Vol. 86, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
February 14, 1979
Pages 815-821
ISOLATION
AND CHARACTERIZATION
Yukihiro Hideshi
OF YEAST PROTEASE B
SANADA, Katsuhisa FUJISHIRO*, TANAKA* and Nobuhiko KATUNUMA
Department of Enzyme Chemistry, Institute School of Medicine, Tokushima University, 770, and *Division of Biological Research, Co., Ltd., Tsu, Mie 514, Japan Received
December
for Enzyme Research, Tokushima, Tokushima Imuraya Confectionary
12,1978 SUMMARY
Protease B was purified from baker's yeast. The final preparation appeared homogeneous by ultracentrifugation and electrophoresis. The S~C,,W value of the enzyme was 3.1 S and its molecular weight was calculated to be 31,000 from the results of sedimentation equilibrium analysis. The amino acid composition of the enzyme was also investigated. The enzyme inactivates phosphogluconate dehydrogenase and uricase, but not malate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase or hexokinase. Three
different
proteases,
B and C have been found the
high
in yeast
purifications
Protease
on acrylamide The present physicochemical
been partially
and Holzer gel paper
[5]
disc
and
properties MATERIALS
the
Hata
purified purified
[4]
et al.
to a homogeneous by Ulane
A,
report
A and C.
by Hata
large-scale
of protease
as protease
et al.
of protease
electrophoresis
describes
designated
[l-3].
and properties
B has also
and Saheki
usually
and Cabib
purification
[l] state [6]. and
B.
AND METHODS
Activated CH-Sepharose 4B, SP Sephadex C-50, QAE Sephadex A-25, and Sephadex G-100 were products of Pharmacia Fine Chemicals Inc. p-Toluene sulfonic acid and 3-(Z-amino-ethyl) indole were obtained from Pierce Chemical Co. Hide Powder and hexokinase (type III) were Azure, D-tryptophan methyl ester, dehydrogenase, obtained from Sigma Chemical Co. Phosphogluconate uricase, and malate dehydrogenase from yeast were purchased from Oriental Yeast Co., Ltd. Alcohol dehydrogenase and glucose-6phosphate dehydrogenase were obtained from Boehringer Mannheim. 0006-291X/79/030815-07$01.00/0 81.5
Copyright All rights
@ 1979 by Academic Press, Inc. of reproduction in any form reserved
Vol.
86,
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Protease B activity was determined as described by Saheki and Holzer [7] using 3% hide powder azure as substrate. One unit of enzyme activity was expressed as the amount producing 1.0 of A590 nm per min per 1.1 ml. The activities of phosphogluconate dehydrogenase, uricase, malate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinase were assayed as described by Bergmeyer [8]. One unit of phosphogluconate dehydrogenase or uricase activity was expressed as the amount producing 1 ymole of product per min at 25'C. Protein concentrations were determined by the biuret method [9] or by the method of Kalcker [lo]. Sedimentation equilibrium studies were carried out as described by Schachman and Edelstein Quantitative amino acid analysis was performed by the [ill. method of Liu and Chang [12]. The D-tryptophan methyl ester affinity column was prepared using activated CH-Sepharose 4B as described in the manufacturer's manual. RESULTS AND DISCUSSION Enzyme Purification was plasmolyzed by Hata water
Two kilograms
at pH 7.0
et al.
[l].
overnight
centrifuged
-
at 10,000
buffer,
rpm for
pH 6.0,
the
was suspended
recentrifuged. by ultrafiltration buffer
for
Sephadex
4 hours.
pH 6.0, the
acetate the
(5.5
Then
0.1
PMlO),
M NaCl
the
same
10 mM Na-phosphate
against
at 15,000 The resulting (3.5
and
to a QAE
and material
C-SO column
and
concentrated
against
overnight
in the
same buffer
with
rotor).
816
the
was applied
and centrifuged
to a SP Sephadex
5 min
to pH
was decanted
was concentrated
dialyzed
(SS-34
rpm for
and dialyzed
solution
The eluate
pH 5.0,
same centrifuge
was applied
the
containing
(Amicon buffer,
XM50)
adjust
were combined,
x 12 cm) equilibrated
same buffer.
filtration
in
(Amicon
column
buffer,
50 ml of
The two supernatants
RC-5 centrifuge
0.1 M NaCl,
The supernatant in
tap
in 350 ml of 10 mM
at 15,000
rotor).
yeast
5N HCl and
in a Sorvall
containing
(SS-34
against
with
was dissolved
same centrifuge
with
to pH 3.8
10 min
baker's
at pH 5 as described
was dialyzed
2N NaOH and centrifuged
precipitate
autolyzed
adjusted
The precipitate
Na-phosphate 6.0 with
and then
The autolysate
and then
(GS3 rotor).
of commercial
was eluted by ultra50 mM rpm for
5 min
supernatant
x 12 cm) equi-
Vol. 86, No. 3, 1979
BIOCHEMICAL
Table Purification
1. 2. 3. 4. 5. 6. 7.
1.
Purification
step
with
washed with
containing (Amicon final
Recovery
ml 2,800 2,800 544 2,000 344 140 4
mg 30,940 23,800 7,400 5,100 1,756 48 3.3
Unit 196.0 616.0 761.6 580.0 817.2 168.0 38.2
Unit/mg 0.006 0.026 0.103 0.114 0.466 3.50 11.6
% 100 314 389 296 417 86 19
with
0.5 M NaCl.
The column
with
with
0.1
the
buffer,
with
to
pattern
PMlO)
results
containing same buffer
and
enzyme was eluted solution
was
and in a collodion
(3 x 112
pH 7.0,
cm)
equi-
and eluted
containing
(Amicon
Representative
are shown in Table Properties
of eluate
by ultrafiltration
and the
column
buffer,
The fraction
was concentrated bag.
G-100
50 mM K-potassium
same buffer.
collodion
(Amicon
a Sephadex
the
The enzyme
PMlO)
with
the protease and in a
on enzyme purification
1.
of Protease of mercuri-protease
B -
Figure B,
1-A shows the
The schlieren
817
to
x 5.5 cm)
pH 7.0, with
to a
was applied
(2.5
buffer,
pH 3.8.
was added
solution
column
pH 5.0,
by ultrafiltration
bag and applied librated
buffer,
with
of eluate
solution
and the
affinity
was
by ultrafiltration
was washed first
0.1 M acetate
concentrated
was eluted
NaCl
0.1 M K-phosphate
M acetate
material
was concentrated
ester
equilibrated
The column
The fraction
of 0.5 M NaCl methyl
pH 5.0.
and then
Then concentrated
a II-tryptophan
then
buffer,
pH 7.0.
the protease
concentration
Yeast Specific activity
buffer,
PMlO).
B in
Total activity
same buffer
0.1 M K-phosphate
Protease
Total protein
50 mM acetate the
of
Volume
Plasmolysis pH 5.0 Activation Acid ppt. QAE Sephadex SP Sephadex Affinity chromat. Sephadex G-100
librated
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ultracentrifugal
pattern
obtained
Vol. 86, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and polyacrylamide disc electrophoretic Fig. 1. Ultracentrifugal patterns of purified protease B. The enzyme was treated with 1 mM mercuric chloride and then passed through Sephadex G-25 to remove excess mercuric chloride. A, Sedimentation velocities were The picture was taken 36 min measured at 60,000 rev/min at 11°C. The protein concentration was after attaining maximal speed. B, Electrophoresis 5.6 mg/ml in 50 mM K-phosphate buffer, pH 7.0. of 50 1J.g of mercuri-enzyme at pH 4.5 in the presence of 4 M urea. The arrow indicates the position of marker dye.
indicates
the
coefficient was 3.1
of the
S.
gel
disc
analysis. by two other
value
methods:
and that
dodecylsulfate
polyacrylamide
water
by Sephadex
of the mercuri-enzyme
818
was also in the weight
by sedimentation
the molecular
gel
at ZO"C,
The molecular
was consistent
as 30,700
chromatography
1-B.
to be 31,000
This
B was estimated
for
electrophoresis
as shown in Fig,
enzyme was determined
determined
calculated
The sedimentation
of the mercuri-enzyme
by polyacrylamide of 4 M urea
component,
peak,
The homogeneity
equilibrium
protease
of a single
of the protein
confirmed presence
presence
electrophoresis.
with
the values
weight G-100
of native column
as 32,000
by sodium
These
values
Vol. 86, No. 3, 1979
BIOCHEMICAL
Table 2.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Amino Acid Composition of Protease B
Amino acids
of residues
No.
g 1OOgprotein
31,000g protein
7.09 2.80 3.16 1.06 12.65 6.85 7.41 6.71 3.20 2.12 7.63 0.76 4.14 0.94 3.61 7.35 4.19 3.84
15.0 5.6 =6 ;:7 29.5 17.9 21.9 14.2 8.6 8.8 26.6 0.9 11.0 2.0 8.6 17.4 7.1 7.2
Lys ine Histidine Arginine Tryptophan Aspartic acid Threoninea Serinea Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
The values are the average of the 24, 48, and 72 hours hydrolyses. aExtrapolated to zero hour hydrolysis.
were much less
than
that
2 shows the amino acid contained
relatively
Inactivation [131, function
of protease
by limited either
protease
Holzer
The purified
enzymes,
gluconate
dehydrogenase B was very
unstable
in 30 min at 25’C. at 25’C.
the physiological
37’C.
B in yeast,
on various
Therefore,
Under these
819
50% of
However, all
conditions,
contain
in an attempt
it
to
we
commercially
actions
from yeast,
and lost
synthase
does not
2 showed its
and uricase
it
and galactosamine.
of pre-chitin
of protease
Figure
in 1.5 min at
out
that
Therefore,
role
Table
Cabib and Ulane
protease
of the preparation
yeast
carried
reported
B was the activation
available
catalytically
Previously,
[14]
[6].
In addition
of glucosamine
A or C activity.
the actions
protease
of the enzyme.
content
the catabolic
examined
by Ulane and Cabib
Enzymes -
proteolysis.
investigate
activity
content
high
of Yeast
andHasilikand
reported
on phospho-
The purified
its
activity
did not experiments one unit
autolose
any
were of
Vol. 86, No. 3, 1979
BIOCHEMICAL
I
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I PHOSPHOGLUCONATE DEHYDROGENASE
URICASE I
100 90 6380 z70 Y ; 60 2 gso ;40 CL 30
100 90
s 80 : 70
30 20
10
1
I 10
I
TIME(MIN)
I
20
TIME(MIN)
Inactivation of phosphogluconate dehydrogenase and uricase by protease B. Phosphogluconate dehydrogenase (0.69 units) and uricase (0.24 units) were incubated with (0) or without Co) 0.12 and 0.15 units, respectively, of protease B in 0.6 ml of K-phosphate buffer, pH 7.0, at 25*C.
Fig..
protease genase vate
B inactivated and 0.12
malate
phosphate that
particular
units
units
of uricase
dehydrogenase,
protein
per minute.
that
specific it
regulation
It
dehydrogenase,
or hexokinase.
B has a rather and thus
of phosphogluconate
alcohol
dehydrogenase,
protease
substrates,
0.04
These action
participates
dehydrodid
not
inacti-
glucose-6results
on intact
suggest protein
in intracellular
in yeast.
ACKNOWLEDGEMENTS We thank R. Kunai for analysis and H. Miyai with acid analytical experiments.
helping with the technical
the ultracentrifugal assistance of amino
REFERENCES 1. Hata, T., Hayashi, R. and Doi, E. (1967) Agric. Biol. Chem. 3J, 150-159 . 2. Lenney, J.F. and Dalbec, J.M. (1969) Arch. Biochem. Biophys. 129, 407-409 3. Hayashi, R., Moore, S. and Stein, W.H. (1973) J. Biol. Chem. 248, 2296-2302 R. and Doi, E. (1967) Agric. Biol. Chem. 4. Hata, T., Hayashi, 2, 357-367 820
Vol.
86,
5.
6. 7. 8. 9. 10. 11. 12. 13. 14.
No.
3, 1979
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Saheki, T. and Holzer, H. (1975) Biochem. Biophys. Acta 384, 203-214 Ulane, R.E. and Cabib, E. (1976) J. Biol. Chem. 251, 3367-3374 Saheki, T. and Holzer, H. (1974) Eur. J. BiochemxJ, 621-626 Bergmeyer, H.U. (1974) Methods of Enzymatic Analysis Vol. 1, Verlag Chemie Weinheim, Academic Press, Inc. New York and London Gornall, A.G., Bardawill, C.S. and David, M.M. (1949) J. Biol. Chem. 177, 751-766 Kalcker, H.M. (1947) J. Biol. Chem. 167, 461-475 Schachman, H.K. and Edelstein, S.J. p73) Methods Enzymol. D), 3-59 -27 (Part Liu, T.Y. and Chang, Y.H. (1971) J. Biol. Chem. 246, 2842-2848 Cabib, E. and Ulane, R. (1973) Biochem. Biophys. Res. Commun. 50, 186-191 Hasilik, A. and Holzer, 1-I. (1973) Biochem. Biophys. Res. Commun. 53, 552-559
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