Download Protease Activity in the Human Erythrocyte

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Protease
Activity
in the
Human
to the
Cell
Localization
By Samir
Proteolytic
activity
erythrocyte
was
membrane.
This
be ascribed
to
the
suspension.
the
soluble
more
active
against
IN
the
which
against
the
could
not
extracted
in
cell
subunits
mole-
experiments
activity
TWO
CIRCUMSTANCES
units of hemoglobin
are
but
not
activity
of
from
dent
on
and
ATP
for
the
membrane-free
was
temperature
an
be
mem-
to O.75-M
protease
or
than
could
cell
by exposure
time
chains
activity
erythrocyte
The
system
beta
The
from
require
confirmed
proteolytic
in degrading
hemolysate.
was
hemoglobin
Burka
chains.
branes.
from
and
R.
active
alpha
hemoglobin
tetrameric
membrane
more
cell
absent
Membrane
Edward
the
leukocytes
totally
of
and
human
to
contaminating
was
Ballas
mature
activity.
Pulse-chase
that
the
localized
portion
than
cules.
in
K.
Erythrocyte:
and
KSCN.
dependid
activity.
was
hemoglobin
or the alphaand beta-chain
to attach
to the human
red cell membrane.
known
not
energy-generating
subFirst,
as the red cell ages in the peripheral
circulation,
small but progressively
amounts
of hemoglobin
become
bound
to the cell membrane.’
Second,
in which
there
is unbalanced
synthesis
of the alpha
and beta subunits
increasing
in conditions
of hemoglo-
bin, the
instance
In the
molecules
the
chains
produced
it is not known
membrane
in excess
what role,
plays
erythrocyte.
in
However,
thalassemia
syndromes,
chains
to the membrane
cells.3
The
manner
membrane
and
senescence
the
in which
the
erythrocyte
and
the
hemoglobins
within
the
proteins.
The
findings
indicate
From
the
Medical
Cardeza
College.
Foundation
26. 1978;
accepted
July
by USPHS
Presented
San
Diego.
Address
Philadelphia.
©
Blood.
1979
Vol.
for
Pa.
Supported
Grant
in preliminary
(‘alif.
December.
reprint
requests
to the
erythrocyte
of
of protein
human
erythrocyte
unknown.
does
subunits,
Research,
Department
Proteolytic
to the cell
a part
in
possibility
we
hemoglobin
contain
and
of
to the
of abnormal
In order
to investigate
this
human
erythrocyte
to degrade
the
the
of excess
hemoglobin
destruction
of red
attachment
remain
first
to
circulating
cells
and have been localized
that
these
enzymes
might
play
Hemazologic
January
normal
for degradation
hemoglobin
and hemoglobin
to the red cell membrane.
Philadelphia.
Submitted
that
the
is characteristic
that attachment
role in premature
responsible
hemoglobin
chains.79
ability
of the mature
activity
directed
against
this activity
is confined
of
which
reacts
processes
cell membrane.2’3
of hemoglobin
death
situation,
enzymes
are present in human
erythroid
membrane.56
It has been
suggested
destroying
excess
have studied
the
and
second
there
is no question
plays a prominent
is not clear,
normal
the
in
also attach
to the
if any, attachment
proteolytic
virtually
Medicine.
all of
Jefferson
9, 1979.
AM-13431.
form
at the 20th
Annual
Meeting
of the American
Society
of Hematology.
1977.
to S. K. Ballas,
M.D.,
Department
of Medicine.
Jefferson
Medical
College.
Pa. /9107.
by Grune
& Stratton.
53. No. 5 (May),
1979)
Inc.
ISSN
0006-4971/79/5305-0008$Ol.0O/O
875
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876
BALLAS
MATERIALS
Erythrocytes
three
the
from
times
310-mOsm
in
membranes
hemolysate
buffer,
were
in 10-12
7.4,
until
hemoglobin
reticulocytes
from
hemoglobin
subunits
Enzyme
assay
membrane
M),
tube
then
pH 7.4, and
g for
in 10-20
frozen
the amount
result
were
also carried
to the
20
and thawed
and
method
mm.
volumes
of protein
The
washed
of Dodge,’#{176}
membrane-free
of 20-mOsm
three
times
phosphate
and suspended
was determined.”
and
a high
preparing
proportion
of
membrane-free
form.’2
The
alpha
and beta
on CM-Sephadex.’3
counter
intact
was determined
0.75-M
ofAipha
and
preparation
by column
of
Beta
globin
degraded
The
mercaptoethanol.’4
at intervals
acid
of the
fragments
added
supernatant
to
was
radioactivity.’2
in the assay
of protease
up to 24
were
to acid-soluble
assay
to acid-soluble
of Morrison
cells,
incubation
mixture.
activity
so that
and Neurath4
in a final
Assays
was designed
denaturization
the samples
of Lowry.”
The
by incubating
concentration
for use as the enzyme
of
of 0.75
were
centrifuged
extract.
The
enzyme
extract
human
M of potassium
at 40,000
amount
of protein
was used in the assay
g
for
30
in the
system
used as a control.
Hemoglobin
by
chromatography
and
I ml
or
5 X
the results.
red blood
KSCN
hemolysate
(approximately
at 37#{176}C,
and
of substrate
of enzyme.
by the method
by the method
with
chains
centrifugation,
amount
substrate
was aspirated
membrane-free
system
incubated
the protein
not influence
of human
of
1 .5 ml of 20% tricholoracetic
Following
as a source
I 8 hr at 0-4#{176}C.Following
previously,
water;
to determine
was extracted
supernatant
were
of the original
cells
2 ml
or hemoglobin
an energy-generating
the tubes
of radioactive
activity
contained
hemoglobin
protein.
Activity
and the clear
Following
cells containing
described’2
to the carbonmonoxy
3 ml,
containing
as percentage
or components
Separation
blood
chromatography
in 1 ml of ice-cold
ofProtease
for
of
substrate,
would
cells,
supernatant
mix
the incubation
proteolytic
thiocyanate
volume
scintillation
on degradation
Extraction
the hemoglobin
by column
undegraded
out using
during
washed
as previously
.5 mg of substrate
placed
is expressed
to depend
by incubating
[‘4C]leucine
a final
of a master
were
in a liquid
The
isolated
were
converting
0.5-1
to precipitate
described
membranes
of the labeled
aliquots
red blood
40,000
The
separated
in
10 l
addition
protein
at
washed
by centrifugation
according
System
system,
and
Following
counted
centrifugation
separated
lysing
were
of
after
were
suspension,
hr 0.5-mi
were
After
membranes
buffer,
presence
them
Assay
The
7.4.
the
was obtained
in the
hemolysate
mm,
colorless.
by
METHODS
blood
pH
BURKA
of Substrate
Labeled
The
and
AND
venous
buffer,
sedimented
ml of Krebs-Ringer
Preparation
each
phosphate
was removed,
pH
l0’
30 cc of heparinized
AND
the
Chains
acid-acetone
on CM-52
method,
in the presence
the
of 8-M
alpha
and
beta
subunits
were
urea.”
RESULTS
The
bin
initial
within
studies
the
red
assay
system
freeze-thawed
observed
in
hemoglobin
the
cell.
containing
either
hemoglobin-free
a
classic
in the
membrane-free
activity
determined
blood
lysate
human
hemolysate
in degrading
substrate
location
Labeled
normal
red cell
cell-free
erythroid
was
present
was
that
can
degrade
incubated
in the
hemoglodescribed
human
hemolysate
or a suspension
membranes.
No proteolytic
activity
system,’2
cell
tetrameric
of activity
substrate
indicating
is of extremely
in the
assay
hemoglobin,
that
low
system
or alpha
proteolysis
activity.
there
and
of
was
was
beta
of
When
only
very
little
hemoglo-
bin chains,
during
a period
of 24 hr at 37#{176}C(Fig.
1). However,
when
the same
substrates
were incubated
in the presence
of a freeze-thawed
membrane
suspension, approximately
20% of the tetrameric
hemoglobin
was degraded
during
24 hr,
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ERYTHROCYTE
MEMBRANE
PROTEASE
877
ACTIVITY
0
w
ID
4
HEMOGLOBIN
E.1
LI
50
#{176}“
CHAIN
Fig. 1 .
globin and
CHAIN
,9H
Degradation
hemoglobin
of radioactive
hemosubunits
by erythro-
cyte
components.
The
proportion
of radioactive
I-.
to acid-soluble
the presence
25
bars
indicate
the
substrate
degraded
fragments
when
of membrane-free
incubated
in
hemolysate
or hemoglobin-free
membrane
for 24 hr at
37”C. The amount
of radioactive
substrate
in
the assay system.
whether
tetrameric
hemoglobin or hemoglobin
chains. was 0.5 mg
-.2
0
and
there
was
even
greater
activity
in degrading
These
findings
indicate
that the activity
hemoglobin
or its subunits
is confined
active
against
freezing
hemoglobin
and
thawing,
activity
to the
were
incubated
subunits.
it was
Since
not
possible
inner or outer
surface
in the same
assay
hemoglobin
the
from
of the
system
membranes
these
directly
related
the
amount
of membrane
suspension
of tetrameric
hemoglobin
degradation
2). Degradation
to the amount
had
membrane.
there
was
in the absence
rate of substrate degradation
Omission
decrease
alpha
chains
of membrane
cell
studies
or alpha
beta
fragmented
the
in the assay
was
increased,
chains
also
as noted
of membrane,
was also time-dependent
against
20%-25%
hemoglobin,
greater
the
than
by
protease
above,
was
system.
the
rate of degradation
that of alpha
chains.
taken to ensure that the experiment
was carried
out under
amount
of substrate
did not limit the rate of the reaction.
The
at 0#{176}C.
did not
against
of
Care
8
Li
0
Li
4
a:
U)
a’
a
U)
Fig. 2.
Degradation
bin and
hemoglobin
amounts
of erythrocyte
Conditions
of incubation
Fig. 1.
of radioactive
hemoglosubunits
by increasing
membrane
susp#{149}nsion.
were
as described
for
beta
was
conditions
in which
the
These
findings
suggest
ID
Li
0
4
a:
of
(Fig.
minimal.
and did not proceed
As
rate
increased
of AlP or the energy-generating
system from the assay mixture
enzyme
activity.
Figure
2 also illustrates
that although
the activity
chains
exceeds
that
was, on the average,
1).
surface
of the cell.
or hemoglobin
subunits
system
and
been
to localize
suspension
in the assay
(Fig.
to degrade
native
and is particularly
However,
when intact
cells
no substrate
degradation,
suggesting
that the protease
is not present
on the exterior
The ability
of red cell membranes
to degrade
hemoglobin
was
subunits
of the human
red
to the cell membrane
0
MEMBRANE
VOLUME
(ml)
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BALLAS
878
4C
,
,
‘
AND
BURKA
I
ID
Li
ID
4
a:
H
30
Q
(:
Fig.
of KSCN
cxRBC membranes
in degrading
radioactive
hemoglobin and hemoglobin
chains at 37’C.
Equal amounts
of substrate
were
placed
in the incubation
system
and assayed
as described
in the
Methods
section.
The shaded area
at the bottom
of the figure shows
the level of degradative
activity
in
KSCN
extracts
of membrane-free
hemolysate.
tracts
3
of
Activity
.
normal
that the membrane-associated
human
beta chain.
In order
to ensure
that
Li
-
#{149}
2
/
a:
H
I
0
.-..--.----.
I
.-
.-
-
-
a’
,
/
,
Hb
,
‘
‘
--
0
- -
4
8
2
TIME
proteolytic
the
activity
protease
has
activity
6
20
24
(hrs)
a particular
attributed
affinity
to the
for the
human
red
cell
membrane
was not due to contamination
with other
formed
elements
of the blood,
an experiment
was carried
out in which
the initial
blood
sample
was enriched
40-fold
with leukocytes.
Before
being
separated
into membrane
and membranefree
hemolysate
human
and
blood
from
a
was
patient
used
enriched
with
in
the
with
chronic
difference
between
the
rates
contained
was minor
no added
activity
leukocytes,
in degrading
proteolytic
leukocytes
assay
collected
myelocytic
system,
leukemia.
of substrate
normal
heparinized
by differential
degradation
There
centrifugation
was
no
by the control
significant
samples,
which
and those that were enriched.
In both samples
hemoglobin
and greater
activity
in breaking
there
down
hemoglobin
chains.
Membrane-free
hemolysate
from
either
leukocyte-poor
or
leukocyte-enriched
samples
caused
no substrate
degradation.
Thus,
the proteolytic
activity
attributed
to human
erythrocyte
membranes
cannot
be ascribed
to contammating
leukocytes.
As shown
membranes,
0.75-M
3, proteolytic
from
cell
proteolytic
activity
in 0.75
extracts
containing
directed
against
of
the
membranes
of
red
hemoglobin
is confined
approach
susceptible
[‘4C]leucine
an energy-generating
be extracted
from
by a final
cell
was
used
red
cell
of
progressively
when
incubated
findings
confirm
or its subunits,
to the cell
blood
concentration
membrane
a period
of 24 hr. Thiocyanate,
did not cause
degradation.
These
and
alone
that
which
membrane.
to confirm
that
alpha
and
beta
to the membrane
protease.
Intact
red blood cells
for 1 hr at 37#{176}C
in order to label the hemoglobin.
At the end of 1 hr the cells were washed
membrane-free
hemolysate
and membranes
were
washed
free of hemoglobin.
Both
suspension
could
hemolysates,
M thiocyanate,
different experimental
chains
are not equally
were incubated
with
activity
membrane-free
Thiocyanate
hemoglobin
over
of the substrates,
is extractable
A
not
thiocyanate.
degraded
with any
red
in Fig.
but
were
system
then
in medium
containing
cold leucine;
the
were separated,
and the membranes
the membrane-free
hemolysate
and a
reincubated
in a Krebs-Ringer
for 3 hr at 37#{176}C,
and
synthesized alpha and beta chains
attached
in the membrane
chromatography
of the globin
chains
on CM-52.’5
In confirmation
the
was
fate
buffer
of the
newly
determined
by
of earlier
studies
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ERYTHROCYTE
MEMBRANE
PROTEASE
ACTIVITY
879
10
V.)
2
NORMAL
-
4
Fig.
alpha
8
)(
‘S-
;
.
-.-.-
.
6
c:*
-‘--‘-.>.L------
!
__
to isolated
,
membranes
for 3 hr at
during
incubation
37’C.
Hemoglobin
i:
was labeled
in the intact
cell
during
an initial incubation
for
hr at 37’C.
The membranes
3
1-#{149}
C)
-
2
-I
4
isolated
and washed
until
of hemoglobin
and then
incubated
in fresh
buffer.
At
intervals
during
the
second
,(‘_‘
2
I,-
I
incubation
C -‘
0
‘
I
60
120
T IM E
there was virtually
in the incubated
alpha and
the period
beta
of
240
medium,
from the
increase
of labeled
hemolysate.
indicating
that
the
membrane.
Selective
in the alpha/beta
0.60 to 1.22 during
during
chains
remaining
membrane
hemoglobin
The fate
chains
attached
to the membrane
incubation
there
was a slight
tachment
progressive
pronounced
human
beta
80
the
amounts
of
and beta chains
attached
to
the
were determined.
alpha
labeled
.
(m in )
no degradation
membrane-free
incubation
from
I
loss
to acid-soluble
of the newly
was not the same
but insignificant
reflected
of beta
attached
the 3-hr period of incubation,
longer
incubations
are more susceptible
an
fragments
synthesized
(Fig. 4). During
increase
in the
degradation
degradation
ratio of chains
red cell
were
free
“t2
4
Amounts
of labeled
beta chains
remain-
4.
and
ing attached
#{149}
and
not
disat-
chains
resulted
in a
to the membrane
was
more
(Table
1). These
findings
suggest
to degradation
by the membrane-bound
effect
that
that
protease.
DISCUSSION
Although
earlier
mature
erythrocytes
human
erythrocytes,’
proteolytic
activity
is confined
solely
studies
erythrocyte
enzymes.’5
activity
in the soluble
Table
1.
had
indicated
that
and suggested
that
the present
studies
in the mature
erythrocyte
to the membrane
site,
Alpha
Recent
portion
/ Beta
1
Ratio
of Nascent
Chains
of Chase
(hr)
3
After
Pulse
Labeling
Alpha/Beta
(cpm)
0
0.60
1
0.81
2
1.03
3
2
activity
was
present
in
studies
by Hanash
and Rucknagel
found proteolytic
of immature
red blood cells, but pointed
out that this
Time
Experiment
proteolytic
it was present
in the stromal
fraction
of
extend
these findings
and indicate
that
is absent
from the cell cytoplasm
and
as is true of a large
number
of other
1.22
0
0.70
2
1.19
4
1.73
0
0.70
24
1.76
and Chase
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880
BALLAS
activity
did not
was absent
specifically
immature
protease
red cells
be
from
look
the cytosol
of mature
erythrocytes.’6
for the presence
of enzyme
activity
erythroid
cells, no conclusions
activity
reported
in this study
of differing
degrees
of maturity
necessary
to determine
Rucknagel
is similar
this.
degree
when
directed
However,
enzyme
since
the
Studies
in rabbit
reticulocytes
indicate
amino
acid loses this ability
to withstand
meric
studies
present
alpha
hemoglobin
reported
hemoglobin
that beta
particular
studies
chains
to
here,
molecules
chains
have
susceptibility
associates,
membrane,
of
indicate
and
beta
normal
that
also
Hanash
in
isolated
This
the
is
circulating
containing
proteolytic
a
subunits
of
polypeptide
more
of
is of low
molecule.
that hemoglobin
intrinsic
cellular
are
and
the presence
erythrocytes
hemoglobin
the
chains,
by
hemoglobin
erythrocyte.’7
substituted
The
stability
these workers
membranes
of
required
of mature
tetrameric
with
hemoglobin,
known
normal
reported
neither
consistent
activity.’8
the
the
here,
system
for activity.
proteolytic
activity
against
BURKA
can be drawn
as to whether
or not the
is also present
in immature
cells. Studies
of
separated
by gradient
centrifugation
will
to the one reported
ATP or an energy-generating
The membrane-associated
Since
in the
AND
susceptible
than
tetra-
degradation
in which
by the
degradation
membrane-associated
of the individual
protease.
The
chains
of nascent
attached
a particular
of beta
to isolated
membranes
was
susceptibility
to proteolytic
chains
explains
the finding
monitored,
indicate
degradation.
This
of DeSimone
and
who observed
betas chains
that after
disappeared
attachment
of hemoglobin
more rapidly
than did
S molecules
alphaA
chains.’9
to the
Since
the present
studies,
as well as those of DeSimone,
were done in cells from patients
with balanced
globin
synthesis,
and there
is no pool of free beta chains
present
in
erythrocytes,
this indicates
molecule
to the membrane
that following
the beta chain
chain.
This might
occur either
after breakdown
of the molecule
while
into
membrane.
mode
Since
the
membrane
remains
rate of degradation
of attachment
of
relationships.
The fact that
exact
attachment
is degraded
of the tetrameric
more rapidly
the molecule
is still in the
individual
chains
following
of
binding
between
individual
to
the
hemoglobin
hemoglobin
the alpha
tetrameric
attachment
form or
to the
hemoglobin
unknown,
it is not possible
to determine
of the beta chain
is due to the particular
hemoglobin
than
and
whether
spatial
membrane
or
to specific
subunits
are
degraded
the
the increased
arrangements
enzyme-substrate
more
rapidly
than
tetrameric
hemoglobin
suggests
that
the activity
of the membrane-associated
enzyme
may
have
special
application
in conditions
(such
as the thalassemia
syndromes)
in which
there
is unbalanced
globin
synthesis.
Two lines of evidence
suggest
that proteolytic
enzymes
play a role in protecting
the erythrocyte
against
injury.
First,
with the cell
of cells
from
the excess
membrane
patients
alpha
before
with
chains
being
3-thalassemia
produced
lost from
in 3-thalassemia
the cell.2 Second,
after
labeling
become
during
of the
associated
incubation
hemoglobin
with
radioactive
amino
acids,
the excess
alpha
chains
are lost from the cell, presumably
by proteolysis.7’t
These
findings
suggest,
but do not prove, that proteolytic
enzymes
within
the red cell destroy
excess
hemoglobin
chains
and thus may protect
the cells
from
the deleterious
has been
suggested
effects
that
of attachment
altered
degrees
contribute a degree of control
to the
thalassemia,79’2#{176}
but there is no factual
of these
of proteolytic
severity
evidence
chains
to the cell
activity
of clinical
to support
with
membrane.3
the red
It
cell may
hemolytic
syndromes
this contention.
in
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ERYTHROCYTE
The
MEMBRANE
factors
responsible
erythrocyte
remain
lular enzymes
and
may
contribute
removal
PROTEASE
for
senescence
poorly
understood.
alterations
in both
to this
of senescent
process.2’23
hemoglobin
chains
premature
destruction
to
and
death
Decreases
structure
The
erythrocytes
system
may be partially
due
membrane
that is associated
881
ACTIVITY
of
membrane
by the
lesions
macrophages
maintenance
The
remains
activity
poorly
the
membrane
of red
blood
in
cells3
thalassemia
supports
globin
significance
synthesis
understood.
the soluble fraction of human
erythroid
cell
istics
cytosol
of rabbit
from
that
found
in the
than
one proteolytic
Further
experimental
enzymes
are
erythroid
enzyme
studies
responsible
cell and
unbalanced
globin
synthesis
on
localization of a protease to the
that
the red cell
membrane
of hemoglobin
and
proteolytic
and thalassemic
activity
also
erythroid
contributes
enzymes
activity
erythroid
the
life
differs
in several
reticulocytes,’t
there
membrane
significantly
and
of
of the
the
be more
erythroid
cell.
which
enzyme
or
its subunits
red
mature
participates
charactermay
within
in ameliorating
span
cell.9
in human
erythroid
cells
that has been reported
in
precursors9
play
to
Since
in the maturing
enzymes
to the cell
of excess
contributes
possibility.
of hemoglobin
role these
to eventual
of protein
attachment
within
the maturing
mammalian
will be necessary
to define
precisely
for degradation
what
lead
reticuloendothelial
syndromes
this
of the proteolytic
Since the proteolytic
circulating
of intracelcell membrane
that
to attachment
of small amounts
with cell aging.’
The fact that
of balanced
and
normal
of the
activity
has been found
in crude
lysates
of both normal
cell precursors,
it has been suggested
that this proteolytic
to the
the
in the concentrations
and function
of the
the
cell.
the
effects
of
However,
erythrocyte
does
in the intracellular
the
indicate
catabolism
its subunits.
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1979 53: 875-882
Protease activity in the human erythrocyte: localization to the cell
membrane
SK Ballas and ER Burka
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