CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
A COMPARISON OF PHOSPHOSERINE PHOSPHATASE
1\
IN TWO ALLELIC SERINE REQUIRING MUTANTS
OF NEUROSPORA CRASSA
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Snowdy Denise Dodson
/
June, 1980
The Thesis of Snowdy Denise Dodson is approved:
Charles R. Spotts, J.bh.D.
~cUB. Maxwell, Ph.D.
California State University, Northridge
ii
ACKNOWLEDGMENTS
I wish to thank Dr. Joyce Maxwell for her tireless
and patient guidance during the preparation of this thesis.
Her encouragement got me started and kept me going and
made this effort a reality.
I also deeply appreciate the thoughtful participation of Dr. Corcoran and Dr. Spotts as members of my
graduate committee.
Dr. Sandra Jewett merits special thanks for the
loan of special equipment and also for her constructive
criticism of my laboratory technique.
iii
TABLE OF CONTENTS
Page
v
LIST OF FIGURES .
LIST OF TABLES.
ABSTRACT.
• .
vi
.
.
. vii
. .. . . . . . ....
INTRODUCTION . •
1
5
MATERIALS AND METHODS •
• ••.
...
.
• .
..
5
• • • •
5
MAINTENANCE AND GROWTH OF NEUROSPORA CULTURES . •
5
HARVESTING AND PROTEIN EXTRACTION •
6
STRAINS USED.
CHEMICALS .
.
.
.
.
.
• • • • .
....
ENZYME ASSAY . • . • • .
PROTEIN DETERMINATION
. .
FINAL CURVES • . . . . .
.
.
13
.
.
13
.." ..." • •
.. .... ............
...
.......... - .
........ ......
STATISTICAL CALCULATIONS.
RESULTS .
.
DISCUSSION . • .
REFERENCES . .
8
~
iv
18
22
37
43
LIST OF FIGURES
Figure
Page
1.
Pathways of serine biosynthesis . • .
2.
Phosphoserine phosphatase activity of
ser-3 from experiment two • • . . • .
3.
2
11
Phosphoserine phosphatase activity for
ST74A, the prototrophic strain used
for comparison with ser-3 . • . •
....
15
Spectrophotometric determination of
inorganic phosphate . . . • • • •
...
.
17
5.
Comparison of phosphoserine phosphatase
activities in experiment four • • • •
. . .
.
20
6.
Phosphoserine phosphatase activities in
experiment one . • . . • • • • • • , •
.
.
24
7.
Comparison of phosphoserine phosphatase
activities in experiment three . • . . • • . •
28
8.
Phosphoserine phosphatase activity of
ser-3 in experiment two . . . • • •
4.
9.
10.
• •
.
.. ..
31
Second determination of phosphoserine
phosphatase activity of ser-3 in
experiment two . . . • • . • . • • . •
....
33
Comparison of phosphoserine phosphatase
activities in experiment five . • • .
v
36
LIST OF TABLES
Table
1.
2.
Page
Phosphoserine phosphatase specific activities
of ser-3 and ser(JBMS) salted protein
extracts . . . • . . . . . . •
21
Spontaneous phosphate production in two
phosphoserine samples • . . . . . . .
26
vi
ABSTRACT
A COMPARISON OF PHOSPHOSERINE PHOSPHATASE
IN TWO ALLELIC SERINE REQUIRING MUTANTS
OF NEUROSPORA CRASSA
by
Snowdy Denise Dodson
Master of Science in Biology
The phosphorylated pathway has been proposed as the
major source of serine in Neurospora crassa (Sojka & Garner,
1967).
Chuck (1980) demonstrated that a serine deficient
mutant of Neurospora crassa, ser(JBM5), which is isogenic
with its progenitor prototrophic strain, shows a marked
decrease in its phosphoserine phosphatase specific activity
when compared to the activity of its prototrophic strain.
This lowered activity in the enzyme catalyzing the terminal
step of the phosphorylated pathway of serine biosynthesis
in a serine requiring mutant which has a single gene difference compared to its prototrophic strain supported the
phosphorylated pathway as the major source of serine in
vii
Neurospora crassa.
The present study assayed the phos-
phoserine phosphatase activities of two allelic serine
requiring mutants of Neurospora crassa, ser-3 and ser{JBMS).
In all assays, both mutants had significantly lower phosphoserine phosphatase specific activities than did their
respective prototrophic strains.
This finding lends fur-
ther support to the phosphorylated pathway as the principal
route of serine biosynthesis in Neurospora crassa.
viii
INTRODUCTION
Three pathways have been proposed for the biosynthesis of serine (Fig. 1).
involves three
enzymes:
The phosphorylated pathway
phosphoglyceric acid dehydro-
genase which converts 3-phosphoglyceric acid, an intermediate in glycolysis, into 3-phosphohydroxypyruvic acid;
phosphoserine transaminase which transaminates 3-phosphohydroxypyruvic acid into 3-phosphoserine; and phosphoserine
phosphatase which dephosphorylates phosphoserine to serine.
The non-phosphorylated pathway involves two enzymes:
gly-
ceric acid dehydrogenase which converts glyceric acid to
hydroxypyruvic acid; and serine transaminase which transaminates hydroxypyruvic acid to serine.
The third pathway,
which is thought to arise from tricarboxylic acid cycle
intermediates, produces serine by way of glyoxylate and
glycine.
This pathway involves two enzymes:
glyoxylate
transaminase which transaminates glyoxylate to glycine; and
serine transhydroxymethylase which converts glycine to
serine.
Sojka and Garner (1967) investigated serine biosynthesis in Neurospora crassa.
They concluded that the
phosphorylated pathway was the main pathway for serine
biosynthesis in Neurospora.
They based their conclu-
sions on the higher activity shown by the enzymes of the
1
2
GLUCOSE
t
t
23
H 0 POCH ~H ·COOH
2
-~
.. -Pi ..
OH
3-phosphoglyceric acid
NA.t'l
HOC~:·COOH
glyceric acid
~
~NADH
NA.tl NADH
H o POCH ~ ·COOH
2 3
2
HOCH
2
0
3-phosphohydroxypyruvic acid
H203POCH2~H·COOH
-P·I
~ ·COOH
·
0
hydroxypyruvic acid
...
NH
2
3-phosphoserine
serine
't
~H2 ·COOH
NH
2 glycine
t
OCH·COOH
glyoxylic acid
Figure 1.
Pathways of serine biosynthesis.
3
phosphorylated pathway as opposed to the enzymes of the
non-phosphorylated pathway.
Support for the phosphorylated
pathway as the principal means of serine biosynthesis has
been reported in bacteria (Pizer, 1963; Umbarger, Umbarger &
Siu, 1963; Pizer, Ponce-De-Leon & Michalka, 1969; Nelson &
Naylor, 1971; Ponce-De-Leon & Pizer, 1972) and also for
Saccharomyces (Ulane & Ogur, 1972).
However, earlier work
by Wright (1951) suggested that serine biosynthesis in
Neurospora involves the conversion
of glyoxylic acid to
glycine which is then converted to serine.
This conclu-
sion, which supports the third pathway outlined above, is
based on the fact that she found that a serine-glycine
dependent mutant of Neurospora grew better on glyoxylate or
glycine than on serine.
Thus, there is a conflict between
the pathway proposed by Wright and the pathway suggested by
Sojka and Garner as the main method of serine biosynthesis
in Neurospora.
One way to resolve this conflict and demonstrate
the major serine biosynthetic pathway would be to find a
serine requiring mutant in Neurospora and then to show that
it differs in a specific enzyme activity in one of the
pathways.
In two serine requiring mutants investigated,
ser-4 (DW 110) and ser-2 (JBM4-13), no enzyme deficiencies
were demonstrable (Maxwell, 1970; Kline, 1973).
Recently,
Chuck (1980) demonstrated that a serine deficient mutant
of Neurospora crassa, ser(JBMS), which is isogenic with its
4
prototrophic strain, shows a marked decrease in its phosphoserine phosphatase specific activity when compared to
the activity of its progenitor prototrophic strain.
Chuck's study was the first demonstration of an enzyme
defect in a serine auxotroph of Neurospora crassa.
His
findings supported Sojka and Garner's contention that the
phosphorylated pathway is the major source of serine in
Neurospora.
The present study examines the question of whether
ser-3, a serine requiring mutant of Neurospora which is
allelic with ser(JBMS)
(Maxwell et al., 1978) has a phos-
phoserine phosphatase specific activity that is similar to
the phosphoserine phosphatase activity in ser(JBMS).
If
one gene controls one enzyme and if one allele produces
changes in an enzyme, then an allelic mutant (in the same
gene) should result in a defect in the same enzyme.
Such a
finding would lend strong support for the phosphorylated
pathway as the main route of serine biosynthesis in Neurospora.
Evidence shall be presented below indicating that
ser-3 and ser(JBMS) are similar in their decreased phosphoserine phosphatase specific activities compared to their
respective prototrophic strains.
MATERIALS AND METHODS
STRAINS USED
Four different strains of Neurospora crassa were
used during these studies.
Mary Mitchell, who was associ-
ated previously with the Division of Biology at the
California Institute of Technology, supplied strain Cl0215300-4-2A.
This nutritionally prototrophic strain grows
colonially at temperatures greater than 32°C (Cl02t) and has
albino conidia (15300 al-2).
Strain Cl02-15300-4-2A
ser(JBM5) is a serine requiring mutant derived from
uv-
irradiated conidia of the previous strain by Dr. Joyce B.
Maxwell and others (1978).
Two additional strains used in
this study were FGSC#l213 ser-3 (47903), a serine requiring
mutant, and ST74A, a prototrophic strain, both available
from the Fungal Genetics Stock Center at Arcata, California.
CHEMICALS
All compounds used were reagent grade except for
the Tris [2 amino-2(hydroxyrnethyl)-l, 3 Propanediol] which
was practical grade.
MAINTENANCE AND GROWTH OF NEUROSPORA CULTURES
Serine deficient mutants were maintained on agar
slants of either Horowitz complete medium (1947) or Vogel's
minimal medium N (1956) supplemented with 1 mg/ml L-serine
5
6
and 2 per cent (w/v) sucrose.
The two prototrophic strains
were maintained on agar slants of unsupplemented minimal
medium containing 2 per cent sucrose.
Cultures used for the protein extracts were grown
in 125 ml Erlenmeyer flasks containing 20 ml of Vogel's
minimal medium N, 2 per cent sucrose, 10 mM glycine and
10 mM sodium formate.
Each flask was inoculated with 0.2
ml to 0.5 ml of dense conidial suspension prepared by adding sterile water to conidiating cultures grown on agar
slants.
The stationary cultures were then incubated for
three days at 25°C.
HARVESTING AND PROTEIN EXTRACTION
The mycelial pads were harvested on a Buchner funnel.
To prevent protein breakdown, care was taken to keep
the harvested pads on ice as much as possible.
To deter-
mine their wet weight, the pads were squeezed dry between
paper towels, then weighed on a Sartorius digital analytical balance, model 2400.
The pads were then ground with
sea sand using an ice cold mortar and pestle.
After grind-
ing, the extract was brought up to 4x (4 ml buffer per 1 gm
wet weight of mycelium) by adding an appropriate amount of
Tris buffer.
(Unless stated otherwise, all buffer used in
these experiments is 0.1 M Tris-HCl, pH 7.5.)
Sand and
cellular debris were removed from the mixture by centrifugation for 20 min at 12100g in a refrigerated Sorvall RC-5B
7
centrifuge.
In order to free the extract from indigenous
phosphates which interfere with the phosphatase assay, the
resultant supernatant was placed in another centrifuge tube
and brought up to lOx
(10 ml solution per 1 gm wet weight
of mycelium) with 100 per cent saturated (NH4) 2 so4 in Tris.
This procedure produced a 70 per cent saturated (NH4)2S04
solution which precipitates most of the proteins in the
extract.
Next the mixture was stirred, allowed to sit for
10 min on ice, and then centrifuged at 12100g for 20 min.
The supernatant was discarded, and the surface of the pellet
was washed with 1 ml of 1M Tris-HCl, pH 7.5.
Preliminary
experiments showed that the supernatant from the salting
procedure did not show any enzyme activity.
After gently
bringing up the pellet into 4x Tris, adding 6x· 100 per cent
saturated (NG4) 2 so4 in Tris, and letting the mixture sit on
ice for 10 min, it was again centrifuged at 12100g for 20
min.
The pellet was brought up to lOx in Tris, placed in
5/8" dialysis bags and dialyzed in the cold (4°C) in three
changes of one liter of Tris (pH 7.5 at room temperature)
allowing at least three hours between each change of buffer.
Dialysis bags were cleaned with EDTA and sodium
bicarbonate and provided by Dr. Sandra Jewett.
Alterna-
tively, crude (i.e. unsalted) samples of each extract were
simply dialyzed to remove contaminating phosphates.
The
first two sets of extracts were not stirred during dialysis;
the last two were.
All but the first group of extracts
8
were dialyzed in a covered flask to prevent evaporation due
to the air movement caused by the fan in the cold room.
After dialysis, the samples were placed in labeled test
tubes and stored in the freezer at -l9°C.
ENZYME ASSAY
Each protein extract was thawed quickly under running water, then centrifuged at 12100g at 4°C for 15 min.
The protein extract was diluted to lOOx with buffer.
Phos-
phoserine phosphatase activity was assayed by testing for
the amount of inorganic phosphate formed from phosphoserine
in the presence of the protein extract as described by Ames
(1966).
150
The reaction mixture consisted of 0.6 mmoles Tris,
~moles
of MgCl2, and 60
volume of 5.1 ml.
~moles
phosphoserine in a final
The phosphoserine substrate solution was
adjusted to pH 6.9-7.0 by dissolving the phosphoserine in
buffer and neutralizing the solution with concentrated NaOH.
Both the extract and the reaction mixture were incubated at
25°C before the reaction was begun.
The reaction was
started by the addition of the lOOx protein extract to the
reaction mixture.
All reaction tubes were brought to a
final volume of 9 ml by adding appropriate amounts of distilled water.
Several reaction tubes, each containing dif-
ferent volumes of protein extract in Tris, were assayed for
each of the protein extracts.
·In the first two experiments,
only three different volumes were used for each protein
extract (1.2 ml, 1.8 ml, and 2.4 ml).
In all subsequent
9
assays, another volume (0.6 ml) was added.
All reactions
were run in a water bath ranging in temperature from 24°C to
26°C.
Timed samples were taken from each mixture.
In the
first two assays, the samples were removed at 7, 15, and 21
min after initiation of the reaction.
In almost allof these
assays, the 7 minute point was higher than was expected
relative to other time points making it difficult to draw a
good straight line through zero.
The initial value obtained
by extrapolation to zero time was 0.1
~0Da 15 .
~OD815
rather than 0
This was especially true at the highest concentra-
tion of protein extract (0.05 to 0.1 mg protein/ml) leading
to speculation that perhaps a two-step reaction was involved
The 7, 15 and 21 minute samples might have been on the plateau of the curve while the exponential activity occurred
during the early part of the reaction.
To check this possi-
bility, an assay was run on the first crude ser-3 extract
containing 0.09 mg protein/ml taking samples at 1 min intervals during the first 8 min of the reaction.
The results
(Fig. 2) indicated a linear reaction through zero.
In order
to monitor the kinetics of the initial enzyme reaction,
several samples were taken during the first few minutes
after the initiation of all subsequent experiments.
This
permitted the determination of the linearity of the reaction.
Also from this point forward a pipetteman pipetter
was used for greater accuracy in sampling. To stop the reaction, 0.9 ml of each timed sample was added to 0.1 ml of
10
Figure 2.
Phosphoserine phosphatase activity of ser-3 from
experiment two.
The reaction mixture contained 0.6 rnrnoles
Tris-HCl (pH 7.5), 150
~moles
MgCl2, 60
~moles
phosphoser-
ine, and 0.2832 mg protein in a final volume of 9 ml.
reaction was started by the addition of crude protein
extract.
At one minute intervals, 0.9 ml aliquots were
removed and assayed for inorganic phosphate.
The
11
0.4
E
c
....
It)
QO
t:i
0
. 0.2 -
TIME (min)
12
50 per cent (w/v) trichloroactic acid (TCA) .
In prelimin-
ary experiments, 15 per cent (w/v) TCA proved to be too low
a concentration to stop the reaction completely; the use
of a 50 per cent solution corrected this problem.
For each
assay, samples were also taken from a set of three controls; all controls contained the same concentrations of
Tris and MgCl2 as the extract assay tubes and were brought
up to the same volume with distilled water.
The phospho-
serine blank had phosphoserine and no enzyme added; the
Tris blank had neither enzyme nor phosphoserine; the enzyme
blank contained enzyme but no phosphoserine.
Control sam-
ples were taken at the end of each assay; these samples
were treated in the same way as those from the timed assay.
The samples were then centrifuged for 10 min in a
clinical centrifuge, and 0.7 ml of the resultant supernatant was added to 2.3 ml of a mixture of one part 10 per
cent (w/v) ascorbic acid to 6 parts 0.42 per cent (w/v)
ammonium molybdate in 1 N H2S04.
Inorganic phosphate
released from the phosphoserine phosphatase reaction produces a blue complex with molybdate in the presence of
ascorbic acid.
After this mixture was incubated in a water
bath for 20 min at 45°C, its absorbance was read at 815 nm
in a Perkin-Elmer Coleman 124 double beam spectrophotometer.
The readings from the controls were subtracted from the raw
data in the following way:
the Tris blank value was sub-
tracted from that of the enzyme; the resultant figure was
13
added to the phosphoserine blank; this final value was then
subtracted from the readings for the enzyme assays.
These
corrected optical density readings were plotted versus time
(Fig. 3).
For the purpose of comparison, a standard phos-
phate curve was run using varying concentrations of
KH2P04 (Fig. 4).
A volume of 3 ml containing 0.07
~moles
of inorganic phosphate gave a reading of 0. 5 at 815 nm •.
This figure is comparable to the published data (Ames,
1966).
In a typical assay of prototrophic protein extract,
from 0.042
~moles/ml
to 0.169
~moles/ml
qf phosphate is
released as a product from cleavage of phosphoserine; the
amount varies with the volume of protein extract assayed.
PROTEIN DETERMINATION
The total amount of protein in each of the extracts
was determined using the Biuret reaction (Gornall,
Bardawill & David, 1949).
on the lOx extracts.
duce a standard curve.
These determinations were done
Bovine serum albumin was used to proProtein concentrations in the
extracts were estimated from comparison with this standard
curve.
FINAL CURVES
The final curves used for comparison between the
prototrophic strains and serine deficient mutants were
drawn by plotting the milligrams protein from the protein
determination versus the slope (change in optical density
14
Figure 3.
Phosphoserine phosphatase activity for ST74A,
the prototrophic strain used for comparison with ser-3.
The extract is from experiment three.
The reaction mixture
contained 0.6 mmoles of Tris-HCl (pH 7.5}, 150
MgCl2, 60
~moles
~moles
of
phosphoserine, and varying amounts of pro-
tein in a final volume of 9 ml.
The reaction was started
by the addition of salted protein extract.
At 1, 2, 3, 4,
5, 15, and 22 minutes, 0.9 ml aliquots were removed and
assayed for inorganic phosphate.
15
PROTEIN (mg/ml>
C>-<>0.043
....... 0.032
...... 0.021
6-60.011
0.2
5
10
15
TIME (min)
20
16
Figure 4.
phosphate.
Spectrophotometric determination of inorganic
Varying concentrations or KH2P04 (1 x 10- 8 to
1 x 10- 7 moles) in a final volume of 3 ml were assayed for
phosphate activity.
17
0.6 -
0.4
-
E
~
....
II)
QO
.::::.•
0
•
0.2
5
MOLES PHOSPHATE (xlO-S)
10
18
per minute) of the curves obtained from the enzyme assays.
A representative plot of these calculated data can be seen
in Fig. 5.
STATISTICAL CALCULATIONS
The mean and standard deviation of the specific
activities of the mutant and prototrophic extracts in each
experiment were calculated in the following way.
the slope
(~ODa1s/min)
First,
was determined for each of the
points plotted for a particular extract.
These slopes were
divided by the number of milligrams protein in the extract
to produce figures for specific activity
protein).
(~OD815/min/mg
These specific activities were entered into a
calculator to determine the mean and standard deviation
for each extract (Table 1).
19
Figure 5.
Comparison of phosphoserine phosphatase activi-
ties in experiment four using data from salted protein
extracts of ser-3 and ST74A, a prototrophic strain.
20
0.04
·-E
1:
.........
E
0.03
...
1:
an
00
•
.:::::.
0
•
<I
0.02
0.01
....._.ser-3
e---eprototroph
0.1
PROTEIN
0.2
(mg)
@
'
TABLE 1. Phosphoserine phosphatase specific activities of ser-3 and ser(JBM5)
salted protein extracts.
Mutant
examined
Experiment
number
Specific activity (60D 815 /min/mg protein)*
Prototrophic strain
Mutant
ser (JBM5)
1
0.242 + 0.01 (9)
0.069 + 0.02 (9)
ser-3
2
0.238 + 0.02 (9)
0.087 + 0.01 (9)
ser-3
3
0.330 + 0.07 ( 2 8)
0.109 + 0.02 ( 28)
ser-3
4
0.348 + 0.06 (28)
0.151 + 0.04 (27)
ser (JBM5}
5
0.416 + 0.07 (28)
0.193 + 0.07 (23}
-
-
-
-
*Mean values are given. Plus or minus (+) values are standard deviations.
Numbers in parenthesis indicate sample sizes.
N
1-'
22
RESULTS
As stated previously, the primary purpose of this
study was to test whether ser-3 and ser(JBMS) are similar
in displaying lower phosphoserine phosphatase specific
activities than the respective prototrophic strains from
which each mutant was derived.
Thus, the first set of
assays were done on ser(JBMS) in order to see whether previous results (Chuck, 1980) could be repeated by an independent investigator and also for use as a basis of comparison with the later ser-3 assays.
As can be seen in Fig. 6, these data support the
earlier findings
(Chuck, 1980) in that they showed a marked
difference between the phosphoserine phosphatase specific
activity of the mutant and that of the prototrophic strain.
This result is consistent with the hypothesis that ser(JBMS)
requires serine due to its deficient phosphoserine phosphatase activity.
Further support for this hypothesis
would be the demonstration that the same deficiency exists
in ser-3 compared to its prototrophic strain.
Ser-3 is
an independently isolated (Dubes, 1953) allele of ser(JBMS)
(Maxwell et al., 1978).
The next set of assays were done on ser-3 extracts
using ST74A as a prototrophic strain for comparison.
Although the methods of assay were the same as those used
23
Figure 6.
Phosphoserine phosphatase activities in experi-
ment one using data from salted protein extracts of
ser(JBM5) and the prototrophic strain from which it was
derived.
24
.___. ser(JBMS)
...... prototroph
0.04
·-E
&::
'E
....00
0.03
&::
loft
c•
c•
~
0.02
0.01
0.1
PR 0 TEIN (mg)
0.2
25
for ser(JBMS), there was one difference in the protein
extractions.
After the first centrifugation, the 100
per cent saturated (NH 4 )2so 4 was mistakenly added to both
the ser-3 and prototrophic pellets before they were brought
up in Tris.
In spite of this rough treatment, the results
were essentially identical to the ser(JBMS) results.
As
can be seen in Table 1, there was a marked difference
between the specific activities of the prototrophic
(0.238 + 0.02
~on
~on
815 /min/mg
protein) and ser-3 (0.087 + 0.01
815 /min/mg protein) phosphoserine phosphatases.
Also,
in the second experiment, ser-3 had a phosphoserine phosphatase specific activity that was only slightly higher
than the ser(JBMS) specific activity in the first experiment; and the specific activities for the two prototrophic
strains were nearly identical (Table 1) •
At this point, a peripheral experiment was done in
an attempt to clarify an inconsistency that had appeared
in the values for the phosphoserine blanks.
These values
had varied significantly from experiment to experiment.
There were two bottles of phosphoserine kept in the lab
refrigerator.
Small amounts of phosphoserine solution had
been prepared for each assay without discrimination between
the two bottles.
In order to account for this variability,
samples from each bottle were assayed for spontaneous
phosphate production as outlined above in the Materials
and Methods section.
From these results (Table 2), it was
26.
TABLE 2.
Spontaneous phosphate production in two
phosphoserine samples.
Time (min)
Optical density at 815 nm
Sample #1
Sam121e #2
l.Oml
0.5ml
l.Oml
0.5m.
7
0.065
0.11
0.1
0.2
15
0.062
0.11
0.105
0.2
21
0.07
0.11
0.105
0.195
observed that sample number two gave readings proportional
to the volume of solution assayed.
This reagent was used
for all of the subsequent enzyme assays.
At the same time,
it was decided to prepare the phosphoserine solutions in
large enough batches to last throughout a particular set
of assays in order to insure more consistent phosphoserine
blank values.
All phosphoserine solutions were stored in
the refrigerator to prevent spontaneous breakdown.
The next set of assays was a repeat of the initial
ser-3 experiment using freshly prepared salted extracts
(Fig. 7).
This second ser-3 experiment produced results
different from those in the first two experiments.
Although
there was still a significant difference between the prototrophic and mutant enzyme specific activities, both
values were higher than those obtained using the first
ser-3 extracts and, thus, also higher than those of the
ser(JBM5} extracts (Table 1).
27
Q
Figure 7.
Comparison of phosphoserine phosphatase activi-
ties in experiment three using data from salted protein
extracts of ser-3 and ST74A, a prototrophic strain.
•
28
0.04
·-E
1:
........
E
0.03
1:
an
.....
00
c•
e•
~
0.02
0.01
...._.... ser-3
·-prototroph
0.1
PR 0 TEIN (mg)
0.2
29
It was thought likely that the variation between
the two sets of results was due to technical differences
between the ser(JBM5) and ser-3 experiments.
For instance,
it was probable that improvements in extraction and assay
techniques could have occurred from the time of the first
run to that of the final ser-3 experiment.
Alternatively,
the difference might reflect random variation typical of
crude enzyme preparations.
As a test of whether the increase in enzyme activity might be due to a change in technique or in reagents,
the first ser-3 salted extract, which had been kept frozen,
was assayed again using the changes in technique and
reagents which had been instituted for the second ser-3
assays.
A comparison of the original salted ser-3 assays
(Fig. 8) and the "improved" assays on the same extract
(Fig. 9) showed little if any difference in values from one
to the other.
Thus, it was concluded that the variability
was probably not caused by changes in technique or in
reagents.
Next it was thought best to rerun both the
ser(JBM5) and the ser-3 experiments (including the prototrophic strains) doing the harvesting and extraction for
both at the same time and the assaying for both using the
same reagents and techniques.
In this way, it was hoped
that the treatment of the four preparations would be as
similar as possible.
When this was done, the results
30
Figure 8.
Phosphoserine phosphatase activity of ser-3 in
experiment two.
The reaction mixture contained 0.2 mrnoles
Tris-HCl (pH 7.5), 50
~moles
of MgC1 2 , 20
~moles
of phos-
phoserine, and varying amounts of protein in a final volume
of 3 ml.
The reaction was started by the addition of
salted protein extract.
At 7, 15, and 21 minute, 0.9 ml
aliquots were removed and assayed for inorganic phosphate.
31
PROTEIN (mg/ml)
0.061
....... 0.046
......... 0.031
0--<>
o. 4
E
~
...
It)
00
c .•
0
0.2
5
10
TIME (min)
15
20
32
Figure 9.
Second determination of phosphoserine phospha-
tase activity of ser-3 in experiment two.
The reaction
mixture contained 0.6 mmoles Tris-HCl (pH 7.5), 150
of MgC1 2 , 60
~moles
~moles
of phosphoserine, and varying amounts
of salted protein extract in a final volume of 9 ml.
The
reaction was started by the addition of protein extract.
At 1, 2, 3, 4, 5, 15, and 22 minutes, 0.9 ml aliquots were
removed and assayed for inorganic phosphate.
33
0.4
PROTEIN (mg/m I)
o--o 0.061
...._. 0.046
---0.031
~0.015
0.2
5
10
TIME (min)
15
20
34
in Fig. 10 and Fig. 5 were obtained for ser(JBMS) and
ser-3 respectively.
Although in both ser(JBMS) and ser-3.
there was still a difference between prototrophic and
mutant specific activity, it was much less marked than in
the previous experiments.
Also in the first experiment the
ser(JBMS) enzyme had shown lower specific activity than the
ser-3 phosphoserine phosphatase specific activities in
experiments two and three; in experiments four and five the
situation had reversed itself with ser(JBMS) showing the
greater specific activity (Table 1).
@
'
35
Figure 10.
Comparison of phosphoserine phosphatase acti-
vities in experiment five using data from salted protein
extracts of ser(JBM5) and the prototrophic strain from which
it was derived.
36
0.04c
E
·'E
0.03
c
....
It)
CIO
c•
e•
<I
0.02
0.01
•-• ser(JBMS)
e-e prototroph
0.1
PR 0 TEIN (mg)
0.2
DISCUSSION
The present study has clarified some aspects of
serine biosynthesis in Neurospora.
The results have shown
that ser-3 and ser(JBMS) are qualitatively similar in their
decreased phosphoserine phosphatase specific activities
when compared to their respective prototrophic progenitor
strains.
Because ser(JBMS) differs from its progenitor by
the single gene associated with its serine dependence, one
may conclude that the serine requirement is due to its
decreased phosphoserine phosphatase specific activity.
Because phosphoserine phosphatase catalyzes the terminal
step in the phosphorylated pathway of serine biosynthesis,
it follows that this pathway must be the main source of
serine under the conditions of this study.
This evidence
supports the theory of Sojka and Garner (1967) but conflicts
with that of Wright (1951) who suggested that the glyoxylate
pathway is the major source of serine in Neurospora.
One
possible resolution of this conflict is that different
serine pathways are activated depending on the medium on
which the organism is grown.
Ulane and Ogur (1972) indi-
cate that two serine-glycine auxotrophs of Saccharomyces
are serine-glycine independent when grown on acetate
because the glyoxylate pathway is derepressed in acetate
medium and supplies the glycine and serine required for
37
38
growth.
However, the phosphorylated pathway is the main
source of serine and glycine for the same mutants grown on
glucose containing media.
Beremand and Sojka (1977) have
found support for similar regulation in the bacterium, Rhodopseudomonas capsulata.
There is also strong evidence that
environmental factors influence pathway selection in serine
biosynthesis for Pseudomonas (Hepenstall & Quayle, 1970;
Harder & Quayle, 1971).
Another question that has been investigated during
the course of this study concerns the nature of the genetic
lesion in ser-3.
How is it that this mutant is serine
requiring in vivo but still shows in vitro phosphoserine
phosphatase activity that varies from one third to one half
that of the prototrophic strain?
There are several possible
explanations for the nature of this lesion.
One theory is that any serine requiring mutant would
show a decrease in phosphoserine phosphatase specific activity.
This unlikely possibility was eliminated by a fellow
investigator who looked at ser-1, a Neurospora mutant that
is non-allelic with ser-3, and found that it showed greater
phosphoserine phosphatase specific activity than a related
prototrophic strain (Feldman, unpublished data).
A second possibility is that the defect is in a
regulator gene.
If this were so, ser-3 wouldproduce reduced
amounts of the same phosphoserine phosphatase as the prototrophic strain.
Purification of the mutant and prototrophic
39
phosphoserine phosphatases would produce a convergence in
their respective specific activities.
In this study, sup-
port for a defect in a regulator gene for ser-3 phosphoserine phosphatase stems from the observation that the specific
activity of the ser-3 enzyme increased sequentially from one
experiment to the next.
One explanation for this behavior
is that the purification techniques of this investigator
improved with time, producing a purer enzyme for each succeeding assay.
Thus, as extraneous proteins were removed
from the extracts, the mutant enzyme activity approached the
levels of the prototrophic enzyme activity.
This conver-
gence in specific activities is consistent with what would
be expected if the lesion occurred in a regulator gene controlling the level of enzyme production.
A variation of
this theory is that there is a time lag for switching on the
structural gene in the mutant; this alternate hypothesis
could be tested by assaying protein extracts from younger
cultures.
If the defect were in the structural gene for phosphoserine phosphatase, then ser-3 would produce a defective
enzyme in quantities similar to the normal enzyme levels
produced by the prototrophic strain.
In this case, purifi-
cation of the prototrophic and mutant enzymes would result
in a divergence of their respective specific activities.
variation of this theory is that the mutant enzyme is more
sensitive to the environment than the prototrophic enzyme.
A
40
If the mutant enzyme were more sensitive to the environment
than the trototrophic enzyme, then one would expect that an
improvement in purification technique would result in
increases in the specific activity on the mutant enzyme; the
specific activity of the less sensitive prototrophic enzyme
would remain relatively stable.
The fact that this was the
case in the present study provides support for the idea that
ser-3 produces a defective phosphoserine phosphatase.
It
should be noted that this same evidence can be used to support the previous theory that ser-3 has a defective regulator gene.
Another explanation for the variation in ser-3
phosphoserine phosphatase specific activity might be that
the fractionation of the extracts differed from one experiment to another such that there was a variable amount of
non-specific protein in each extract.
But if this were
true, one would expect to see the same amount of variability
in the prototrophic specific activity as seen in the mutant.
In this study, the mutant enzyme activity showed greater
variation fron one experiment to the next than did the prototrophic specific activity (Table 1).
A defective structural gene might provide one
explanation for the fact that although ser-3 requires serine
in vivo, it can have in vitro phosphoserine phosphatase
activity that is up to one half that of the prototrophic
enzyme.
Perhaps the defective enzyme is unable to function
under cellular conditions.
Preliminary experiments by Beck
41
(unpublished data) indicate that the mutant phosphoserine
phosphatase is much more sensitive to changes in pH than
is the prototrophic enzyme.
Another possibility is that
the Km of the enzyme is changed by the mutation so that it
can not bind substrate at the low concentrations found in
the cell but can bind it at the relatively high experimental concentrations.
Determination of the Km of the
ser-3 enzyme would resolve this question.
Temperature is
another environmental variable that could be tested.
It is also possible that an organized enzyme system
is necessary for cellular serine production in Neurospora.
If the enzyme aggregate was not functional, the cell could
contain each of the individual enzyme activities required
for each of the steps in the pathway but still be unable to
produce serine.
This would explain why ser-3 is auxo-
trophic for serine in the cell but has active phosphoserine
phosphatase in vitro.
Wagner and others (1966) have found
evidence suggesting an organized enzyme system for the synthesis of valine and isoleucine in Neurospora.
A similar
situation has been described in malic dehydrogenase mutants
of Neurospora (Munkres & Woodward, 1966).
Also, the fact
that ser-3 is a "leaky" mutant in that if allowed to sit
long enough it will eventually grow on minimal medium is
suggestive evidence for the hypothesis that an enzyme aggregate may be involved in its serine biosynthesis.
Perhaps
an organized enzyme system is necessary for normal in vivo
p '
42
growth, but the unaggregated enzymes can synthesize serine
if given enough time.
Thus, the evidence from this study could be used to
support any of several theories concerning the nature of
the lesion in ser-3.
Full resolution of this question will
require isolation and purification of both the mutant and
prototrophic enzymes and comparison of their specific
activities.
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