- Email: [email protected]
Effect of fructose 1,6-bisphosphate on the activity of liver pyruvate kinase aftter limited proteolysis with cathepsin B
BIOCHEMICAL
Vol. 110, No. 2, 1983 January 27, 1983
AND BIOPHYSICAL
Pages
EFFECT OF FRUCTOSE 1,6-BISPHOSPHATE KINASE AFTER LIMITED Noboru Faculty Received
RESEARCH COMMUNICATIONS
Nakai,*
Yutaka
ON THE ACTIVITY
682-687
OF LIVER PYRUVATE
PROTEOLYSIS WITH CATHEPSIN B
Fujii,
Kyoichi
Kobashi
and Jun'ichi
Hase
of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyam-shi, Toyama 930-01,JAPAN December
10, 1982
Treatment of rat liver-type pyruvate kinase with rabbit liver cathepsin B at pH 7.0 caused loss of activity in the standard assay with 0.6 mM of phosphoenolpyruvate. The modified enzyme exhibited about 10% of the original activity when assayed with 2.0 mM of the substrate. No detectable change in the subunit molecular weight of the enzyme occured during inactivation. Cn addition of 4 uM fructose 1,6-bisphosphate the activity of the treated enzyme was restored to that of the original enzyme. Limited proteolysis of the enzyme by cathepsin B appears to enhance the requirement for the positive effector, fructose 1,6-bisphosphate. Limited
proteolysis
of enzyme molecules
considered
to be an initial
contribute
to the regulation
is defined
as the hydrolase
arginine-p-nitroanilide
of protein
stage
for
hemoglobin
(l),
glycolysis
such as glucokinase,
most protein
substrates
it appears
shown
cathepsin
cleavage
result
of the limited
subunit
molecule Pyruvate
catalyzes
without
and to modify
loss
may also
Cathepsin
to inactivate
fructose
kinase
Fru-Pz
that
is
B (EC 3.4.22.1) o-N-benzoyl-DL-
a-N-benzoyl-DL-arginine-g-naphtylamide
and has been reported
that
proteinases
cl-N-benzoyl-L-arginineamide,
and pyruvate
We have
tissue
degradation
of enzyme activity.
(BAPA),
(1)
with
to act
(Fru-Pn)
rabbit for
fructose
in
aldolase
1,6-bisphosphatase
B inactivated
near
involved
(2).
as a peptidyldipeptidase
of the activity
proteolysis
some enzymes
1,6-bisphosphate fructose
and
With
(3-5).
muscle
aldolase
for
l-phosphate
the carboxyl-terminal
region
as a of the
(6). kinase
(ATP:
a key reaction
*Present address: Department Fukui 910-11 JAPAN
pyruvate
2-0-phosphotransferase,
in glycolytic
pathway,
of Chemistry,
0006-291X/83/020682-06$01.50/0 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
682
Fukui
EC 2.7.1.40)
and the activity Medical
School,
of the liverMatsuoka,
Vol. 110, No. 2, 1983
type
enzyme,
Fru-Pz
but
BIOCHEMICAL
not
the muscle-type
enzyme,
RESEARCH COMMUNICATIONS
has been shown
to be affected
by
(7). In the present
pyruvate greatly
AND BIOPHYSICAL
kinase enhanced
paper
for
we report
the allosteric
by limited
that
the requirement
effector,
proteolysis MATERIALS
with
fructose cathepsin
of the liver-type 1,6-bisphosphate
is
B.
AND METHODS
NADH, Fru-Pz and BAPA were obtained from Boehringer-Mannheim. Reagents: Phosphoenolpyruvate cyclohexylammonium salt and sodium ADP were purchased All other reagents were of the highest grade from Oriental Yeast Co. Japan. available.
Preparation of enzymes: Rat liverand muscle-type pyruvate kinases were prepared from the tissues of the animals fed on a high carbohydrate diet (8) according to the method of Harada --et al. (9). The enzymes preparations showed a single band on polyacrylamide slab gel electrophoresis in the presence of SDS. Specific activities of liverand muscle-type of enzymes were 200 and 240 unitsjmg protein, respectively. These preparations were dissolved in 10 mM potassium phosphate buffer, pH 6.0, containing 5 mM each of 2-mercaptoethanol and MgS04, and then dialyzed aginst the same buffer in order to remove Fru-Pz completely. Rabbit muscle-type pyruvate kinase was obtained from Boehringer-Mannheim and was dissolved and dialyzed as described above. Cathepsin B from rabbit liver was prepared as described (6), the specific activity was 2.5 unitsfmg protein. Assay of enzymes: Except where otherwise indicated, pyruvate kinase activity was measured with a coupled assay system using lactate dehydrogenase (rabbit muscle-type: Boehringer-Mannheim) similar to that described by Harada --et al. The assay mixture contained final concentrations of 50 mM Tris-HCl (6). buffer, pH 7.4, 100 mM KCl, 5 mM MgS04, 2 mM ADP, 0.2 mM NADN, 2 mM phosphoenolpyruvate and 4.6 units of lactate dehydrogenase in a total volume of 1.0 ml. The decrease in absorbance of NADH at 340 nm was measured at 30°C, using a Gilford 250 multiple spectrophotometer. One unit of pyruvate kinase was defined as the amount of enzyme that caused the oxidation of 1 umole of NADH per min. The activity of cathepsin B was measured with 0.1 M sodium phosphate buffer, pH 6.5, containing 0.8 mM BAPA, 1 mM each of 2-mercaptoethanol and EDTA at 37'C Absorbance of p-nitroaniline released from BAPA by cathepsin B was (1) measured at 410 nm using a Hitachi 100-10 spectrophotometer. One unit of cathepsin B was defined as the amount of enzyme that released 1 pmole of p-nitroaniline per hr.
The reactin of pyruvate Treatment of pyruvate kinase with cathepsin B: kinase with cathepsin B was carried out at 30°C in 0.2 M triethanolamineacetate buffer at indicated pHs in the presence of 5 mM each of Z-mercaptoethanol and EDTA. Protein determination: of Lowry --et al. (10)
using
Protein concentrations bovine serum albumin
were determined as a standard.
by the method
Electrophoresis: SDS polyacrylamide slab gel electrophoresis was carried by the method of Laemmli (11). The gel was stained with 0.2% Coomassie brilliant blue and scanned at 580 nm with a Gilford 250 multiple spectrophotometric gel scanner. 683
out
Vol. 110, No. 2, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Figure after
1 shows the effect
incubation
pyruvate
with
kinase
at 3O'C.
cathepsin
was stable
Modification
The activities cathepsin
and rabbit
of phosphoenolpyruvate cathepsin below
by cathepsin
B exhibited to that
of the original
for 0.17
mM and that
of the
enzyme
the activity
From Lineweaver-Burk
phosphoenolpyruvate
type
(Fig.
in the presence treated.enzyme
of curve (Fig.
with
ZC),
treated
6
7
6
a Hill
coefficient
and in the presence
of of
of the untreated
was calculated B was 0.27
10’
PH
Fig. 1. Loss of activity of liver-type pyruvate kinase, measured under standard assay condition, on digestion with cathepsin B at several pHs. Rat liver pyruvate kinase (0.225 unit/ml) was incubated with (closed circles) and without (open circles) cathepsin B (0.05 unit/ml) as described in MATERIALS AND METHODS. Relative inactivation is shown by the line (-X-). 684
modified
the Km of the native
cathepsin
9
was
of 4 pM Fru-P2
-100
5
with
kinase
to that
of 4 pM Fru-P2 with
of the concentration
pyruvate
ZB),
by
shown).
The addition
was identical
plots
not affected
enzyme activity
activation;
a hyperbolic
pH.
of phophoenolpyruvate
10% of the original
remarkable
1.0 mM phophoenolpyruvate
not
The enzyme
of 2.0 x1$4substrate.
caused
(data
30 min
at neutral
were
as a function
2.4.
Liver
for
B was rapid
at concentrations
approximately
system
enzyme.
in Fig.
activity
in the presence
kinase
incubation
kinases
above
kinase
buffer.
after
pyruvate
described
pyruvate
little
to the assay
1.2 similar
pH region
muscle
is shown
B showed
0.6 mM, but
observed
at neutral
of liver
pyruvate
B in triethanolamine-acetate
the condition
The activity
of liver
of the enzyme by cathepsin
of rat B under
of pH on the activity
mM.
enzyme to be
Vol. 110, No. 2, 1983
old _,_
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
_-*----- *./---a 2.0
1.0
fPEP,mMl
-1
l/(PEP,mhll
-05
1 0
1 05
logfPEP,mhV)
Fig. 2. Effect of Fru-Pz on the inactivated liver-type pyruvate kinase with cathepsin B (0.05 unit/ml) at pH 7.0 for 2 hr at 30'. (A); The modified enzyme (closed circles) and the original enzyme (open circles) were assayed at various concentrations of phosphoenolpyruvate in the presence (solid line) and the absence (dotted line) of 4 uM Fru-Pa. Other assay conditions were as described in MATERIALS AND METHODS. (B); Lineweaver-Burk plots obtained from data of (C); Replots of data from (A) for calculation of the (A). Hill coefficients.
In
weight
order
to
of the
gel
electrophoresis
lar
weight
were
determine
whether
enzyme subunits, (Fig. observed
3). during
cathepsin
the modified
B
caused
a change
enzyme was subjected
No significant
changes
in
to SDS slab
the subunit
the modification.
Fig. 3. SDS polyacrylamide (15%) slab gel electrophoretic patterns of the native and modified pyruvate kinases. was treated with cathepsin Liver pyruvate kinase (0.61 rag/ml) B (0.05 mg/ml) at pH 7.0. Cathepsin B, native pyruvate kinase and 50% and 80% "inactivated" enzymes were analyzed dn SDS slab gels. (A); Cathepsin B, (B); native enzyme, (C) and (D); 50% and 80% "inactivated" enzymes, respectively. 685
in molecular
molecu-
Vol. 110, No. 2, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION The pH optimum for
hydrolysis
of synthetic
as hemoglobin tentatively
of cathepsin
(1).
of fructose
modification
of liver
Thus,
the difference
enzymes may depend which
may also
kinase
activity. Under
also
the lack
the standard
assay liver
of 1.0 mM substrate. kinase,
enzyme completely of the proteolytic
buffer was 5.0
for
observed reactivated Limited
appears dependent
modification
kinase with
the native
a change
modifier, kinase
mM) of
B showed
Unexpectedly, in
that Fru-Pz.
by cathepsin
The nature B is now under
ACKNOWLEDGEMENT thank Professor T. Tanaka and his colleagues University for their helpful suggestion with crystalline rat liver-type pyruvate kinase. B.L. Horecker of Roche Institute of Molecular suggestion in preparation of the manuscript.
of School of respect to We are also Biology for
REFFERENCES 1. 2. 2: 5.
Otto, K. (1971) in Tissue Proteinases (Barrett, A.J. and Dingle, J.J., eds), pp. l-28, North Holland Publishing Co., Amsterdam. Nakashima, K. and Ogino, K. (1974) J. Biochem. 2, 355-365. Aronson, N.N.Jr. and Barrett, A.J. (1978) Biochem. J. 171, 759-765. Bond, J.S. and Barrett, A.J. (1980) Biochem. J. 189, 17-25. Hannappel, E., MacGregor, J.S., Stephen, D. and Horecker, B.L. (1982) Arch. Biochem. Biophys. 214, 293-298. 686
of
makes the
investigation.
We wish to Medicine, Osaka preparation of indebted to Dr. his advice and
the
the presence
at the COOH-terminus
in conformation
on the allosteric of pyruvate
cathepsin
of Fru-Pz
probably
enzyme.
pyruvate
(2.0
enzyme.
by the addition
at
proteins,
B on muscle
with
the
of these
concentration
treated
for
the native
modification
high
was
effective
of the substrate
with
such
In contrast,
for
of cathepsin
proteolysis,
to cause
stability
structure
condition
pyruvate
(2).
6.5
proteins
and that
B was most
proteolytic
of effect
for
region;
of aldolase (6),
by cathepsin
on the quarternary
enzyme was fully
pyruvate
the inactivation
the pH of greatest
10% of the activity
modified
kinase
in pH optima
explain
phosphoenolpyruvate, about
is
pH for
1,6-bisphosphatase pyruvate
to be acidic
such as BAPA and 4.5
to be 5.3 in a citrate
modification
pH, which
substrates
The optimal
determined
neutral
B has been reported
Vol. 110, No. 2, 1983
6. 7. 8. 9. 10. 11.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
K. and Hase, J. (1978) Biochem. Biophys. Nakai, N., Wada, K., Kobashi, Res. Communs. 83, 881-885. Bailry, E., Stirpe, F. and Taylor. C.B. (1968) Biochem. J. 108, 427-436. Tanaka, T., Harano, Y., Sue, F. and Morimura, H. (1967) J. Biochem. 62, 71-91. Harada, K., Saheki, S. Wada, K. and Tanaka, T. (1978) Biochim. Biophys. Acta 534, 327-339. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Laemmli, U.K. (1970) Nature (London) 227, 680-685.
687
Vol. 110, No. 2, 1983 January 27, 1983
AND BIOPHYSICAL
Pages
EFFECT OF FRUCTOSE 1,6-BISPHOSPHATE KINASE AFTER LIMITED Noboru Faculty Received
RESEARCH COMMUNICATIONS
Nakai,*
Yutaka
ON THE ACTIVITY
682-687
OF LIVER PYRUVATE
PROTEOLYSIS WITH CATHEPSIN B
Fujii,
Kyoichi
Kobashi
and Jun'ichi
Hase
of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyam-shi, Toyama 930-01,JAPAN December
10, 1982
Treatment of rat liver-type pyruvate kinase with rabbit liver cathepsin B at pH 7.0 caused loss of activity in the standard assay with 0.6 mM of phosphoenolpyruvate. The modified enzyme exhibited about 10% of the original activity when assayed with 2.0 mM of the substrate. No detectable change in the subunit molecular weight of the enzyme occured during inactivation. Cn addition of 4 uM fructose 1,6-bisphosphate the activity of the treated enzyme was restored to that of the original enzyme. Limited proteolysis of the enzyme by cathepsin B appears to enhance the requirement for the positive effector, fructose 1,6-bisphosphate. Limited
proteolysis
of enzyme molecules
considered
to be an initial
contribute
to the regulation
is defined
as the hydrolase
arginine-p-nitroanilide
of protein
stage
for
hemoglobin
(l),
glycolysis
such as glucokinase,
most protein
substrates
it appears
shown
cathepsin
cleavage
result
of the limited
subunit
molecule Pyruvate
catalyzes
without
and to modify
loss
may also
Cathepsin
to inactivate
fructose
kinase
Fru-Pz
that
is
B (EC 3.4.22.1) o-N-benzoyl-DL-
a-N-benzoyl-DL-arginine-g-naphtylamide
and has been reported
that
proteinases
cl-N-benzoyl-L-arginineamide,
and pyruvate
We have
tissue
degradation
of enzyme activity.
(BAPA),
(1)
with
to act
(Fru-Pn)
rabbit for
fructose
in
aldolase
1,6-bisphosphatase
B inactivated
near
involved
(2).
as a peptidyldipeptidase
of the activity
proteolysis
some enzymes
1,6-bisphosphate fructose
and
With
(3-5).
muscle
aldolase
for
l-phosphate
the carboxyl-terminal
region
as a of the
(6). kinase
(ATP:
a key reaction
*Present address: Department Fukui 910-11 JAPAN
pyruvate
2-0-phosphotransferase,
in glycolytic
pathway,
of Chemistry,
0006-291X/83/020682-06$01.50/0 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
682
Fukui
EC 2.7.1.40)
and the activity Medical
School,
of the liverMatsuoka,
Vol. 110, No. 2, 1983
type
enzyme,
Fru-Pz
but
BIOCHEMICAL
not
the muscle-type
enzyme,
RESEARCH COMMUNICATIONS
has been shown
to be affected
by
(7). In the present
pyruvate greatly
AND BIOPHYSICAL
kinase enhanced
paper
for
we report
the allosteric
by limited
that
the requirement
effector,
proteolysis MATERIALS
with
fructose cathepsin
of the liver-type 1,6-bisphosphate
is
B.
AND METHODS
NADH, Fru-Pz and BAPA were obtained from Boehringer-Mannheim. Reagents: Phosphoenolpyruvate cyclohexylammonium salt and sodium ADP were purchased All other reagents were of the highest grade from Oriental Yeast Co. Japan. available.
Preparation of enzymes: Rat liverand muscle-type pyruvate kinases were prepared from the tissues of the animals fed on a high carbohydrate diet (8) according to the method of Harada --et al. (9). The enzymes preparations showed a single band on polyacrylamide slab gel electrophoresis in the presence of SDS. Specific activities of liverand muscle-type of enzymes were 200 and 240 unitsjmg protein, respectively. These preparations were dissolved in 10 mM potassium phosphate buffer, pH 6.0, containing 5 mM each of 2-mercaptoethanol and MgS04, and then dialyzed aginst the same buffer in order to remove Fru-Pz completely. Rabbit muscle-type pyruvate kinase was obtained from Boehringer-Mannheim and was dissolved and dialyzed as described above. Cathepsin B from rabbit liver was prepared as described (6), the specific activity was 2.5 unitsfmg protein. Assay of enzymes: Except where otherwise indicated, pyruvate kinase activity was measured with a coupled assay system using lactate dehydrogenase (rabbit muscle-type: Boehringer-Mannheim) similar to that described by Harada --et al. The assay mixture contained final concentrations of 50 mM Tris-HCl (6). buffer, pH 7.4, 100 mM KCl, 5 mM MgS04, 2 mM ADP, 0.2 mM NADN, 2 mM phosphoenolpyruvate and 4.6 units of lactate dehydrogenase in a total volume of 1.0 ml. The decrease in absorbance of NADH at 340 nm was measured at 30°C, using a Gilford 250 multiple spectrophotometer. One unit of pyruvate kinase was defined as the amount of enzyme that caused the oxidation of 1 umole of NADH per min. The activity of cathepsin B was measured with 0.1 M sodium phosphate buffer, pH 6.5, containing 0.8 mM BAPA, 1 mM each of 2-mercaptoethanol and EDTA at 37'C Absorbance of p-nitroaniline released from BAPA by cathepsin B was (1) measured at 410 nm using a Hitachi 100-10 spectrophotometer. One unit of cathepsin B was defined as the amount of enzyme that released 1 pmole of p-nitroaniline per hr.
The reactin of pyruvate Treatment of pyruvate kinase with cathepsin B: kinase with cathepsin B was carried out at 30°C in 0.2 M triethanolamineacetate buffer at indicated pHs in the presence of 5 mM each of Z-mercaptoethanol and EDTA. Protein determination: of Lowry --et al. (10)
using
Protein concentrations bovine serum albumin
were determined as a standard.
by the method
Electrophoresis: SDS polyacrylamide slab gel electrophoresis was carried by the method of Laemmli (11). The gel was stained with 0.2% Coomassie brilliant blue and scanned at 580 nm with a Gilford 250 multiple spectrophotometric gel scanner. 683
out
Vol. 110, No. 2, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS Figure after
1 shows the effect
incubation
pyruvate
with
kinase
at 3O'C.
cathepsin
was stable
Modification
The activities cathepsin
and rabbit
of phosphoenolpyruvate cathepsin below
by cathepsin
B exhibited to that
of the original
for 0.17
mM and that
of the
enzyme
the activity
From Lineweaver-Burk
phosphoenolpyruvate
type
(Fig.
in the presence treated.enzyme
of curve (Fig.
with
ZC),
treated
6
7
6
a Hill
coefficient
and in the presence
of of
of the untreated
was calculated B was 0.27
10’
PH
Fig. 1. Loss of activity of liver-type pyruvate kinase, measured under standard assay condition, on digestion with cathepsin B at several pHs. Rat liver pyruvate kinase (0.225 unit/ml) was incubated with (closed circles) and without (open circles) cathepsin B (0.05 unit/ml) as described in MATERIALS AND METHODS. Relative inactivation is shown by the line (-X-). 684
modified
the Km of the native
cathepsin
9
was
of 4 pM Fru-P2
-100
5
with
kinase
to that
of 4 pM Fru-P2 with
of the concentration
pyruvate
ZB),
by
shown).
The addition
was identical
plots
not affected
enzyme activity
activation;
a hyperbolic
pH.
of phophoenolpyruvate
10% of the original
remarkable
1.0 mM phophoenolpyruvate
not
The enzyme
of 2.0 x1$4substrate.
caused
(data
30 min
at neutral
were
as a function
2.4.
Liver
for
B was rapid
at concentrations
approximately
system
enzyme.
in Fig.
activity
in the presence
kinase
incubation
kinases
above
kinase
buffer.
after
pyruvate
described
pyruvate
little
to the assay
1.2 similar
pH region
muscle
is shown
B showed
0.6 mM, but
observed
at neutral
of liver
pyruvate
B in triethanolamine-acetate
the condition
The activity
of liver
of the enzyme by cathepsin
of rat B under
of pH on the activity
mM.
enzyme to be
Vol. 110, No. 2, 1983
old _,_
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
_-*----- *./---a 2.0
1.0
fPEP,mMl
-1
l/(PEP,mhll
-05
1 0
1 05
logfPEP,mhV)
Fig. 2. Effect of Fru-Pz on the inactivated liver-type pyruvate kinase with cathepsin B (0.05 unit/ml) at pH 7.0 for 2 hr at 30'. (A); The modified enzyme (closed circles) and the original enzyme (open circles) were assayed at various concentrations of phosphoenolpyruvate in the presence (solid line) and the absence (dotted line) of 4 uM Fru-Pa. Other assay conditions were as described in MATERIALS AND METHODS. (B); Lineweaver-Burk plots obtained from data of (C); Replots of data from (A) for calculation of the (A). Hill coefficients.
In
weight
order
to
of the
gel
electrophoresis
lar
weight
were
determine
whether
enzyme subunits, (Fig. observed
3). during
cathepsin
the modified
B
caused
a change
enzyme was subjected
No significant
changes
in
to SDS slab
the subunit
the modification.
Fig. 3. SDS polyacrylamide (15%) slab gel electrophoretic patterns of the native and modified pyruvate kinases. was treated with cathepsin Liver pyruvate kinase (0.61 rag/ml) B (0.05 mg/ml) at pH 7.0. Cathepsin B, native pyruvate kinase and 50% and 80% "inactivated" enzymes were analyzed dn SDS slab gels. (A); Cathepsin B, (B); native enzyme, (C) and (D); 50% and 80% "inactivated" enzymes, respectively. 685
in molecular
molecu-
Vol. 110, No. 2, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
DISCUSSION The pH optimum for
hydrolysis
of synthetic
as hemoglobin tentatively
of cathepsin
(1).
of fructose
modification
of liver
Thus,
the difference
enzymes may depend which
may also
kinase
activity. Under
also
the lack
the standard
assay liver
of 1.0 mM substrate. kinase,
enzyme completely of the proteolytic
buffer was 5.0
for
observed reactivated Limited
appears dependent
modification
kinase with
the native
a change
modifier, kinase
mM) of
B showed
Unexpectedly, in
that Fru-Pz.
by cathepsin
The nature B is now under
ACKNOWLEDGEMENT thank Professor T. Tanaka and his colleagues University for their helpful suggestion with crystalline rat liver-type pyruvate kinase. B.L. Horecker of Roche Institute of Molecular suggestion in preparation of the manuscript.
of School of respect to We are also Biology for
REFFERENCES 1. 2. 2: 5.
Otto, K. (1971) in Tissue Proteinases (Barrett, A.J. and Dingle, J.J., eds), pp. l-28, North Holland Publishing Co., Amsterdam. Nakashima, K. and Ogino, K. (1974) J. Biochem. 2, 355-365. Aronson, N.N.Jr. and Barrett, A.J. (1978) Biochem. J. 171, 759-765. Bond, J.S. and Barrett, A.J. (1980) Biochem. J. 189, 17-25. Hannappel, E., MacGregor, J.S., Stephen, D. and Horecker, B.L. (1982) Arch. Biochem. Biophys. 214, 293-298. 686
of
makes the
investigation.
We wish to Medicine, Osaka preparation of indebted to Dr. his advice and
the
the presence
at the COOH-terminus
in conformation
on the allosteric of pyruvate
cathepsin
of Fru-Pz
probably
enzyme.
pyruvate
(2.0
enzyme.
by the addition
at
proteins,
B on muscle
with
the
of these
concentration
treated
for
the native
modification
high
was
effective
of the substrate
with
such
In contrast,
for
of cathepsin
proteolysis,
to cause
stability
structure
condition
pyruvate
(2).
6.5
proteins
and that
B was most
proteolytic
of effect
for
region;
of aldolase (6),
by cathepsin
on the quarternary
enzyme was fully
pyruvate
the inactivation
the pH of greatest
10% of the activity
modified
kinase
in pH optima
explain
phosphoenolpyruvate, about
is
pH for
1,6-bisphosphatase pyruvate
to be acidic
such as BAPA and 4.5
to be 5.3 in a citrate
modification
pH, which
substrates
The optimal
determined
neutral
B has been reported
Vol. 110, No. 2, 1983
6. 7. 8. 9. 10. 11.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
K. and Hase, J. (1978) Biochem. Biophys. Nakai, N., Wada, K., Kobashi, Res. Communs. 83, 881-885. Bailry, E., Stirpe, F. and Taylor. C.B. (1968) Biochem. J. 108, 427-436. Tanaka, T., Harano, Y., Sue, F. and Morimura, H. (1967) J. Biochem. 62, 71-91. Harada, K., Saheki, S. Wada, K. and Tanaka, T. (1978) Biochim. Biophys. Acta 534, 327-339. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Laemmli, U.K. (1970) Nature (London) 227, 680-685.
687