Vol.
86,
February
No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 982-987
28, 1979
Amino Acid
Substitution
of Mating
Factor
Structure-Activity Yoshihiro
Masui*, Hiroshi
*The
Peptide
Protein
Minoh, **Central
Research
January
Summary :
Analogs
Chino*
Foundation
Japan
Institute,
Mishimagun,
Naoyoshi Sakakibara*
Research
Osaka 562,
Shimamotocho,
Received
Tanaka**,
and Shumpei
Institute,
cerevisiae:
Relationship
Takaharu Kita**
of Saccharomyces
Suntory
Ltd.
Osaka 618,
Japan
4,1979 of the mating
factor
of Saccharomyces cerevisiae, Trp'11 13 His'-Trp3-Leu-Gin-Leu6-Lys7-Pro8-Gly-Gin-Pro -Met"-Tyr , from which amino acids were eliminated or substituted for other amino acid, were synthesized. These analogs showed lower biological activity than the natural mating factor if assayed after 6 hours incubation with a-mating type cells of S. cerevisiae. However, if assayed after 24 or 48 hours Incubation, the situation changed, i.e. the analogs in which Leu' or Lys7 were replaced by the corresponding D-isomer, showed higher mating factor activity than the unsubstituted mating factor. The same result was obtained with the analogs in which Met was replaced by norleucine. INTRODUCTION The mating C&mating
type
and a-mating
factor cells,
type
of Saccharomyces which
cells
is
(1).
cerevisiae
indispensable Its
amino
is
to the mating acid
sequence
Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr firmed
by the chemical As was reported
degraded
by both&-
and a hepta-peptides both
of which
our studies mating sequential
factor
were
synthesis in our
by cleavage inactive
to determine activity elimination
of the peptide paper
type
cells
of a peptide
as mating the minimum
(6),
factor. sequence
the changes
of amino
acid
982
process
of 5.
by
betweenS(to be
3, 4) and was con-
6).
the mating
factor
cerevisiae
Besides,
a hexa-
Leub and Lys',
during
of the peptide
the course required
activity
from either
was
into
bond between
of biological
residues
0006-291X/79/040982-06$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
(S),
(3,
produced
was determined (2,
previous
and a-mating
a peptide
amino-
for
of the
due to the or carboxyl-
Vol. 86, No. 4, 1979
terminal
BIOCHEMICAL
ends of the mating
observations
stimulated
substitution
on the mating
The present analogs
factor
our
paper
peptide
interest factor
factor
differed
to explore
dramatically.
the effects
The
of amino
acid
activity.
describes
of the mating
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the properties
of chemically
of 5. cerevisiae
along
with
synthesized
their
biological
activities. MATERIALS and METHODS Biological activity of the mating factor analogs was assayed by incubating 2. cerevisiae X2180 lA, a-mating type cell, at 27°C for 6, 24 and 48 hours in the presence of the peptide to be examined (4). The activity was expressed as the minimum amount of a peptide necessary to induce the morphological changes in the test organisms. Syntheses of peptides were carried out according to the conventional solution procedure under the conditions described in the previous paper (6). RESULTS and DISCUSSION Analytical
Properties
mating
factor
the effect
were
of substituting
amino
residues
sized
was checked
on the biological
summarized
Substitution
effect
Trp
1
on its
residue
or leucine
natural
mating
activity
(Table
Substitution acid
Factor
amino
acid
of
to examine
factor
by other
of each peptide analysis
syntheand
and Rf on cellulose
amino
acids
As the dodecapeptide
plates
Replacement
with
(6),
but
replacement
the
and by D-tryptophan,
to l/1,000
- l/10,000
lysine
to a complete
led
of
to have little
by phenylalanine
peptides,
activity
devoid
tridecapeptide
was anticipated
Substitution
active its
-
as the natural
activity.
reduced
of that loss
of the of the
IA).
of His' reduced (Table
chemically
in the mating
rotation
number
I.
less
factor.
large
Homogeneity
of optical
by other
biological
-A
synthesized
chromatography,
was as active
gave slightly
tyrosine
layer
of Mating
of Trp'
sarcosine
activity
in Table
acid
activity.
The values
of Trp
substitution
amino
by thin
analysis.
N-terminal
Factor
of 2. cerevisiae
acids
were
of Mating
analogs
amino
element
of Analogs
IB).
of Mating its
activity Furthermore,
Factor
-
to l/lDO,OOD
Substitution - l/l,OOO,OOO
the replacement
983
of His2
of His2
of its
by any other
original
by D-histidine
BIOCHEMICAL
Vol. 86, No. 4, 1979
Table Amino
Properties
I. Acid
and Biological
Optical
Replacement*
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
[o(]25D
Activity
Rotation
of
Mating
Factor
Rf**
(c=O . 5 , AcOH)
Analogs Biological
A
B
C
Activity***
0.59
0.74
6
w/ml Natural
Mating
A. Trp'
was
Factor
-35.6"
substituted
by
[Phe']
-36.1"
0.56
0.73
7
x 10
[Sar']
-47.0"
0.46
0.65
1.3
x 102
[Leu']
-38.5"
'3.62
0.76
3
x lo3
Uyr’l
-36.1"
0.59
0.73
1.5
x 104
[Tv’ 1 Bz des
Trp'
[LYA B. His2
was substituted
0.59
0.75
8
x 104
0.64
0.82
4
x lo5
-35.5"
0.46
0.61
3
x 108
by
UYS21
-36.5"
0.42
2.5
x lo5
[Phe2]
-35.9"
0.61
4
x lo5
[Leu2]
-43.0"
0.67
9
x 106
[D-His2]
-39.8
3
x 108
C. Trp3
was
substituted
0.69
0.81
by
[Ala31
-41.6"
0.55
0.69
1
x 102
[Phe3]
-42.7"
0.65
0.74
1.4
x lo2
[Leu3]
-45.1"
0.59
0.74
2.3
x lo2
[Vr31
-34.6"
0.53
0.73
8
x 1D2
[W31
-38.2"
0.49
0.64
2.1
x lo4
[Gly31
-37.2"
0.53
0.64
2.9
x lo5
VW31
-40.9"
0.51
0.62
5
x 105
des
-35.4"
0.57
0.73
5
x lo8
Trp3
D. Pro8,
Pro ",
[Nleu
E.
-48.0" -52.5"
Met
12
12 ]
and Tyr
13
were
substituted
by
-38.1"
0.64
0.78
2
x lo2
[D-Tyr13]
-37.3"
0.63
0.74
1.5
x 104
[GUY"]
-18.5"
0.57
0.74
1.5
x 104
My81
-25.7"
0.55
0.72
2
x lo4
[Phe13] [Tyr-NH21
-36.2" -43.0"
0.64 0.59
0.69 0.74
3
x lo4
6
x lo4
[Va112]
-41.8"
0.63
0.77
1
x 105
-27.0"
0.60
0.76
1.5
x lo4
-37.0"
0.59
0.74
4
x 108
-26.9"
0.60
0.75
1.1
x 105
-36.1"
0.57
0.68
3
x lo8
-21.4"
0.59
0.72
2.4
x lo6
Leu6
and
Lys'
were
[D-Leu6] des Trp'
CD-Leu6]
[D-Lys7] des
Trp'
[D-Lys7]
[D-Leu6,'D-Lys7]
substituted
by
* Trp'-His2-Trp3-Leu-Gln-Leu6-Lys7-Pro8-G1y-Gln-Pro **A l-BuOH:AcOH:H 0 (4:1:5, upper phase) B 1-BuOH:AcOH:P$ridine:H 0 (15:3:10:12) C I-BuOH:AcOH:H O:n-Hexage (4:1:5:0.5, *** Minimum amounz needed 7 to induce the type cells.
984
11
upper phase) morphological
-Met12-Tyr13
changes
in
a-mating
Vol.
86,
No.
4, 1979
BIOCHEMICAL
gave an inactive
peptide.
mating
factor
factor
activity.
though
the des Trp
removal Substitution
results
conclusion
dodecapeptide
resulted
of Trp3
of Mating
into
two groups
on biological
hydrophobic
side
chains
and others
but replacement extremely amino
by amino
low activity
acid
acids
sequence
with
Substitution
of Pro*,
ment of Pro*
or Pro
original
activity.
activity
to l/16,000
D-tyrosine,
Pro ",
11
with
These
the slight
changes
the amino
acid
residues
of its
of Leub and Lys'
substitution
of Leu'
loss
of activity. Though
active
of either
as the natural
for
slightly, with
having
an identical
the absence
of Trp'
and the hydropho-
of Mating which
Factor
-
showed
l/2,000
also
Replace-
or norleucine
lowered
gave much less ID.
by the
its
active
From these
substitution
by others
of the
of Tyr 13 with
Replacement
factor
activity
activity.
by valine
induced
The
peptides
not Trp-His, for
carrying chains.
its
yielded
in Table
of Mating
by D-leucine
The same was true devoid
tridecapeptide,
Factor
yielded
Tyr 13 or Metl*
the dodecapeptide
(6).
were
results
even
of any one of reflected
in the
activity.
Substitution
replacement
group
amide
in the mating
biological
side
reduced
gave peptides
of conformation
those
hydrophilic
and Tyr13
are summarized
the
of the activity
Namely,
except
and tyrosine
factor,
of Trp3
factor
respectively.
results
loss
dodecapeptide
and l/15,
that,
as mating
As the
of Met12
by the evidence
of replacement
group
Substitution
mating
to the effect
be essential Met12
for
Trp3
of His-Trp,
glycine
phenylalanine
peptides.
decrease
could
needed
of the
of substituting
of the latter
the sequence
His2
The result
former
the mating
of the tryptophan
-
carrying
IC).
that
supported
activity.
of the
(Table
showed no activity, bicity
acids
COMMUNICATIONS
of the peptide
active
according
acids
strongly
a complete
Factor
amino
by amino
part
was fully
of the peptide
RESEARCH
suggest
is further
other
substitution
BIOPHYSICAL
an essential
This
can be divided with
These
constitutes
of His2
AND
-As
a less
active
of the peptide for
shown in Table
resulted
the replacement
of the N-terminal complete
98.5
peptide.
abolition
IE,
Combined
in complete
of Lys7 by D-lysine. Trp
residue
of its
was as biological
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
48
24
6
HOURS Fig.
1.
Effect
Relative
activity
of each analog mating
activity
on Mating as a ratio
at the time
factor
needed
. , [D-Leu6]; mating
of Incubation was expressed
of assay
at 6 hours
O , [D-Lys7];
Factor
to the amount
incubation. A)
Activity.
of minimum
amount
of natural
0, natural; and h, [D-trp']
[D-Tyr13];
factors.
was observed
when its
of Amino Acid
Substitution
Leu'
or Lys7 was substituted
by D-leucine
or
D-lysine. Effect
The substitution gave a less
active
incubation assayed the
of any amino
at 27°C (Table after
cleavage
24 and 48 hours
incubation.
of the peptide
toward
degradation
contrast,
their
mating
6 hours
corresponding by a-mating factor
incubation,
a peptide
having
is
type enough
but fairly D-lysine
in place
loss
the
is degraded
by of
more resistant 1,
6 pg/ml
biological
activity
of Lys7 lost
were
replacement
in Fig.
of its of its
6 hours
factor
gave peptides
expression
rapid
986
mating
As shown
always
when peptides
Leu6 and Lys7,
cells.
Factor factor
after
changed
Since
D-isomer
for
mating
was assayed
situation
bond between
of Mating
in natural
activity
But the
one with
the natural
when its
Degradation
residue
I).
either
after
peptide
on the acid
of
activity
followed.
no activity
In even
Vol. 86, No. 4, 1979
after
72 hours
BIOCHEMICAL
incubation,
activity
than
the original
with
test
organisms.
the
in which
II-leucine
two peptides
and,
replaced
The possibility
that
factor
D-amino
derived
be excluded
analogs paper
is
Leu'
the
acid
when assayed could
or norleucine
biological
after
24 hours
be drawn
about
replaced
Met12,
low activity
expressed
incubation the peptides
although
by the mating
in them is due to the contaminating
by competetion data).
COMMUNICATIONS
these
gradually.
from L-isomers
(unpublished
factor
RESEARCH
had more potent
The same conclusion
degraded
having
BIOPHYSICAL
as a result,
mating
were
analogs
AND
of respective between
Analysis
now in progress
D-amino
the original
acid
using
the conditions
of these
described
natural
preparations
and substituted
of the mode of function
factor mating could
mating mating
factor factor
in the preceeding
(7). REFERENCES
1. 2.
Levi,
J.D.
Stotzler,
(1956 ) D.,
Nature
Kl itz,
H.,
177,
753 - 754.
and Duntze,
W. (1976)
Eur.
J. Biochem.
69,
397 - 400. 3.
Tanaka, Acad.
4.
Tanaka, 82,
T.,
Kita,
H.,
Murakami,
T.,
and Narita,
K. (1977)
Proc.
Japan
H.,
Murakami,
T.,
and Narita,
K. (1977)
J. Biochem.
H. (1977)
J.
53, 67 - 70. T.,
Kita,
1688 - 1687.
5.
Tanaka,
6.
Masui,
7.
Tanaka,
Kita,
T.,
and Kita,
Y., Chino, H. (1977) T.,
PI., Sakakibara, Biocnem.
and Kita,
S.,
Biophys.
H. (1978)
Biochem. Tanaka,
82, T.,
1689 -1693. Murakami,
T.,
and
Res. Commun. 78, 534 - 538. Biochem.
1319 - 1324.
987
Biophys.
Res. Commun. 83,
86,
February
No.
4, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages 982-987
28, 1979
Amino Acid
Substitution
of Mating
Factor
Structure-Activity Yoshihiro
Masui*, Hiroshi
*The
Peptide
Protein
Minoh, **Central
Research
January
Summary :
Analogs
Chino*
Foundation
Japan
Institute,
Mishimagun,
Naoyoshi Sakakibara*
Research
Osaka 562,
Shimamotocho,
Received
Tanaka**,
and Shumpei
Institute,
cerevisiae:
Relationship
Takaharu Kita**
of Saccharomyces
Suntory
Ltd.
Osaka 618,
Japan
4,1979 of the mating
factor
of Saccharomyces cerevisiae, Trp'11 13 His'-Trp3-Leu-Gin-Leu6-Lys7-Pro8-Gly-Gin-Pro -Met"-Tyr , from which amino acids were eliminated or substituted for other amino acid, were synthesized. These analogs showed lower biological activity than the natural mating factor if assayed after 6 hours incubation with a-mating type cells of S. cerevisiae. However, if assayed after 24 or 48 hours Incubation, the situation changed, i.e. the analogs in which Leu' or Lys7 were replaced by the corresponding D-isomer, showed higher mating factor activity than the unsubstituted mating factor. The same result was obtained with the analogs in which Met was replaced by norleucine. INTRODUCTION The mating C&mating
type
and a-mating
factor cells,
type
of Saccharomyces which
cells
is
(1).
cerevisiae
indispensable Its
amino
is
to the mating acid
sequence
Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr firmed
by the chemical As was reported
degraded
by both&-
and a hepta-peptides both
of which
our studies mating sequential
factor
were
synthesis in our
by cleavage inactive
to determine activity elimination
of the peptide paper
type
cells
of a peptide
as mating the minimum
(6),
factor. sequence
the changes
of amino
acid
982
process
of 5.
by
betweenS(to be
3, 4) and was con-
6).
the mating
factor
cerevisiae
Besides,
a hexa-
Leub and Lys',
during
of the peptide
the course required
activity
from either
was
into
bond between
of biological
residues
0006-291X/79/040982-06$01.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
(S),
(3,
produced
was determined (2,
previous
and a-mating
a peptide
amino-
for
of the
due to the or carboxyl-
Vol. 86, No. 4, 1979
terminal
BIOCHEMICAL
ends of the mating
observations
stimulated
substitution
on the mating
The present analogs
factor
our
paper
peptide
interest factor
factor
differed
to explore
dramatically.
the effects
The
of amino
acid
activity.
describes
of the mating
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the properties
of chemically
of 5. cerevisiae
along
with
synthesized
their
biological
activities. MATERIALS and METHODS Biological activity of the mating factor analogs was assayed by incubating 2. cerevisiae X2180 lA, a-mating type cell, at 27°C for 6, 24 and 48 hours in the presence of the peptide to be examined (4). The activity was expressed as the minimum amount of a peptide necessary to induce the morphological changes in the test organisms. Syntheses of peptides were carried out according to the conventional solution procedure under the conditions described in the previous paper (6). RESULTS and DISCUSSION Analytical
Properties
mating
factor
the effect
were
of substituting
amino
residues
sized
was checked
on the biological
summarized
Substitution
effect
Trp
1
on its
residue
or leucine
natural
mating
activity
(Table
Substitution acid
Factor
amino
acid
of
to examine
factor
by other
of each peptide analysis
syntheand
and Rf on cellulose
amino
acids
As the dodecapeptide
plates
Replacement
with
(6),
but
replacement
the
and by D-tryptophan,
to l/1,000
- l/10,000
lysine
to a complete
led
of
to have little
by phenylalanine
peptides,
activity
devoid
tridecapeptide
was anticipated
Substitution
active its
-
as the natural
activity.
reduced
of that loss
of the of the
IA).
of His' reduced (Table
chemically
in the mating
rotation
number
I.
less
factor.
large
Homogeneity
of optical
by other
biological
-A
synthesized
chromatography,
was as active
gave slightly
tyrosine
layer
of Mating
of Trp'
sarcosine
activity
in Table
acid
activity.
The values
of Trp
substitution
amino
by thin
analysis.
N-terminal
Factor
of 2. cerevisiae
acids
were
of Mating
analogs
amino
element
of Analogs
IB).
of Mating its
activity Furthermore,
Factor
-
to l/lDO,OOD
Substitution - l/l,OOO,OOO
the replacement
983
of His2
of His2
of its
by any other
original
by D-histidine
BIOCHEMICAL
Vol. 86, No. 4, 1979
Table Amino
Properties
I. Acid
and Biological
Optical
Replacement*
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
[o(]25D
Activity
Rotation
of
Mating
Factor
Rf**
(c=O . 5 , AcOH)
Analogs Biological
A
B
C
Activity***
0.59
0.74
6
w/ml Natural
Mating
A. Trp'
was
Factor
-35.6"
substituted
by
[Phe']
-36.1"
0.56
0.73
7
x 10
[Sar']
-47.0"
0.46
0.65
1.3
x 102
[Leu']
-38.5"
'3.62
0.76
3
x lo3
Uyr’l
-36.1"
0.59
0.73
1.5
x 104
[Tv’ 1 Bz des
Trp'
[LYA B. His2
was substituted
0.59
0.75
8
x 104
0.64
0.82
4
x lo5
-35.5"
0.46
0.61
3
x 108
by
UYS21
-36.5"
0.42
2.5
x lo5
[Phe2]
-35.9"
0.61
4
x lo5
[Leu2]
-43.0"
0.67
9
x 106
[D-His2]
-39.8
3
x 108
C. Trp3
was
substituted
0.69
0.81
by
[Ala31
-41.6"
0.55
0.69
1
x 102
[Phe3]
-42.7"
0.65
0.74
1.4
x lo2
[Leu3]
-45.1"
0.59
0.74
2.3
x lo2
[Vr31
-34.6"
0.53
0.73
8
x 1D2
[W31
-38.2"
0.49
0.64
2.1
x lo4
[Gly31
-37.2"
0.53
0.64
2.9
x lo5
VW31
-40.9"
0.51
0.62
5
x 105
des
-35.4"
0.57
0.73
5
x lo8
Trp3
D. Pro8,
Pro ",
[Nleu
E.
-48.0" -52.5"
Met
12
12 ]
and Tyr
13
were
substituted
by
-38.1"
0.64
0.78
2
x lo2
[D-Tyr13]
-37.3"
0.63
0.74
1.5
x 104
[GUY"]
-18.5"
0.57
0.74
1.5
x 104
My81
-25.7"
0.55
0.72
2
x lo4
[Phe13] [Tyr-NH21
-36.2" -43.0"
0.64 0.59
0.69 0.74
3
x lo4
6
x lo4
[Va112]
-41.8"
0.63
0.77
1
x 105
-27.0"
0.60
0.76
1.5
x lo4
-37.0"
0.59
0.74
4
x 108
-26.9"
0.60
0.75
1.1
x 105
-36.1"
0.57
0.68
3
x lo8
-21.4"
0.59
0.72
2.4
x lo6
Leu6
and
Lys'
were
[D-Leu6] des Trp'
CD-Leu6]
[D-Lys7] des
Trp'
[D-Lys7]
[D-Leu6,'D-Lys7]
substituted
by
* Trp'-His2-Trp3-Leu-Gln-Leu6-Lys7-Pro8-G1y-Gln-Pro **A l-BuOH:AcOH:H 0 (4:1:5, upper phase) B 1-BuOH:AcOH:P$ridine:H 0 (15:3:10:12) C I-BuOH:AcOH:H O:n-Hexage (4:1:5:0.5, *** Minimum amounz needed 7 to induce the type cells.
984
11
upper phase) morphological
-Met12-Tyr13
changes
in
a-mating
Vol.
86,
No.
4, 1979
BIOCHEMICAL
gave an inactive
peptide.
mating
factor
factor
activity.
though
the des Trp
removal Substitution
results
conclusion
dodecapeptide
resulted
of Trp3
of Mating
into
two groups
on biological
hydrophobic
side
chains
and others
but replacement extremely amino
by amino
low activity
acid
acids
sequence
with
Substitution
of Pro*,
ment of Pro*
or Pro
original
activity.
activity
to l/16,000
D-tyrosine,
Pro ",
11
with
These
the slight
changes
the amino
acid
residues
of its
of Leub and Lys'
substitution
of Leu'
loss
of activity. Though
active
of either
as the natural
for
slightly, with
having
an identical
the absence
of Trp'
and the hydropho-
of Mating which
Factor
-
showed
l/2,000
also
Replace-
or norleucine
lowered
gave much less ID.
by the
its
active
From these
substitution
by others
of the
of Tyr 13 with
Replacement
factor
activity
activity.
by valine
induced
The
peptides
not Trp-His, for
carrying chains.
its
yielded
in Table
of Mating
by D-leucine
The same was true devoid
tridecapeptide,
Factor
yielded
Tyr 13 or Metl*
the dodecapeptide
(6).
were
results
even
of any one of reflected
in the
activity.
Substitution
replacement
group
amide
in the mating
biological
side
reduced
gave peptides
of conformation
those
hydrophilic
and Tyr13
are summarized
the
of the activity
Namely,
except
and tyrosine
factor,
of Trp3
factor
respectively.
results
loss
dodecapeptide
and l/15,
that,
as mating
As the
of Met12
by the evidence
of replacement
group
Substitution
mating
to the effect
be essential Met12
for
Trp3
of His-Trp,
glycine
phenylalanine
peptides.
decrease
could
needed
of the
of substituting
of the latter
the sequence
His2
The result
former
the mating
of the tryptophan
-
carrying
IC).
that
supported
activity.
of the
(Table
showed no activity, bicity
acids
COMMUNICATIONS
of the peptide
active
according
acids
strongly
a complete
Factor
amino
by amino
part
was fully
of the peptide
RESEARCH
suggest
is further
other
substitution
BIOPHYSICAL
an essential
This
can be divided with
These
constitutes
of His2
AND
-As
a less
active
of the peptide for
shown in Table
resulted
the replacement
of the N-terminal complete
98.5
peptide.
abolition
IE,
Combined
in complete
of Lys7 by D-lysine. Trp
residue
of its
was as biological
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
48
24
6
HOURS Fig.
1.
Effect
Relative
activity
of each analog mating
activity
on Mating as a ratio
at the time
factor
needed
. , [D-Leu6]; mating
of Incubation was expressed
of assay
at 6 hours
O , [D-Lys7];
Factor
to the amount
incubation. A)
Activity.
of minimum
amount
of natural
0, natural; and h, [D-trp']
[D-Tyr13];
factors.
was observed
when its
of Amino Acid
Substitution
Leu'
or Lys7 was substituted
by D-leucine
or
D-lysine. Effect
The substitution gave a less
active
incubation assayed the
of any amino
at 27°C (Table after
cleavage
24 and 48 hours
incubation.
of the peptide
toward
degradation
contrast,
their
mating
6 hours
corresponding by a-mating factor
incubation,
a peptide
having
is
type enough
but fairly D-lysine
in place
loss
the
is degraded
by of
more resistant 1,
6 pg/ml
biological
activity
of Lys7 lost
were
replacement
in Fig.
of its of its
6 hours
factor
gave peptides
expression
rapid
986
mating
As shown
always
when peptides
Leu6 and Lys7,
cells.
Factor factor
after
changed
Since
D-isomer
for
mating
was assayed
situation
bond between
of Mating
in natural
activity
But the
one with
the natural
when its
Degradation
residue
I).
either
after
peptide
on the acid
of
activity
followed.
no activity
In even
Vol. 86, No. 4, 1979
after
72 hours
BIOCHEMICAL
incubation,
activity
than
the original
with
test
organisms.
the
in which
II-leucine
two peptides
and,
replaced
The possibility
that
factor
D-amino
derived
be excluded
analogs paper
is
Leu'
the
acid
when assayed could
or norleucine
biological
after
24 hours
be drawn
about
replaced
Met12,
low activity
expressed
incubation the peptides
although
by the mating
in them is due to the contaminating
by competetion data).
COMMUNICATIONS
these
gradually.
from L-isomers
(unpublished
factor
RESEARCH
had more potent
The same conclusion
degraded
having
BIOPHYSICAL
as a result,
mating
were
analogs
AND
of respective between
Analysis
now in progress
D-amino
the original
acid
using
the conditions
of these
described
natural
preparations
and substituted
of the mode of function
factor mating could
mating mating
factor factor
in the preceeding
(7). REFERENCES
1. 2.
Levi,
J.D.
Stotzler,
(1956 ) D.,
Nature
Kl itz,
H.,
177,
753 - 754.
and Duntze,
W. (1976)
Eur.
J. Biochem.
69,
397 - 400. 3.
Tanaka, Acad.
4.
Tanaka, 82,
T.,
Kita,
H.,
Murakami,
T.,
and Narita,
K. (1977)
Proc.
Japan
H.,
Murakami,
T.,
and Narita,
K. (1977)
J. Biochem.
H. (1977)
J.
53, 67 - 70. T.,
Kita,
1688 - 1687.
5.
Tanaka,
6.
Masui,
7.
Tanaka,
Kita,
T.,
and Kita,
Y., Chino, H. (1977) T.,
PI., Sakakibara, Biocnem.
and Kita,
S.,
Biophys.
H. (1978)
Biochem. Tanaka,
82, T.,
1689 -1693. Murakami,
T.,
and
Res. Commun. 78, 534 - 538. Biochem.
1319 - 1324.
987
Biophys.
Res. Commun. 83,
