...

from

by user

on
Category:

japan

1

views

Report

Comments

Description

Transcript

from
THEJOURNAL OF BIOLOGICAL
CHEMISTRY
Vol. 256, No. 12, Issue of June 25, pp. 6408-6412, 1980
Printed In U.S.A.
Increased Turnoverof Proteins from the Sarcoplasmic Reticulumof
Dystrophic Chicken Muscle Cellsin Tissue Culture”
(Received for publication, December 28, 1979, and in revised form, February 3, 1981)
Earl M. Ettiennetgy, Kenneth Swartzg,and Robert H. Singer$IJ**
From the Departments of §Physiology and Ivnatomy, University of Massachusetts MedicalSchool, Worcester,
Massachusetts 01 605
Chicken myoblasts were cultured fromthe pectoralis
Evidence that proteins resulting from genetic errors may be
muscles of dystrophic and normal 11-day-old embryos.
selectively degraded at high rates (10,ll)led us toinvestigate
Cells were allowed to grow to fusion (differentiation) the possibility that abnormally functioning proteins contained
and exposedto [“Slmethionine for a short period. Subin the sarcoplasmic reticulum may turn over more rapidly.
sequently, the decay of labeled proteins
in the presence We proposed to investigate the process of development which
of cycloheximide was measured for various cellular
involves myoblast fusion in cell cultures for evidence of abfractions as well as individual proteins isolated from normal protein synthesis and degradation during the differthe sarcoplasmic reticulum and separatedby gel elec- entiation of the sarcoplasmic reticulum, before the onset of
trophoresis. Some dystrophic material showed an inthe pathology. This approach enabled us to examine early
creased decay when compared
to normal material.The events in molecular differentiation for evidence of protein
most significant ( p t0.005) difference was found in a
which may relate to the latter onset of the
M, = 65,000 component of the sarcoplasmic reticulum. abnormalities
This same component accumulates labelat an acceler- disease. A number of separate investigations have reported
ated rate in the presenceof the protease inhibitor leu- various changes in the collective rates of synthesis of dyspeptin. Increased turnover of
this protein, possibly cal- trophic muscle proteins in vivo (12-14). Other studies suggest
sequestrin, may be a manifestation of the genetic dis- higher average rates of degradation (6-8). We focus here on
the unusualkinetics of synthesisand decay of a specific
ease.
protein component of the sarcoplasmic reticulum which may
result from a genetic alteration.
Avian muscular dystrophy is a genetic disease resulting in
MATERIALS AND METHODS
the progressive degeneration of skeletal muscle tissue (l),with
Chemi~ak-[”~S]methionine (500-780 Ci/mmol) was obtained
visible swelling of mitochondria and the sarcoplasmic reticufrom New England Nuclear. Soybean trypsin inhibitor and marker
lum. Similar ultrastructural changes are also seen in cloned proteins
for electrophoresis standard were obtained from Sigma.
muscle from dystrophic human muscle biopsies ( 2 ) . Physio- Ampholines for electrophoresis were obtained from Bio-Rad. Cell
logical changes related to the ultrastructural eventsinclude culture media and supplements were obtained from Microbiological
the partial loss of normal tension development and delayed Associates.
Animals-White Leghorn fertilized eggs were obtained from SPArelaxation (2-5). However, the most significant change is
characterized by progressive atrophy of the muscle (4), which FAS, Inc. Dystrophic eggs were supplied from the Department of
reflects an accelerated degradationof the muscle fiber proteins Animal Genetics, University of Connecticut, Storrs, CN. Eggs were
incubated in a Leahy Manufacturing Co. incubator (Higginsville,MO)
(6-8).
at 38 “C and a relative humidity of 70%.
It is possible that, by the time the pathological aspects are
Cell Cultures-Muscle cell cultures were prepared from the pecapparent, the molecular pathology of the primary defect is toralis muscles of 10- to 12-day-oldchicken embryos according to the
obscured by other secondary manifestations of the disease. procedures of Paterson and Strohman (15). Plating density was usuWe have shown that embryonic dystrophic sarcoplasmic re- ally 2.5 X 10‘ cells/100-mm plate. Cells were grown for the first 24 h
ticulum vesicles showed an increased calcium transport over in minimal essential medium supplemented with 10% horse serum
and 2% embryo extract. All culture plates were precoated with a 0.1normal SR’ vesicles (9). However, there was a decreased m g / d of gelatin solution. Following a change of medium, myoblasts
transport in the adultdystrophic SR vesicles. This observation were allowed to grow to confluence. Fusion was observed to be
emphasized the importance of an investigation into the early initiated within 48 h after plating. Cell in minimal essential medium
events in the development of the muscle pathology. This work deficient in methionine were rountinely labeled with [35S]methionine
extends these observationsto theidentification of differences (20 pCi/ml) final concentration after 70% of nuclei were in multinuin the protein composition of the sarcoplasmic reticulum from cleated fibers. For procedures involving suppression of protein synthesis and determinationof protein half-lives, 0.5 pg/ml, final concennormal and dystrophic chicken muscle.
tration, of cycloheximide was added to each plate following an appropriate pulse with the labeled amino acid.
Isolation of Sarcoplasmic Reticulum-Harvesting of labeled cells
the payment of page charges. This article must therefore be hereby was accomplished by washing four times with Earle’s balanced salt
marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solution, scraped free of the plate with arubberpjlicernan,and
suspended in 1 ml of 0.3 M sucrose and 10 mM imidazole, pH 7.0, at 4
solely to indicate this fact.
.$ Recipient of a grant in aid from the Muscular Dystrophy Asso- “C, with 50 pg/ml of soybean trypsin inhibitor. The cell suspension
was homogenized with a dounce homogenizer. Samples were taken of
ciation of America.
7 Present address, Division of Biology and Medicine, Lawrence the cell homogenate for counting and for SDS-polyacrylamide gel
electrophoresis. Membranes were isolated from the homogenate acBerkeley Laboratory, Berkeley, CA 94720.
cording to procedures published elsewhere (9). One-pl samples of the
* * To whom requests for reprints should be addressed.
’ The abbreviations used are: SR, sarcoplasmic reticulum; SDS, cell homogenates and isolated membrane fractions were spotted onto
glass fiber fdters, and proteins were precipitated by boiling in 10%
sodium dodecyl sulfate.
* The costs of publication of this article were defrayed in part by
6408
Increased Turnover of Protein from Sarcoplasmic Reticulum
6409
trichroloaceticacid.Subsequentwashes
in water and acetone removed any unincorporated label. T h e activity on the filters was then
counted.
SDS-GelElectrophoresis-Samples
were also solubilized in 1%
SDS and 10 mM dithiothreitol, 12.5 mM iodoacetamide, and glycerol/
bromphenol blue for 1 min a t 100 “C. They were subsequently layered
onto a 7.5 to 15% gradient slab gel and subjected to electrophoresis
according to the method of Laemmli (16). Gels were vacuum heat
dried and exposed to Kodak XR-5 x-ray film for autoradiographic
analysis. T h e exposed film wasdeveloped in a KodakX-omatic
processor. ProcessedXR-5negatives were scanned with an Ortec
(4310) microdensitometer. T h e molecularweights of the resolved
protein bands were determined with [,‘“S]methionine labeled
vesicular
stomatitis virus coat proteinsof known molecular weight.
The amount of label in each band is directly proportional to the
optical density of the band and decreases exponentially following a
cold chase in unlabeled methionine. T h e values of optical density a t
hourly intervals for each protein band in the XR-5 negatives were
used to determine thehalf-life of the protein following washout of the
label. Data points were correlated by linear regression analysis, and
SLICE NUMBER (mm)
graphical print-outs on a digital PDP/1104 computer were obtained.
FIG.
1.
Electrophoresis
andautoradiography
of labeled
Two-dimensional Gel Electrophoresis-The method of O’Farrell
muscle microsomes. Chicken embryos after 14 days of incubation
(17)wasused.Pelletedsarcoplasmicreticulummicrosomesfrom
I:‘“S]methionine-labeledmyotubes were suspended in lysis buffer and were labeled in 01’0 with 250 pCi of [““Slmethionine for 6 h during
of muscle membranes
applied to tube
gels for isoelectricfocusing. Electrophoresis conditions which fusionof myoblast and the differentiation
were 600 V for 12 h, then 2000 V for 2 h. T h e isoelectric focusing gels was in process. T h e tissue was excised and washed in saline, and the
Fifty-pg samples wereloaded
were removed from the tubes and the pH gradient was determined
by labeledmicrosomeswereextracted.
a contact electrode (LKB)as a function of gel length. Theywere then onto a 7.5 to 15% gradient polyacrylamide slab gel. T h e gels were
equilibrated for 30 min in a solution containingS I X and subsequently dehydrated and exposed to a Kodak XK-5 x-ray film. The component
overlayed on a 12% polyacrylamide slab gel. Electrophoresis was for running a t approximately 65,000 daltons (arrows) in the dystrophic
4 h a t 150 V according to Laemmli (16). The slab gels were subse- lane has incorporated label a t a greater rate than the normal tissue
quently fixed, dried,and used to expose to KodakXR-5 film at over the 6-h interval. The speckled regionon the graph shows label
incorporated into 1-mm slices
of the lane carrying the normal
S R and
-80°C.
is shifted 1 mm to the left with respect to the dystrophicslices (open
faced). T h e gelswhichwere scanned are shown in the inset (n =
normal, d = dystrophic); the lineson either side ofthe lanes indicate
RESULTS
the position of the molecular weight markers.
Incorporation into Muscle in
Ovo-Dystrophic and normal
pectoralis muscle removed from
14-day-old embryos which
had beenexposed to [:’‘S]methionine for 6 h showed significant and control cultures (Fig. 2.4). However, as shown in Fig. 2B,
incorporation of thelabelintoseveralprominentprotein
at the end of the incorporation, the specific activity of the
bands of sarcoplasmicreticulum microsomes, with M, = label was 3.5 times greater in the dvstrophic SR than in the
100,000, 65,000, 54,000, 46,000, and 36,000 (Fig. 1). The M, = dystrophic homogenate. The amount of label in the normal
100,000 component had the mobility reported for the (Ca2+ SR was 2.7 times greater than in the corresponding homoge+ Mg”)-dependentATPase (18). Componentswith M, = nate. The loss of label from dystrophic and normal SR ap65,000 and 54,000 migrated on gels with the same apparent
peared to bebiphasic.Unlike
the homogenate, the rate of
mobility as the calcium
binding proteins calsequestrin and the initial labeling as well as the subsequent loss of label from
high affinity calcium binding protein, respectively (18). The dystrophic SR is increased over the normalSR.
Cycloheximide-treated Cultures-Cycloheximide (0.5 pg/
othercomponent a t M, = 12,000 may bea hydrophobic
lipoprotein (19) and at M, = 36,000, a glycoprotein (19). These ml) was added to each
cell culture after a labeling period of 6
lattertwoproteinshaveunknownfunctions
in thesarcoh. This concentration effectively inhibits any further incorplasmic reticulum (19). Fig. 1 shows that the protein which poration of label; hence, the results reflect only the decay of
M, = 65,000 from dystrophic cells incorporated three times
the proteins. As was seen in the case of Fig. 2, biphasic decay
rates were obtained for both dystrophic and normal controls
the label than the equivalent band
in the normal controls,
suggesting that “calsequestrin” was synthesized a t a higher (Fig. 3).
rate in dystrophic tissue than controlvalues. Increased labelT h e half-lives of loss of radioactivity in the homogenate
ing was also apparent in other lower molecular weight com- fractions were shown to be 19 h for the dystrophic and 32 h
ponents of dystrophic SR.
for the normal (data given in Table I).
Incorporation of Methionine into Total Protein a n d SarUnlike the homogenates, the 5-h decay
profile of sarcocoplasmic Reticulum of Muscle Cell Cultures-In order to plasmic reticulum from cultures treated with cycloheximide
controls. The
assess whether a putative biochemical defect in dystrophic was biphasic in both dystrophic and normal
of the
muscle may be associated with an increased turnover of the computer-fit half-lives for the fast decaying component
proteins of the sarcoplasmic reticulum, the incorporation of biphasic curve was 1 h for normals and 0.5 h for dystrophic
[,”‘SS]methionineinto pectoralis myotubes in culture was ob- and a proportional increase of20 times longer for the slow
served as well as the subsequent decayof label after replace- decaying components. Thus, some proteinsin the dystrophic
ment of the medium with unlabeled methionine. The
isolated SR are being collectively degraded at nearly twice the rate of
sarcoplasmic reticulum was compared with total homogenate the normal SR.
in both normal and dystrophic
cells with respect to kinetics
of
Decay of Specific Proteinsfrom the Sarcoplasmic Reticulabeling and decay.Cells were harvested at 3-h intervals and lum-Isolated,
labeled
sarcoplasmic
reticulum
described
homogenized. Samples of the total homogenates
were counted above was analyzed on 7.5 to 15% polyacrylamide gradient
and also used to extract the sarcoplasmic reticulum micro- slab gels. Equivalent amountsof four samples, takenat hourly
somes. The uptake of the label into the cell homogenates as intervals after chasing, were loaded into each lane of the gel,
and electrophoresis and autoradiography were performed as
well as its decay appeared nearly identical for both dystrophic
Increased
Turnover
6410
of Protein
from
Sarcoplasmic
described. Fig. 4 shows the resultant profile for decay in the
radioactivity of specific proteins over a 4-h period. Since the
cultures were treatedwith cycloheximide, the gel profiles
represent only degradationof individual protein constitutents
of the SR. The fiim in Fig. 4 was then cut into strips corresponding to the lanes, and the optical density
for each protein
(not saturating the film) was obtained. These values were
then plotted on a semilog scale versus time to arrive at an
estimate of their decayprofiles and determinationof half-lives
as shown in Fig. 5. Table I1 shows values obtained for halflives of each major component
of the SR in normal and
dystrophic cell cultures. In all cases except that of the M , =
100,000 component, the half-lives of the dystrophic proteins
are faster than those for normal controls. The
significant
most
0
A
9 -
-
8 -
0
5
6 -
,o
0
a
4 -
7 -
5 -
1 -
Reticulum
difference in decay rate occursin the M , = 65,000 component
( P < 0.005). All half-lives can be represented as monophasic
components, some of which fit the short lived kinetics obprofiie of the SR and some
served inFig. 5 for the total decay
of which fit the long lived kinetics. Components migratingat
M , = 140,000, 100,000, 68,000, and 54,000 in both dystrophic
and normal controls
show half-lives whichcorrelate well with
the half-lives displayed by the shorter lived populations in
bothnormalanddystrophic
SR andarenot significantly
11).Other minor components
different from each other (Table
present in the gel may represent either precursor forms of
these membrane proteins or specific degradation intermediates of any of the larger protein components.
of LeupeptinTwo-dimensional
Gel
Electrophoresis
treated Cultures-Normal and dystrophic cultures were labeled with ["Slmethionine for 6 h and lysosomal protease
activity in the differentiating myotubes
was blocked bytreatment with leupeptin(20-22). The sarcoplasmic reticulum was
subsequently isolated from each culture and equal amounts
subjected to two-dimensionalgel electrophoresis. The resulting pattern from exposure of the gel on film is shown in Fig.
6. A major feature of the gel is the dramatic amountof label
incorporated into the M , = 68,000 component in dystrophic
cells, as well as a much diminished M , = 65,000 protein.
Normal sarcoplasmic reticulum showed both M , = 68,000 and
65,000 proteins with approximately equal amounts of incor-
TIME (hours1
TABLEI
FIG. 2. Kinetics of methionine incorporationin total homogHalf-lives (t1/3 of
decay for cellular homogenates and
enate and isolated microsomes from normal and dystrophic
sarcoplasmic reticulum components
chick embryo muscle cultures. Pectoralis tissue from 12-day-old
ti:.? f EMS"
chickswasremovedand
plated at IO6 cells/plate. After 3 days of
culture the medium was replaced with medium lacking methionine
and the cultures were labeled for 10h with [35S]methionine(250 pCi/
Homogenate
plate). After10 h, the labelingmediumwasreplacedwithfresh
19.11 f 0.0Ih
Normal
medium and the incubation of plates of cells continued for an addi31.75 f 0.02
Dystrophic
tional 80 h. The cellswere harvested at specific intervalsduring
Sarcoplasmic reticulum
labeling and chasing by washing twice with 0.3 M sucrose, pH 7.0, 10
(short lived)
mM imidazole andthen scraping cellsinto a total volume of 2 ml. The
1.12 f 0.01
Normal
homogenate was assayed on filter paper andwashed with hottrichlo0.57 f 0.02
Dystrophic
roacetic acid ( a )after cellular disruption by dounce homogenization. Sarcoplasmic reticulum
The remaining homogenate was centrifuged according to the proce(long lived)
in
dures previouslydescribed (9). The SR pelletwassuspended
22.45 f 0.01
Normal
extraction solution and aliquots were counted
(B).
In all cases protein Dystrophic
13.8 f 0.01
concentrations weremeasuredcolorimetrically,and
data wereexRoot
mean
square
residual.
pressed as counts per min/pg of protein. - - -, normal cells; -,
n = 4 for each mean value.
dystrophic cells.
2520-
15'-
A
-
f-B
:- - - - -1:1: "'I=-"""
"
--
IO -
- ".
- - - --:""""-I"
4-
3s
-
5-
0
X
z
0
V
-
Half Life = I .I
I
I
I
I
I
2
3
4
I
5 0
TIME (hours)
C
Yo_ g.f3[
Half Life = 13.8
- .
.
I
IO
I"""""1
Half Life = 22.5
2-
-_- - - - -
-
I"-"""T
'.
"*'"""
Half Life = .6
I
I
I
I
J
I
2
3
4
5
9
0
1
2
3
4
TIME (hrs.)
5
FIG. 3. Decay of methionine-labeled proteins in the presenceat 4 "C, with 50 p g / d of soybean trypsin inhibitor. Aliquots of the
cell homogenates and isolated membrane fractions then
werecounted.
of cycloheximide. For the suppression of proteinsynthesisand
determination of protein half-lives,0.5 pg/ml, final concentration,of Loss of label is plotted as a function of time after drug addition and
cycloheximide was added to each plate following a 6-h exposure to normalizedper pg of protein.Computerplotsforeachbiphasic
["S]methionine. Labeled cells were harvested at the indicated inter- component of the projected half-lives (in hours) are includedfor the
normal ( A ) and dystrophic (B)SR, but not for the homogenate (C)
vals subsequent to the addition of the drug by washing four times
with Earle's balanced salt solution, scrapedwith a rubber policeman, where the differencebetween the normal (- - -) anddystrophic
was not significant.
and suspended in 1 ml of 0.3 M sucrose and 10 mM imidazole, pH 7.0, (--)
Increased
Turnover
Protein
offrom
Sarcoplasmic
Reticulum
641 1
Normal (hrs.)
Dystrophic
(hrs.)
0
1
2
4
0
1
2
4
h
h
105
2.3
2.1
0.59
0.27
1.2
1.9
0.35
0.25
0.073.2
1.9
0.86
0.87
12.4
0.0005
2.7
0.43
0.48
4.1
0.025
2.5
0.71
0.65
15.7
0.0015
4.1
2.16
0.67
18.3
5.2
1.76
1.45
140,000
50
Normal
Dystrophic
100,000
Normal
Ilystrophic
40
0.02
68,000
1 0 0
Normal
Dystrophic
n
..
'
P
1 6 8
-65
54
-46
-36
-
65,000
Normal
Dystrophic
x
54,000
2
Normal
Dystrophic
0
c
46,000
0
Normal
Dystrophic
7Y
36,000
Normal
Dvstronhic
FIG. 4. Electrophoresis separation of labeled proteins from
isolated sarcoplasmic reticulum
of normal and dystrophic cells
after cycloheximide treatment.
At the times noted. equal amounts
of samples from Fig. 3 were removed for further analysis by S I X acrylamide gradient slab gel electrophoresis for the purpose of identifiying the decay rates of particular proteins. The dried gel was used
to expose the x-ray film presented here.
ZD
20
10 -
A
M ) K D I L T w l CWPONEWT
I O
NORMAL 7 7 2
=I
WsTRagtlCT'/Z.
Zhn.
I.Shrs.
--
D
0.001
A. DYSTROPHIC
M K D U W COUPONEM
WORYAL
-
T'/2 - 4 . l k a .
OISTWPUICT~/~-P.D~~~.
1
I
0
-0d
6.7 6.5 6.3 6.0 5.8 5.6 4
5.4
.9 5.2
0. NORMAL
x
0
I
2e
20
-
II
10 -
I
c
I
I
I\
-
6DK DALTON
COYPOWENT
NORMAL T'/2
I.? 4hra
DY3TROPUlC
1 h . m
2 7 h
--
I
F
1
I
I
X I K DALTON COYPOYEWT
N O R Y I L T$'2 = I 6 1k n .
DY3TROPMICT'/2. D 2hrs
-
+
:
:
:
:
:
.
.
:
6.5 6.3 6.0
5 8 5.6 5.4 5.2
4.94.7
1
1
I
1
2
3
4
0
1
'
1
2
3
I
1
4
5
TIME (hours)
FIG. 5. Computed degradation half-lives for particular proteins. The exposed bands on the x-ray film from Fig. 4 were scanned
for optical density as described and the densities of each band in
arbitrary absorbanceunits was plotted as a function of time in
cycloheximide. Each molecular weight species is plotted in a separate
panel as noted. Normal points are represented by triangles; dvstrophic points are represented by squares.
PH
FIG. 6. Two dimensional gel pattern of sarcoplasmic reticu-
lum proteins from normal and dystrophic cultures.
Isolated
microsomes labeled with [%]methionine for 6 h from dystrophic ( A )
and normal ( B ) cell cultures were subjected to two-dimensional gel
electrophoresis. Cultures were treated with 8 pg/ml of the protease
inhibitor leupeptin during this time. The nrrou- in A points to a
68.000-dalton component of the dystrophic which is virtually absent
in the normal SK.
higher molecular weights and more acid pH, possibly indicatporation. However,the amount of label incorporated into eaching additional precursor forms.
of these components was dramatically lower than that incorDISCUSSION
porated into the M , = 65,000 component of the dystrophic
tissues. Other significant increases in labeling were evident in
We have shown in Fig. 1 that, during the culturing of cells
specific proteins of the dystrophicgel patterns, particularly at from dystrophic pectoralis of embryonic chicken, the sarco-
6412
Increased
Turnover
Protein
offrom
Sarcoplasmic
Reticulum
plasmic reticulum in general, and a M , = 65,000 component
thereof in particular, exhibited an increased rate of incorporation of [35S]methioninecompared to normal cells. Additionally, this event occurs in cells undergoing differentiation and,
hence, early inthe ontogeny of the muscle tissue. This observation led us to a more detailed kinetic study in muscle cell
cultures, with particular emphasis on the sarcoplasmic reticulum, over a time interval which included the fusion of myoblasts into myotubes and the subsequent synthesis of muscle
proteins.
Our earlier observations that embryonic dystrophic sarcoplasmic reticulum was physiologically and biochemically different from either embryonic normal or adult dystrophic SR
(9) contributed to our exploration of the SR proteins for
evidence of degradation of abnormal proteins. The labeling of
the dystrophic SR proteins showed a higher specific activity
than normal SR despite the fact that the total cellular homogenates in either case were similar. Consistent with the
observation of increased labeling is the fact that dystrophic
sarcoplasmic reticulum shows a higher degradation rate for
constituent proteins than either cellular homogenates or normal SR.
When we examined the comparative degradationof individual protein components of the dystrophic and normal sarcoplasmic reticulum, after separation by gel electrophoresis, we
observed that thegreatest stabilitieswere exhibited by the M ,
= 140,000 and 100,000 components. These proteins provided
aninternalstandard
of controlsuch that any significant
comparative variations in the degradation of other proteins
would suggest a possible candidate for a primary product of
the genetic dysfunction based on evidence for selective degradation of abnormal proteins (10, 11). Confirmation of increased degradation of the M , = 65,000 component ( p <
0.0005) compared to the other components or to normal SR
proteins is consistent with the observed increased incorporation and, hence, an increased turnover. The lower molecular
weight components with increased turnover may be either
degradation productsof the samedefect or additional proteins
from nearby genes affected by the genetic defect. Spradling et
al. (23) havefoundin
Drosophila that the genetic lesion
ocelliless affects the transcription of nearby genes. Alternatively, however, the elevated degradation may not be a primary defect but could be attributable to errors in post-translational modifications such as protein processing, membrane
insertion, or glycosylation. Finally, these proteins may all be
components of a supramolecular complex involved in Ca2’
transport asis the putativecalsequestrin (65,000)component.
If such a complex functioned “abnormally” in the SR membrane, it could conceivably be “wholly” susceptible to enzymatic degradation.
Increased incorporation into theM , = 68,000 component in
the presence of leupeptin (Fig. 6) at theexpense of the 65,000
component suggests that the former may be a precursor of
the latter. The observation that the protein accumulates in
the presence of leupeptin also supports the concept that it is
highly susceptible to protease activity. It is possible that the
larger protein contains a “signal” sequence (24) for insertion
into theSR membrane which may not be
functioning properly
in the dystrophic cells. Both of these proteins are present in
normal cells, perhaps representing normalprocessing activity.
It is worth noting that any abnormality in the biochemical
function of the sarcoplasmic reticulum would result in abnormal Ca’+ metabolism. Since calcium is a key cofactor in
cellular energy metabolism and motility, it is logical that
cellular catabolism and motility would be adversely affected
in amanner consistent withthat observed in the pathology of
dystrophic muscle as described in the Introduction.
REFERENCES
I. Stephen, D. H., Hainey, C., Angello, J.C., Linkhart, T. A., Bonner,
P. H., and White, N. (1977) Pathogenesis of Human Muscular
Dystrophies (Rowland, L. P., ed) pp. 835-855, ExcerptaMedica,
Amsterdam
2. Botelho, S. Y., Beckett, S.B., and Bendler, E. (1960) Neurology
10,601
3. Buchthal, F., Kamieniecka, Z., and Schmalbruck, H. (1974) E x plorato9 Concepts in Muscular Dystrophy (Milhorat, A. T.,
ed) Vol. 11, pp. 526-554, Excerpta Medica Foundation, Amsterdam
4. Desmedt, J . E., and Hainut, K. (1968) Nature (Lond.)217,529
5. McComas, A. J.,and Thomas, H. C. (1968) J. Neurol. Sci. 7, 309
6. Srivastava, U. (1972) Can. J.Biochem. 50,409-415
7. Srivastava, U. (1968) Can. J. Biochem. 46, 35-41
8. Simon, E.J.,Gross, C. S.,
and Lessell, I. M. (1962) Arch. Biochem.
Biophys. 96,41-46
9. Ettienne, E. M., and Singer, R. H. (1978) J.Membr. Biol. 44,195210
10. Goldberg, A. L., and St. John, A. (1976) Annu. Reu. Biochem. 45,
747-803
11. Goldberg, A. L., and Dice, J. F., Jr. (1974) Annu. Reu. Biochem.
43,835
12. Batelle, B.-A., and Florini, J. R. (1973) Biochemistry 12, 635-643
13. Weinstock, I. M., Soh, T. S.,Freedman, H. A., and Culter, M. E.
(1969) Biochem. Med. 2,345-356
14. Ionasescu, V., Zellweger, H., and Conway, T. W. (1971) Arch.
Biochem. Biophys. 144,51-58
15. Paterson, B. M., and Strohman, R.C. (1972) Deu. Biol. 29, 113133
16. Laemmli, U. K. (1970) Nature (Lon& 227,680-685
17. OFarrell, P. H. (1975) J.Biol. Chem. 250,4007-4021
18. MacLennan, D. H. (1974) J . Biol. Chem. 240,980-984
19. MacLennan, D. H., Zubrzycka, E., Jorgensen, A. 0..
and Kalnins,
V. I. (1978) The Molecular Biology of Membranes (Fleischer,
S.,Hatefi, Y., MacLennan, D. H., and Tzagoloff, A., eds) pp.
309-320, Plenum Press, New York
20.McGowan, E., Shafiq, S.,and Strachr-, A. (1976) Exp. Neurol.
50, 649
21. Rourke, A. W. (1975) J. Comp. Physiol. 86, 343-352
22. Goldspink, D. F., and Goldspink, G. (1977) Biochem. J . 162, 191194
23. Spradling, A. C., Waring, G. L., and Mahowald, A. P. (1979) Cell
16,609-616
24. Blobel, G. (1977) International Cell Biology (Brinkley, B. R. and
Porter, K. R.,eds) pp. 318-325. Rockefeller University Press,
New York
Fly UP