Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 13, 1979
Pages 895-906
PROGRESSIVE LOSS OF MITOCHONDRIAL CRFATINE PHOSPHOKINASE ACTIVITY
IN MUSCULAR DYSTROPHY Michael
Mahler*
Department of Pharmacology University of Southern California Los Angeles, California 90033
Received
April
30,1979
SUMMARY: Mitochondria were isolated from the pectoralis and gastrocnemius muscles of chickens with a hereditary muscular dystrophy, and age-matched controls. In the pectoralis, for dystrophic birds aged 0.12, 0.25, 0.55, and 1.55 yr, the creatine phosphokinase activity of the intact mitochondria, expressed in terms of pellet protein, was 69X, 45%, 24%, and 13% as great, respectively, as that of the controls. The corresponding figures for the gastrocnemius were 79X, 46%, 51%, and 28%. The mitochondria from dystrophic muscles exhibited satisfactory respiratory control ratios, P:O ratios, and state 3 respiratory rates. To check whether their apparent loss of creatine phosphokinase activity was due to the presence of increasing amounts of non-mitochondrial pellet protein, the state 3 respiratory rate was used as a mitochondrial marker; the rates per mg protein were similar in mitochondria from normal and dystrophic muscles of each age group.
INTRODUCTION After
a step-like
or impulse-like
by a skeletal
muscle,
its
provocatively
similar
to the response
the rate
of oxygen
consumption
tion
of creatine
(2,
tion
in skeletal
muscle
phosphokinase model Present
(7-15;
address:
Abbreviations:
4-6).
Fig.
of oxygen
We have
This
in the rate
consumption
of a first
apparently
is normally
reaction. cf.
rate
change
therefore rate-limited
prediction
changes
order
changes
of ATP hydrolysis with
system
in parallel
hypothesized
a time
(l-3). with
(5,6)
adds quantitative
Moreover, the concentra-
that
by the mitochondrial detail
course
respiracreatine
to a popular
1).
Department of Physiology UCLA School of Medicine Los Angeles, CA 90024 Cr, creatine; CP, creatine phosphate; CPK, creatine phosphokinase; EGTA, ethylene glycol bis (S aminoethyl ether) N,N, N'-tetraacetic acid. 0006-291X/79/110895-12$01.00/0 895
Copyright All rights
@ 1979
by Academic Press, Inc. in anyform reserved
of reproduction
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
PI
RESEARCH COMMUNICATIONS
ATPases
ATP
ADP
z
I
Cr
CP 4 MITOCHONDRIAL I OUTER I MEMBRANE ADP+kP
1 I
s
INNER MEMBRANE
(T-E) ATP
.+
A)DP
Figure 1. Schematic diagram showing a hypothesized CPK in the control of respiration in muscle. In normal
skeletal
CPK activity
resides
and finally
disappears;
and disappears, gradually mitochondrial
in
so;
to its
of this
embryonic
adult
level.
usually transition,
the BB, MB, and MM, or the MB and MM forms of several
species,
enzyme activities muscle:
in addition
including in adult
man, skeletal
to a high low levels
not
the CPK profile
leaves 1.
it.
Within
this
of MM activity,
regresses an observation
are
thereafter,
extraAt
can be the BB and MB,
to that appreciable
evident
to this
appearance,
the MM isoenzyme.
(21,
fetal-like
dystrophies CPK iso-
in normal
fetal
MB activity, 29-33). state,
made by Hall
Extramitochondrial CPK is a dimer, and two different exist, the so-called brain (B) and muscle (M) types. to form the BB, MB, and MM isoenzymes (16-18).
896
late
of extramitochondrial similar
decreases decreases
In the muscular
(19-28).
of BB activity,
context,
in
isoenzymes
is
then
a relatively
or shortly
entirely
the pattern muscle
level
and in some cases known whether
after
the active
then
increases,
By birth
resides
extramitochondrial
BB activity
MB activity
and MM activity,
of mitochondrial
stage,
the BB isoenzyme.L
simultaneously,
CPK activity
stages
at an early
entirely
or nearly
increases
earlier
muscle
role
It
is
or never
and DeLuca
types of subunit These combine
(34)
vol. 88, No. 3, 1979
has an added and rabbit, This
BIOCHEMICAL
significance: mitochondrial
suggests
that
muscle
of fetal
and dystrophic
trophy.
expect
this
reported
CPK activity
as well.
If muscle
so,
and if extends
described
here
RESEARCH COMMUNICATIONS
in cardiac
absent
isoenzyme
enzyme to be absent,
The experiments
that is
the mitochondrial
skeletal
would
they
AND BIOPHYSKAL
muscle
until
several
of CPK might
the similarity
or progressively were
designed
days after be absent
between
to the mitochondrial
of the mouse
in
fetal
the CPK profiles
isoenzyme, lost,
birth.
one
in muscular
to investigate
this
dyspossi-
bility. MATERIALS
AND METHODS
The Department of Avian Sciences of the University of California at Davis furnished chickens from a line with hereditary muscular dystrophy (line 455) and a paired control line (f/454). White Leghorn chickens obtained locally were also used as controls. For all quantitites measured, the values obtained from these control groups were not significantly different, and have therefore been pooled. The pectoralis and gastrocnemius muscles were homogenized in an ice-cold medium containing 0.35 M mannitol, 10 mM Tris-phosphate (pH 7.2), 0.1 mM EGTA, and 50,000 I.U./g heparin (35,36). Muscles were minced with scissors, put through a tissue press with 1.0 mm holes, weighed, then homogenized by hand in a Dounce-type vessle, using 4-9g tissue per 60 ml of homogenizing medium, and pestles with clearances of 0.30 mm, then 0.20 mm. The homogenate was centrifuged for 10 min at 500g. The supernatant was filtered through three layers of cheesecloth, then centrifuged for 10 min at 8OOOg. The whitish layer adhering to the mitochondrial pellets was removed by swirling, and the pellets were washed with homogenizing medium, combined, resuspended gently in a small glass homogenizer with Teflon pestle, centrifuged for 10 min at SOOOg, and suspended in 0.7-1.5 ml of a medium with the same composition as the homogenizing medium, but without heparin and with 5g/lOO ml dialyzed bovine serum albumin. CPK activity in the intact mitochondria was assayed in the reverse direction (ADP + CP -+ ATP + Cr) at 30°C and pH 7.0, by coupling the CPK reaction to those catalyzed by hexokinase and glucose-6-phosphate dehydrogenase with Calbiothem kits, and measuring the increase in NADPH absorbance at 340 nm with a For mitochondrial protein contents of Gilford recording spectrophotometer. the absorbance change was always linear with time, and for a given l-10 rig/ml, the reaction rate was proportional to the mitochondrial protein preparation, When CP was omitted from the medium, the absorbance change was neglicontent. gible. After solubilization for 30 min at 95'C in 0.9 N NaOH, mitochondrial protein was measured by the method of Lowry et al. (37), using bovine serum albumin as a standard. Spot checks with the method of Bradford (38) gave similar results. Mitochondrial oxygen consumption at 30°C was measured in a Gilson Oxygraph using a medium containing 0.25 M mannitol, (Model KM) with Clark oxygen electrode, 10 mM KCl, 10 mM Tris-HCl (pH 7.2), and 0.1 mM EDTA. During an experiment, the following compounds were added, with the indicated final concentration: 5 mM potassium phosphate buffer (pH 7.2), 3.5 mM a-ketoglutarate, 0.3 mM ADP, and 1.4 mm MgC12. In order to solubilize mitochondrial CPK, mitochondrial suspensions were diluted with a medium containing 0.1 M sodium phosphate buffer (pH S), 10 mM beta mercaptoethanol, and 1 mM EDTA, and incubated overnight. Electrophoresis and visualization of the CPK isoenzymes in this extract were done by the methods of
897
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pectorolis
140
Mitochondriol (%
CPK
activity,
of oge-matched
I.U./mg
control
a.
0 : Control
)
A 1 Dystrophic X I Dystrophic
(I.U./OO, mox
60
-
40
-
20
-
01
0
“““““‘I
0.2
0.4
0.6
0.6
I .o
fi
I.2
,%
of age-matched
11 1.4
11 1.6
control)
11 I.8
-
I 2.0
Age (yr)
140
120
-
Mitochondriol CPK _ (% of age-matched
activity, I.U./ mg control)
-
Gostromemius
b.
0: Control
A:
Dystrophic
X:
Dystrophic
( I.U./O
% of age-matched 4
II
100
0
I 0
I-
n, 0.2
control
1
mox’
I
I 0.4
I
I, 0.6
11, 0.6 1.0 Age (yr)
,
, 1.2
,
, 1.4
,
, 1.6
,
, 1.6
,
Figure 2. Mitochondrial CPK activity during the course of muscular dystr0phy.n , A : CPK activity expressed in terms of mitochondrial protein content; X: CPK activity expressed in terms of state 3 respiratory rate. Bars denote + S.E.M.
2.0
Vol. 88, No. 3, 1979
BIOCHEMICAL
Hall et al. (39). To quantify was done on a muscle homogenate 10 min at 500g.
AND BIOPHYSICAL RESEARCH COhVvtUNlCATlONS
the migration of the MM isoenzyme, electrophoresis prepared as described above, and centrifuged for
RESULTS AND DISCUSSION The CPK activities muscles
of mitochondria
of increasing
as hypothesized,
age are
a large
the course
of muscular
age group
the P:O ratio,
of the mitochondria controls, for
vities which
from
did
of species
not markedly exhibit
decline
muscles
simply
chonclria,
either
Dystrophic
muscle
structures
of various
from
muscles
these
mitochondrial
1.
TABLE
from
those
known
might
it
0.12 0.25 0.55 1.55
ameankS.E.M.
during
3, for
each
3 respiratory
results
These
in
of the
preparations
muscles,
rate
have been relack-
the CPK acti-
of the cor?esponding
decline
to contain thus
and that
activity
of
increasing
seemed possible
this
progressively might
mitochondria
preparations
results
suggest
that
from dystrophic
the integrity
(I.U./mg
amounts that
of the mito-
normal
of degenerating
the pellets
larger
have accounted
from
Pectoralis (yr)
occurs
to those
of the mitochondria
have contained
CPK activity
Age
similar
Similar
control.
of a general
types;
CPK
were
that,
or as isolated. is
protein,
and state
and dystrophic
in CPK activity
--in vivo
ratio,
Even in occasional
respiratory
part
They suggest
CPK activity
values.
normal
different
satisfactory
the progressive was not
both
2.
2 and Fig.
muscles
(41-47).
and dystrophic
in Table
control
literature
from
1 and Fig.
As shown
the dystrophic with
from normal
in mitochondrial
respiratory
control,
were
in Table
dystrophy.
a variety
in respiratory
given
decrease
and consistent
ported
isolated
amounts for
protein)
Gastrocnemius
Normal
Dystrophic
13.9+1.0(4)a 16.4+3.3(5) 28.2?2.7(2) 18.1+0.7(2)
9.6+1.6(4) 7.5?1.4(3) 7.1+0.3(2) 2.4?0.5(5)
Normal 14.5+0.2(2) 13.8?1.7(5) 20.7+0.8(2) 14.9?1.4(3)
(n)
899
of non-
the observed
and dystrophic
mitochondrial
prepared
Dystrophic 11.6+4.1(4) 6.4?0.8(3) 10.5+1.4(2) 4.1+0.6(4)
muscle
Vol. 88, No. 3, 1979
TABLE
2.
BIOCHEMICAL
Respiration
Respiratory
in
control
mitochondria
ratio
Dystrophic
3.9+0.6(4) 2.9?0.3(9) 3.7f0.9(2) 3.3+0.5(2)
b 2.1?0.4(4) 3.5?0.4(2) 3.0+0.4(Z) 2.5?0.2(2)
4.0?0.4(3) 3.6?0.1(9) 3.3*0.4(Z) 3.2f0.1(2)
3.1?0.4(4) 3.6*0.2(Z) 3.2?0.5(2) 3.7?0.0(2)
O/min-mq
decrease test
Normal
Dystrophic
2.1?0.4(5) 3.5*0.3(Z) 4.0*0.4(Z) 2.5iO.3(3)
3.7*0.3(Z) 3.1iO.2(5) 2.4?0.2(2) 3.5?0.7(3)
3.1?0.3(5) 4.OiO.3(2) 3.5?0.4(2) 3.7?0.0(3)
307i64(2) 347?54(6) 322'?20(2) 162f44(3)
364?91(4) 284?55(3) 320*56(2) 299?21(3)
b
we used marker,
or at least
nearly
so, by intact
respiratory
rates
suspensions
prepared
protein. from
muscle,
tory
that
or the amount
With
increasing
amounts in muscle
of fat
(cf.
and nervous
to be available
of pellet
age, 40).
, presumably
would
not
be determined
consistently muscles,
different
in that
3
the
there
of non-mitochondrial
in
of the controls,
in terms
entirely,
2, the state
indicating
of CPK activity
the mitochondria is
essentially
of the absolute
state
the same 3 respira-
protein.
the dystrophic Mitochondrial
tissues
it
To
of the preparations
in the amounts
to that
expressed
rate
muscles.
As shown in Table
and dystrophic differences
relative is
that
were
the pattern
the dystrophic
3 respiratory
protein
normal
the CPK activity rate
from
mitochondria.
no important
dystrophic
protein
on the assumption
from
2 shows
mean?S.E.M.(n)
the state
per mg pellet
Fig.
whether
268i42(4) 255i61(3) 359?24(2) 188+14(3)
Gastrocnemius
protein
also
281?52(4) 231?37(9) 234t6 (2) 240t26(2)
Dystrophic
per mg pellet
a
Dystrophic
Normal
possibility,
probably
rate
Dystrophic
as a mitochondrial
were
respiratory
Normal
Gastrocnemius
in CPK activity
this
muscle
Pectoralis
Normal
4.4i1.4(2) 2.4&0.3(S) 2.7&0.3(Z) 2.4*0.4(3)
dystrophic
Maximal
Dystrophic
Normal
uatom
ratio
and
Pectoralis
Gastrocnemius
a
normal
Normal
Aqe (yr)
0.12 0.25 0.55 1.55
from
P:O
Pectoralis
0.12 0.25 0.55 1.55
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(10,48), does not
muscles
contained
CPK activity and although occur
900
in adipose
large,
has so far no direct cells
increasing been
evidence (15).
It
found
only
appears was possible
BIOCHEMICAL
Vol. 88, No. 3, 1979
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ADP
a ( 1.55yr)
mito
S/20/1978
Dystrophic
odult
b
( 1.55 yr)
Gostrocnemius
248 RCR =3.2
\
L
RCR = 2.2
Figure 3. Oxygen electrode traces obtained with suspensions of mitochondria from a dystrophic chicken aged 1.55 yr. Arrows denote additions of ADP and Mg*. Numbers above traces are respiratory rates, in patom O/min. mg. Vertical and horizontal calibration bars designate 60 patom 0 and 30 set, respectively. that
the pellets
increasing least
isolated
fraction
in part
for
from
the dystrophic
of mitochondria the observed
from
decrease
fat
muscles cells,
contained and that
in CPK activity.
901
a substantial, this
To test
accounted this
at
possibility,
BIOCHEMICAL
Vol. 88, No. 3, 1979
50g of fat This
was processed
produced
a small,
g of tissue
was less
in the usual and did
not
pellets
obtained
fat
than
increase
brownish
10% of that
respiratory after
from
used
rate
mitochondria
for
muscle;
muscle
from muscle.
which
the protein
when this
pellet
in the absence
the addition
dystrophic
to isolate
pellet for
RESEARCH COMMUNICATIONS
of added
yield
per
was suspended
AUP was very
of 0.3 ml? AUP.
We conclude
that
did
significant
amounts
not
contain
low,
the of
mitochondria. There
appear
to be no previously
of mitochondria
from
normal
about
muscles,
than
those
which
are
whether
reported for
this
in Table
3.
apparently
high
values
isolation
not for
muscle
protein,
l-3
of the rabbit between
these
could
substantially
with
possible
amounts
that
To determine
the results
of the MM isoenzyme
of progressively
smaller
amounts
if,
during with
were with
with
Mitochondrial CPK of rat heart and skeletal muscle exhibits equilibrium random kinetics (10,11,13,50). From the general equation (13), it follows that in the absence of products,
v/v max
reported Artefactually
conditionsL.
results
to
summarized
previously
have resulted
our
muscle,
our methods
and those
the the
influenced mitochondria
the mitochondria rapid rate
= XYl(l+x+Y+xY),
where x denotes [Sl]/K, y denotes [S ]/K2, and K1 and K2 are the disThe K 's for CP and ADP sociation constants (K 's) for S an 3 S2. for rat skEleta1 muscle (50), are 4.0 mM and 0.06 & respectibely, and 0.50-0.72 mM and 0.035-0.051 mM, respectively, for rat heart (10,13). The concentrations of CP and AUP in the reverse direction CPK assay used in the present study were 20 mM and 1.2 mM, respectively, from which it can be calculated that v/V was about 0.79 in rat skeletal muscle mitochondria, and 0.93-0.95m?$ rat heart mitochondria. The corresponding values calculated for the previous studies listed in Table III are roughtly similar. All of the measurements appear to have been made at 30°C, and at a pH near 7.
902
for higher
and cardiac
(MM) CPK became associated it
here
(10,11,48,50).
in assay
CPK activity
of appreciable but
values
obtained
skeletal
I.U./mg
and rat,
the CPK activity
are
we applied
to differences
considered
for
the values
mammalian
range
extramitochondrial
We thus
muscles,
for
in the
due simply
procedure,
but
mitochondrial
workers
mitochondrial
by the association
muscle,
values
was species-dependent,
The differences
mitochondria.
from normal
by other
published
skeletal
15 I.U./mg
discrepancy
and cardiac
are
chicken
the most part
skeletal
2.
by the method diffuse,
its
way,
AND BIOPHYSICAL
Vol. 88, No. 3, 1979
BIOCHEMICAL
TABLE 3.
chicken chicken
of
reported
values
for
CPK activity
Reference
pectoralis qastrocnemius
This This
study study
17.5+2.0(13)a 15.2+1.0(12)
This
study
5.6+0.5(4)
skeletal
rabbit
heart
muscle
Sobel et al. This study
skeletal
muscle
Jacobs
et
(1972)
al.
2.9 4.5?0.6(4)
(1964)
Jacobus and Lehninqer Saks This rat
mitochondrial
Tissue
rabbit
rat
Survey
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
heart
et al. study
Jacobs
et
(1977) al.
(1964)
Jacobus and Lehninger Saks This
a.
Muscle
et al. study
1.0 1.6 2.8-4.8 6.8+0.4(3)
(1973)
(1973)
b.
homogenoie
( gastrocnemius
1.0 2.4 2.7fO.5 8.0+0.2(6)
(1973)
1
Mitochondria ( pectoralis
:
1
jjJL
+1‘
-
C.
-
Mitochondria (gastrocnemius)
-i +T Figure ate (a)
4.
Electrophoretic and mitochondrial
analysis extracts
of CPK from a whole-muscle homogen(b,c). Arrow indicates origin.
903
Vol. 88, No. 3, 1979
BIOCHEMICAL
TABLE
4.
Tissue
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Mitochondrial
protein
yields
Mitochondrial
protein
yield
(mq/g
tissue)
Age 0.12
pectoralis, pectoralis, gastrocnemius,
rat skeletal muscle rabbit skeletal muscle rat heart
a
the dystrophic
this
possibility.
fugation
did
Second,
0.55
1.55
yr
0.12?0.01(2) 0.17+0.01(2) 0.32+0.07(2) 0.30+0.02(2)
yr
0.13+0.06(2) O.lOf0.02(5) 0.16'0.04(31 0.19+0.02(5)
0.52+0.06(6) 0.53'0.07(4) 8.2?1.0(3)
muscles.
not
lessen
as shown
almost
the high
lower
isoenzyme,
protein
(46,47)).
yields
genization,
at 0.12
and 0.25
yr are
have given Free
energy
exists
by the rise for
With
Table
by the hydrolysis
the variations toughness variations
If
here,
cells
were
from
gentle. than
reported
in mitochondrial
those
dystrophic
the
of the dystrophic
the mitochondrial
904
be due
in the extent
and maintenance
The might
lower
previously
appears
muscle
considerably
with
migrated
It
relatively
consistent
established
genuine.
used
yields
increasing
of ATP.
normal
was presumably
muscle.
muscles
are
the method
4) were
normal
as MM.
high
that
function
normal
here for
and centri-
extracts
relatively
to the observed muscle
from
fraction
by others
which
(The
caused
a small
against
suspension,
from
reported
procedures.
methods.
The possibility
isolated
reported
other
of washing,
argued
of mitochondrial
CPK activities
(cf.
of evidence
of mitochondria
and only
CPK values in isolation
with
pectoralis
provided
CPK activity
by a hand homogenization
obtained
might
rounds
4, electrophoresis
mitochondrial
to differences
Mitochondrial
two lines
the CPK of mitochondria
mitochondrial
disrupted
the
in Fig.
all
However, additional
First,
as the mitochondrial that
yr
mean+S.E.M.(n)
from
that
0.25
o.12~o.01(4)a0.22+0.05(9) 0.32+0.02(4) 0.38?0.14(3) 0.35+0.18(2) 0.23+0.04(6) 0.29'0.04(4) 0.2710.07(3)
nonnal dystrophic normal dystrophic
gastrocnemius,
yr
results
of homomuscle, CPK activity.
is apparently CPK reaction
entirely is normally
Vol. 88, No. 3, 1979
an obligatory (cf.
Fig.
l),
deterioration
BIOCHEMICAL
step
in the overall
then
the
loss
of muscle
dystrophy. mitochondrial
to determine
of this
as in
possible
that
here
dystrophic
by other
the direction(s)
ATP production
be expected
and structure,
reported
triggered
of oxidative
enzyme might
is likewise
CPK activity
of a degeneration
process
function
it
However,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for
factors.
in which
fact
to cause occurs
the progressive muscle
Further
causality
in muscle a general
in muscular loss
is
of
simply
part
are
necessary
experiments
flows.
ACKNOWLEDGEMENTS Special
thanks
the Department Dr.
Layne,
Dubick,
I also
fellowship
of the Muscular
in his
laboratory
Wayne Bidlack,
Yang for
the use of facilities
of Southern
and William
This
Dystrophy
study
California,
at the University Paul
Geiger,
Seltzer
was supported
for
of of California
Paul
facilities,
of and
and electrophoresis
the use of laboratory
Viitanen,
discussions.
for
on the extraction
Drs.
Paul
Bessman,
of the University
out
thank
Erickson,
and helpful
Samuel
collaboration
and William
Susan
advice
for
CPK, carried
at San Diego. Ennis
due Dr.
of Pharmacology
Norman Hall,
mitochondrial
are
Hochstein, and Michael
technical
by a postdoctoral
Association. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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