg

/0/0~0

10-

|
|

dpm/mg
h

4.104.-

40-

3 -104

~, 3s-

~ , ~ - - - - - -

20

/
mg

Q,.

30-

2.104-,

140~

dpm/mg
440 ~

3qO~

%00 O ~ o ....,.o

2~

1'2

,8

2',

Fig. 3 A - C . Determination of net protein turnover in starving

plasmodia as measured by isotope dilution. Microplasmodia were
grown for 36 h in the presence of radioactive amino acids and then
transferred to starvation medium containing 10 mg/ml cold amino
acids. (A) Increase of the amino acid pool during the period under
study. (B) Decrease of the total protein content in the cultures.
(C) Decrease of the specific radioactivity in the amino acid pool
(9
O), and the total protein (0
~) in the cultures

1.1~

Fig. 2A D. Determination of net protein turnover in growing

plasmodia as measured by isotope dilution. Microplasmodia were
grown in radioactive medium for 36 h arid then transferred to growth
medium containing cold amino acids. (A) Increase of radioactivity
in the medium expressed as dpm/mg amino acids. (B) Increase of
the amino acid pool during the period under study. (C) Increase
of the total protein content in the cultures. (D) Decrease of the
specific radioactivity in the amino acid pool (O
9 and the
total protein (O
e) in the cultures

Plasmodia differentiating in the salts medium

without any amino acid, however, did not release
amino acids as judged by radioactivity and ninhydrin
reaction (without figure). Obviously the transport
systems change depending on the presence of amino
acids in the environment. If amino acids are present,
they are readily released, without external amino acids,
however, they seem to be retained.

3. Turnover of Protein during Growth and Spherulation

The rate of turnover of protein during growth and
spherulation was estimated by an isotope dilution
technique. Microplasmodia were labeled with the
radioactive amino acid mixture for 36 h. They were
then transferred to growth medium or to salts medium
containing 10 mg nonradioactive amino acids/ml medium. The cultures were harvested after the times
indicated in Figure 2 (growth medium) and Figure 3
(salts medium).
Figure 2 gives the rate of turnover of the protein
during growth. Figure 2c shows that the amount of
protein increased about 30 ~ during 6 h growth. The
dilution of specific radioactivity within this time was
30~, too (Fig. 2d). This means in fact that overall
protein turnover during growth must be very low.
In the spherulating cultures, however, the specific
radioactivity of the protein was diluted to half the

32
original value (Fig. 3c). Half the amount of protein
was degraded, too (Fig. 3b). This indicates a very
high rate of turnover. At least 70 % of the originally
existing protein must be degraded during spherulation,
and, simultaneously, 50 % of the protein present after
24 h differentiation must be synthesized during that
time. This calculation disregards the fact that amino
acids of the degraded protein could be reused so that
the value of 50 % may be even too low.
The amino acid pool of microplasmodia starving
in nonnutrient salts medium supplemented with amino
acids expanded to about 125 % of its original size
(Fig. 3a), the specific radioactivity, however, decreasing about 60 %. The loss of specific radioactivity
took place mainly during the first 6 h of starvation
during which time considerable amounts of amino
acids were released into the medium (Fig. I a). Whether
this is the only explanation for the big decrease of
specific radioactivity, or whether uncomplete labeling
during the 36 h prelabeling time or own de novo synthesis of amino acids are responsible, too, is not yet
known.

4. Evidence for Multiple Amino Acid Pools

Obtained by Density Labeling of Enzymes
A further indication for multiple pools came from
density labeling experiments. Microplasmodia were
grown in growth medium supplemented with normal
or with deuterated amino acids (10 mg/ml) instead of
tryptone. Extracts of thes e plasmodia with an addition
of /Lglucuronidase from beef liver as an internal
density marker were analyzed on RbCl-density gradients by centrifugation at 250000 x g for 65 h.
Figure 4 shows the results of the density labeling
experiment for the enzymes glutamate dehydrogenase
(Fig. 4a) and glucose-6-phosphate dehydrogenase
(Fig. 4 b). In every diagram two independent gradients
are given together to facilitate an estimation of the
proportion of "old" light enzyme to newly synthesized
denser enzyme protein. It is evident that the portion
of preexisting light enzyme after the labeling period
is higher for glucose-6-phosphate dehydrogenase than
for glutamate dehydrogenase. But the density increase
indicates a degree of deuteration (calculated according
to H u e t al., 1962) of 37% for glucose-6-phosphate
dehydrogenase, whereas the degree of deuteration for
glutamate dehydrogenase was only 27 ~o- This difference of the density increase cannot be interpreted as
the consequence of a differential turnover: on the
contrary, the existing differential turnover ought to
produce an even higher degree of deuteration for
glutamate dehydrogenase than for glucose-6-phosphate dehydrogenase. The different rates of deuteration, just opposite to values to be expected from the

Arch. Microbiol.,Vol. 117 (1978)

n

"1.37
' 1.36

100

~ 2

3'o

4'o

10C

k,N,

30

,t\
k

4b

FLN,

Fig.4A and B. Differentiallabeling of two enzymes, glutamate

dehydrogenaseand glucose-6-phosphatedehydrogenase,in the same
extractsof Physarumpolycephalum.Microplasmodiaweregrownfor
24 h in the presence of either 10 mg 1H- or 2H-aminoacids/mgof
medium, extracts were made and analysedby equilibrium density
gradient centrifugation in RbCi-gradients, with /3-glucuronidase
added as internal densitystandard. Each diagramshowsthe results
from the "light" and "heavy" extracts superimposed, the open
symbols refer to gradients made from extracts from plasmodia
grown on tH-amino acids, the filled symbols to the ones from
plasmodia grown on 2H-amino acids. 9 9 density gradient of
the RbCl-gradient(uppercurve); 0
9 fi-glucuronidaseactivity;
zx zx activityof the enzymeunder investigation:(A) glutamate
dehydrogenase,(B) glucose-6-phosphatedehydrogenase

ratio between old an newly made enzyme, can be

explained only on the base of a different compartimentation and labeling of the pools which deliver the
amino acids for the synthesis of the cytoplasmic
enzyme glucose-6-phosphate dehydrogenase and for
the enzyme glutamate dehydrogenase active in the
mitochondria.

DISCUSSION
The size and composition of the amino acid pool
changes depending on the choice of the medium by
which the spherulation of Physarum was induced. But
in each case the first answer to stress conditions is a
depletion of the amino acids out of the pool.
The relative amounts of the individual amino acids
change very much in both induction systems. Therefore
it is not possible to determine the rate of synthesis
of protein only after labeling with one single amino
acid, e.g. leucine (Sauer et al., 1970) without making
corrections. The different changes of the concentration of each amino acid in the pool will provide
different apparent rates of turnover of the same protein
depending on the special amino acid used. Examples

G. Wendelberger-Schieweg and A. Hfittermann: Amino Acid Pool and Protein in Physarumpolcephalum

for this have been found in other organisms, e.g. rat

liver (Poole et al., 1969), ChIamydomonas reinhardtii
y-1 (Hoober, 1972).
To gain at least a foothold for the interpretation
of incorporation data the specific radioactivity of the
amino acid pool during a labeling period with radioactive amino acids was determined. A similar study
was carried out by Sauer et al. (1970), and Henney
and Maxey (1975) for Physarum flavicomum. But,
since the labeled amino acids were diluted by a great
amount of unlabeled amino acids only in the growth
medium, but not in the amino acid-free salts medium,
these authors labeled with markedly different specific
radioactivities of amino acids. Thus the specific radioactivities of the pools were not comparable. Therefore always 10 mg unlabeled amino acids/ml salts
medium were added in our experiments. If radioactive
amino acids were present in the medium, they were
taken up with rather slow saturation kinetics. Equilibrium between the specific radioactivity of the pool
and the medium was not reached even after 24 h of
incubation with the labeled amino acids. This situation
is rather different from the one observed for bacteria
(e.g. Escherichia coli, Oxender, 1972) or rat liver cells
(e.g. Mortimer et al., 1972; Partridge and Jefferson,
1975; Urban et al., 1976).
Two explanations can be offered for this different
behaviour of the slime mold amino acid transport.
It may either be that even in the presence of external
added amino acids still considerable amino acid
synthesis may occur, since the organism is able to
synthesize most of its amino acids (Daniel et al., 1963).
Another factor involved may be a high selectivity of
the membrane. Slime molds, as other amoeboid
organisms, are separated from their environment only
by their membranes, which implies special properties
in their part.
The experiments carried out via radioactive labeling
and extracting the whole plasmodia necessarily neglected the possibility of multiple pools located in
different compartments within the plasmodia, as was
shown to be the case for other organisms, e.g. Saccharornyces (Wiemken and DOrr, 1974), Chlarnydomonas (Stegemann and Hoober, 1974), or rat liver
cells (Mortimore et al., 1972). Indications for multiple
pools existing within the microplasmodia were obtained in this study in two different ways: first by
measuring the kinetics of the amino acid uptake,
which, being biphasic, can only be explained by
multiple pools. This assumption is confirmed by the
density labeling data (Fig. 4) which deliver strong
evidence that the two enzymes in question must have
been synthesized from amino acids of different pools.
The most striking difference in the area of protein
metabolism between growing and differentiating plas-

33

modia is the difference in protein turnover. Via isotope

dilution experiments, which circumvent the problems
of different and multiple pools and thus reveal reliable
data, no protein turnover was detectable in growing
plasmodia. The loss in specific radioactivity in the
protein was exactly matching the net increase due to
net protein synthesis. During starvation induced
differentiation, however, the decrease in specific radioactivity of the protein to half the value at the beginning indicates that at least 50 ~ of the protein
present after 24 h of starvation was synthesized during
this period. This rather high de novo synthesis takes
place during a period when the total mass of protein
decreases to half the original value. Thus the conditions
are quite similar to those which have been reported
for higher animal (Schimke and Doyle, 1970) and plant
cells (Huffaker and Peterson, 1974).
This unexpected high rate of de novo synthesis of
protein during differentiation yields a new point of
view for studies pertaining the control of this morphogenesis at the level of protein synthesis. It is now
quite clear that synthesis of proteins during spherulation is not such a unique event as was thought to be
so when the occurrence of differential protein synthesis was demonstrated during this differentiation
for the first time (Hiittermann et al., 1971; HOttermann and Gebauer, 1972) in such a primitive morphogenesis. On the background of the new information,
it is necessary to compare the rate of synthesis of a
given enzyme during both stages, growth and differentiation, to demonstrate an altered enzyme synthesis
without any doubt, which in turn can be taken as an
indication for an evolvement of this protein in the
regulation of this morphogenesis.
Acknowledgements. The work was supported by grants from the
Deutsche Forschungsgemeinschaft.

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Received October 19, 1977