- Email: [email protected]
The purification and characterization of an inhibitor of protein synthesis from the muscle of dystrophic mice and homozygous control mice
Journal of the Neurological Sciences, 1982, 56 : 311-326
311
Elsevier Biomedical Press
THE PURIFICATION AND CHARACTERIZATION OF AN INHIBITOR OF PROTEIN SYNTHESIS FROM THE MUSCLE OF DYSTROPHIC MICE AND HOMOZYGOUS CONTROL MICE
M O N R O E W. C H I N - S E E and D. M c E W E N N I C H O L L S *
Department of Biology, York University, 4700 Keele Street, Downsview, M3J IP3 Toronto (Canada) (Received 8 February, 1982) (Accepted 7 April, 1982)
SUMMARY
Homogenates of hindlimb muscle were obtained from homozygous ( + / + ) control mice and dystrophic (dy/dy) mice of the ReJ 129 strain and subjected to purification by ion exchange chromatography, gel filtration, centrifugation on sucrose gradients, and sodium dodecyl sulphate gel electrophoresis. A proteinaceous material inhibitory to protein synthesis that was detected previously in fractions from dystrophic muscle (Petryshyn and Nicholls 1978) was present at reduced levels of activity in muscle fractions from homozygous controls. Although the specific activity after the sucrose gradient step was similar in the 2 groups of animals, the activity per animal was 3 times higher in the dystrophic mice. Evidence is presented to support the view that the inhibitory material is identical in the 2 groups and also that the active protein, of an approximate molecular weight of 120 000, can be denatured by heat and 2-mercaptoethanol to the less active form that was described previously, of an approximate molecular weight of 70 000.
INTRODUCTION
Although the earliest studies of dystrophic mice carried out in vitro reported increases in protein synthesis, more detailed investigations questioned these reports and showed that, provided corrections are made for cell size and for DNA content, the synthesis of total proteins is not changed (Kitchin and Watts 1973; Hayashi et al. 1975). These studies can be affected by differences in the transport This investigation was supported by the Muscular Dystrophy Association of Canada and the Natural Sciences and Engineering Research Council of Canada, Grant 3489. M. W. Chin-See held a Predoctoral Fellowship of the Muscular Dystrophy Association of Canada. * To w h o m reprint requests should be addressed. 0022-510X/82/0000-0000/$02.75 © Elsevier Biomedical Press
312 of amino acids into the precursor pool and by differences in the amount of proteil~ and in the species of labelled protein that is measured in the cell. Thus Srivastaw~ (1972) studied labelling in cell-free preparations (polyribosomes) and reported a decrease in myosin labelling, no change in actin labelling and an increase in tropomyosin labelling. From an early stage until the disease is well-advanced (e.g. by 90 days of age) the animals fail to thrive and the wasting of the hindlimb is accompanied by a marked decrease in myofibril content and a marked decrease in the protein content of soluble cytosolic fractions (Srivastava 1968; Petryshyn and Nicholls 1976). Muscle wasting may be due to decreased protein synthesis or to increased protein breakdown, and the results of Spargo et al. (1979) suggest that both of these changes are taking place owing to the use of amino acids for energy metabolism rather than for protein synthesis. In cell-free studies of the machinery for protein synthesis in this laboratory it was found that the cytosolic fraction, which contains most of the elongation factor 1 and 2 activity (i.e. the pH 5 supernatant fraction), exhibited changes in activity during altered states of hormone balance and of growth (Girgis and Nicholls 1973; Nicholls et al. 1975; Petryshyn et al. 1977; Nicholls et al. 1977; Kuliszewski and Nicholls 1980). These changes were chiefly due to elongation factor 1 activity (Young and Nicholls 1978). In the case of dystrophic mice, this cytosolic fraction of hindlimb muscle, but not other tissues, showed a pronounced decrease in the capacity to support protein synthesis (Petryshyn and Nicholls 1976). However, the lower incorporation with dystrophic preparations was not due to altered activity of elongation factors, ribonuclease, proteolytic enzymes, GTP, or sulphydryl reagents, but it was attributable to the presence of activity that was inhibitory to protein synthesis. The inhibitory activity was partially purified by gel filtration and ion exchange chromatography and was found to be sensitive to pronase and insensitive to heat, trypsin, ribonuclease A, deoxyribonuclease I and phospholipase C (Petryshyn and Nicholls 1978). When muscle from the tittermate controls was studied no inhibitory activity could be detected in the pH 5 supernatant fraction nor after gel filtration but it could be detected after DEAE-cellulose chromatography. Since the littermate control group contained heterozygotes (dy/+ ) as well as homozygotes (+ / + ), it was not known whether the inhibitory activity in the littermate controls was due to the presence of the heterozygous dystrophic animals. A preliminary experiment showed that the pH 5 supernatant fraction from homozygous controls behaved the same in protein synthesis as the pH 5 supernatant fraction from heterozygous controls. However, even if the inhibitor were present in a more purified fraction (i.e. after DEAE-cellulose chromatography) in the homozygous control animals, it was not known whether the inhibitory activity was the same as that from dystrophic mice. The following experiments were carried out to resolve these questions.
313 MATERIALSAND METHODS ATP, GTP and creatine phosphate as their sodium salts, creatine phosphokinase (rabbit muscle type E.C. 2.7.3.2), acetyl phenylhydrazine, micrococcal nuclease, papain, pronase P, ribonuclease A, and trypsin were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Hemin chloride and Biogel P150 (50 150 mesh) were obtained from Calbiochem, Los Angeles, CA, U.S.A. Chloramine-T trihydrate (N-chloro-p-sulfonamide, sodium salt) was from Aldrich Chemical Co., Milwaukee, WI, U.S.A. Diethylaminoethyl cellulose (microgranular DE 32) was purchased from Whatman. L-[U-laC]Leucine (339 Ci/Mol) and carrier-free [1251]sodium iodide (16.05 mCi//~g iodine) were obtained from Amersham, Oakville, Ont., Canada. All other chemicals were of reagent grade, and were obtained as described previously (Girgis and Nicholls 1971 ; Young and Nicholls 1978).
Animals Male dystrophic mice (dy/dy) and control animals (+ /+ ) of the strain ReJ 129 were obtained from Roscoe B. Jackson laboratory, Bar Harbor, ME, U.S.A. These animals were maintained with free access to food (fox cubes, Master Feeds Inc.) and water until they were 60 to 70 days old. New Zealand white rabbits (2.5 kg) were supplied by Rabbits Unlimited, Stouffville, Ont., Canada.
Preparation of reticulocyte lysates The procedure for the preparation of rabbit reticulocytes was similar to that of Hunt et al. (1972), except that the rabbits were injected subcutaneously for 5 consecutive days with 2.5 ml of a 1~ (w/v) acetylphenylhydrazine solution to induce anaemia and made further anaemic by collecting 20 ml blood from the marginal ear vein on the first, third and fifth days. Three days after the final injection, the blood was collected and the reticulocytes washed, lysed and stored at -70°C.
Assay for inhibitory activity using reticulocyte lysate The following components were contained in a final volume of 65 pl: 10 mM Tris-HC1 (pH 7.8 at 25 °C), 2 mM magnesium acetate, 76 mM KC1, 1.0 mM ATP, 0.2 mM GTP, 30/~M of each of 19 amino acids (cysteine was excluded according to Hunt et al. 1972) 5 mM creatine phosphate, 5 #g creatine phosphokinase, 25 pM hemin chloride, 25 #1 of freshly thawed reticulocyte lysate, and 15 #1 of buffer or extract to be tested for inhibitory activity. 0.125 pCi of [J4C]leucine was added together with unlabelled leucine to give a final concentration of 30 pM. The reaction mixture was incubated for 30 min at 30°C, and the reaction was terminated by applying 10/~1 aliquots of the reaction mixture to 15 mm squares of 3 MM filter paper (Whatman) and then soaking in ice-cold 10~o (w/v) trichloroacetic acid
314 containing 1~o (w/v) leucine. The filter squares were prepared tbr counting by heating for 5 min at 95°C in fresh 5~i trichloroacetic acid containing 1';i', carrier leucine and then consecutive washes with cold 5~/o trichloroacetic acid, ethanol, and acetone. After drying for 1-2 min, the filters were stirred at 30 °C in 1J~°"/,,(v/v) H202 for 2 min to decolorize and were washed with ethanol and acetone as before. The filters were dried and counted in 8 ml of toluene-based scintillation fluid. Counting efficiency was approximately 85°/0 and was corrected for quenching by the external standardization method. Percent inhibitory activity was expressed as follows: incorporation with buffer - incorporation with inhibitor x 100. This assay incorporation with buffer was approximately 20 times more sensitive than the rat liver ribosome assay used previously (Petryshyn and Nicholls 1978).
Determination of protein concentration Non-collagen protein concentration of the soluble protein fractions was measured by the method of Lowry et al. (1951) with bovine serum albumin as a standard. For samples free of nucleic acids, protein concentration was determined at A2t0 according to Tombs et al. (1959).
Preparation of pH 5 supernatant fraction All of the work was performed at 2-4 °C. Mice were killed by cervical dislocation, and the hindlimbs were quickly removed and placed on ice, and the muscles were removed, weighed and minced with a pair of chilled scissors. The muscle mince was homogenized in a glass homogenizer with 15-20 complete passes of a Teflon pestle in 3 vol. of buffer A consisting of 0.25 M sucrose, 50 mM Tris-HC1 (pH 7.8 at 25 °C), 80 mM potassium acetate, 6 mM magnesium acetate and 10 mM 2-mercaptoethanol. The preparation of the pH 5 supernatant fraction from the muscle was as described previously (Petryshyn and Nicholls 1976).
Radioiodination of purified inhibitor Iodination of control and dystrophic inhibitor with [1251]iodidewas performed essentially as described by Greenwood and Hunter (1963). Fractions from the sucrose gradient step of inhibitor purification that demonstrated peak activity (13-13.5~ (w/v) sucrose) from both control and dystrophic preparations were pooled and concentrated by lyophilization. The freeze-dried residues were subsequently redissolved in 50/~1 of double-distilled water, and the protein concentration of each sample determined by method of Lowry et al. (1951). The protein was iodinated in 1.5 ml Eppendorf tubes by mixing 1 mCi of [12SI]sodium iodide, 10 #1 of 0.5 M phosphate buffer (pH 7.5 at 25 °C), 2-5/~g of protein contained in 10 t~t of 0.05 M phosphate buffer (pH 7.5 at 25 °C) and 10 #1 of a 14.2 mM solution of chloramine-T
315 trihydrate in 0.05 M phosphate buffer (pH 7.5 at 25°C). The reaction was immediately terminated by adding 25 /~1 of a 131.5 mM solution of sodium metabisulphite in 0.05 M phosphate buffer (pH 7.5 at 25 °C). Residual [~25I]iodine was diluted by adding 50/~1 of a 60.3 mM solution of potassium iodide in 0.05 M phosphate buffer (pH 7.5 at 25 °C). The entire contents of the tube were transferred to a 1 x 10 cm Sephadex G-50 gel column previously equilibrated with 0.05 M phosphate buffer (pH 8.6 at 25 °C). The column also was pretreated by passing through 1 ml of 2% (w/v) bovine serum albumin in phosphate buffer (pH 8.6 at 25 °C) and washing with 20 ml of the same buffer. This pretreatment minimized loss of labelled protein due to non-specific adsorption. Elution was carried out at a rate of 0.5 ml/min with 0.5 M phosphate buffer (pH 8.6 at 25°C). Material eluting between 1.5 and 5.5 ml was collected directly into vials containing 50 mg of crystalline bovine serum albumin in 1 ml of elution buffer as carrier protein. This fraction was found to contain most of the labelled protein. RESULTS
Chromatograph), The pH 5 supernatant fractions from control and dystrophic muscles were subjected to DEAE-celtulose chromatography. Elution of bound proteins with buffer containing a salt gradient resulted in a profile showing prominent protein peaks. The profiles were similar for both control and dystrophic preparations (Fig. 1). When the individual fractions were assayed for inhibitory activity using the lysate system, 2 regions of inhibitory activity could be seen. One peak occurred at 0.30 M KC1 as previously reported for dystrophic mice (Petryshyn and Nicholls 1978), and another peak was seen at 0.05 M KC1. The present investigation describes studies using only the former peak. The elution of the inhibitory material occurred at the same ionic strength in the control preparation as it did in the dystrophic preparation. The ~ inhibition, however, was much lower in the control preparation than it was in the dystrophic preparation.
Gel filtration Protein fractions that contained the inhibitory activity were obtained following DEAE-cellulose chromatography and were subjected to Biogel P150 gel filtration. A single protein peak was seen to elute near the void volume. On assaying for inhibitory activity, peak activity in both control and dystrophic preparations were seen to coincide with the leading edge of the main protein peak (Fig. 2) which was 0.6 mg/ml for controls and 2.0 mg/ml for dystrophic. The ~o inhibition was markedly lower in the control preparations.
316 I
I
I
I
60
04"12'0 ° ~40 P-.
20
0 0
I0
I --
20 30 FRACTION NO, I
I
I
--0
4O
I
B
~4
20 o
4O
03
P.. 0~
/.0 20
0 0
FRAC~ON N O
Fig. 1. Chromatography of the pH 5 supernatant fraction obtained from (A) dystrophic and (B) control muscle. 300 mg of the dystrophic and control wildtype pH 5 supernatant fractions were chromat0graphed separately on a 2.5 x 9 cm column of DEAE cellulose equilibrated in buffer B [50 mM Tris-HCt (pH 7.8 at 25°C) containing 10~ glycerol]. Bound protein was eluted at a flow rate 0.4 ml/min, using a linear gradient of 0-1 M KC1 contained in 200 ml of buffer B. 5 ml fractions were collected; Inhibitory activity was assayed using 3 #g of protein from each fraction. O, dystrophic protein; ©, control protein ; A, dystrophic inhibitory activity; A, control inhibitory activity.
Sucrose gradient centrifugation When the inhibitory activity from the control and dystrophic fractions obtained by gel filtrations (fractions 5-8) was subjected to centrifugation on individual 10-20% (w/v) linear sucrose gradients, the peak inhibitory activities from both dystrophic and control material were seen to coincide at the same sucrose concentration (Fig. 3). The activity however was markedly reduced in the control preparation compared to the dystrophic preparations (i.e. approximately 20 U/fraction for control compared to 100 U for dystrophic). Table 1 summarizes the results obtained when the inhibitory activity was
317 A
I
BSA
I
I
1 10G
-20
•
56
Q.
5
I
B
I0 FRACTION NOI
BSA
I
15
I
1 I 0 0 --
-- 2 . 0
50
-- /.0 o~ Q.
0
0
0
5 FRACTION
I0 NO
15
Fig. 2. Gel filtration of the D E A E cellulose fractions eluting at 0.3 M KC1 obtained from (A) dystrophic and (B) control muscle. Protein fractions containing inhibitory activity that were eluted from the D E A E cellulose column by between 0.25 and 0.38 M KC1 were pooled and concentrated by ultrafiltration in an Amicon cell fitted with a PM-10 membrane. This protein was filtered on a 1 × 55 cm column of Biogel PI50 equilibrated with buffer B. The column was eluted with buffer B at a flow rate of 0.2 ml/min. The void volume was 4.5 ml and 2 ml fractions were collected. Bovine serum albumin (BSA) eluted in fraction 7. Inhibitory activity was assayed using 15/~1 aliquots from each fraction. Symbols as in Fig. 1.
purified by the procedures discussed above. Although the dystrophic material yielded more than 6 times as much total activity (units) as the control material, the specific activity was approximately equal after step 4 (about 600 U/mg protein). Despite the low inhibitory activity in the initial pH 5 supernatant fraction obtained from control mice, the percent yield of activity recovered from control sources compared to the activity recovered from dystrophic sources improved at each step of purification. Thus the final yield upon sucrose gradient centrifugation was 3 ~ for control preparations but only 1~o for dystrophic preparations. When the inhibitory activity was recalculated per pair of hindlimbs, the ratio of the units/dystrophic mouse to the units/control mouse fell from 8.5 at step 1 to 3.3 at step 4.
t
I 0
A
I---~
C
IOC
r
2
8C
i
k
~-- 6 c .,,a
~ 40
20
I0
15 % SUCROSE
20
Fig. 3. Sucrose gradient centrifugation of inhibitory material obtained after gel filtration from dystrophic and control muscle. Inhibitory preparations (1.3 mg protein) were obtained from fractions 5-8 of the Biogel P150 gel filtration step. The fractions were pooled and concentrated by ultrafiltration as described under Fig. 2. The control and dystrophic preparations (0.5 ml in buffer B) were layered on a 4.5 ml linear gradient of 10-20~ (w/v) sucrose in buffer C [50 mM Tris-HCl(pH 7.8 at 25°C), 80 mM potassium acetate~ 6 mM magnesium acetate and 10 mM 2-mercaptoethanol]. The gradient was centrifuged at 0°C for 16 h at 105000 x g in a Beckman SW 65 rotor ray 6.4 cm and 0.3 ml fractions were collected from the bottom of the tubes. The sucrose density of each fraction was determined with a refractometer at 24 °C. Protein synthesis inhibitory activity in each fraction was assayed using 15/~1 aliquots. The sucrose gradient was calibrated with the following molecular weight markers: ovatbumin, 43000 (O); bovine serum albumin, 68 000 (A); and 7-globulin, 155 000 (G). A, dystrophic inhibitory activity; A, control inhibitory activity. TABLE 1 PURIFICATION OF INHIBITORY ACTIVITY FROM NORMAL AND DYSTROPHIC MICE a Purification step
1. pH 5 supernatant
+/+ dy/dy
2. DEAE cellulose
+/+ dy/dy
3. Biogel P150
+/+ dy/dy
4.10 209o sucrose gradient
+/+ dy/dy
Soluble Total Specific protein b activityc activity (mg) (units) (units/mg)
Yield Activity Ratio activity (units/hindlimbs) (units/dystrophic hindtimbs)/ (units/control hindllmbs)
300 300
1500 26 300
5 88
I00 100
375 3180
8.5
5.0 16
132 2090
26 130
9 8
33 253
7.7
2.0 11
75 1000
39 91
5 4
19 122
6.4
45 286
663 550
3 1
11 35
3.3
0.068 0.52
The recovered activity values for control and dystrophic inhibitory material are representative of 3 separate experiments using pooled material obtained from 4 control and 8 dystrophic animals per experiment. b Soluble protein was estimated as described in Materials and Methods for the following: at the ion exchange step, pooled fractions eluting between 0.25 and 0.38 M KC1; at the gel filtration step, pooled fractions eluting between fractions number 5 and 8; at the equilibrium centrifugation step, fractions occurring between 12.50 and 13,50Wo(w/v) sucrose. c Total activity was estimated by assaying 5-15 #g of protein from pooled inhibitory material at various stages of purification. One unit (U) of activity is defined as the amount of protein needed to effect 50~o inhibition of protein synthesis after 30 min of incubation at 30°C in a standard lysate translation system.
319 This could be explained in several ways. For example, each purification step in the dystrophic preparation may be removing proteolytic enzymes which digest the inhibitory substance or may be removing unrelated inhibitory material present. Alternatively, the 'dystrophic' inhibitor may be more labile than the 'control' inhibitor, and hence, more 'dystrophic' inhibitor is inactivated at each purification step. Possibly there may be a masking protein (an 'anti-inhibitor') normally present in control muscle, and decreased in dystrophic muscle, but which is successively stripped away at each purification step.
Characterizatton of inhibitor Molecular weight determination of active inhibitory mater&l Estimations of the molecular weight of active inhibitory material from control and dystrophic muscle were made by comparing the sedimentation in sucrose density gradients with that of several marker proteins and the results suggested a molecular weight of 115 000 _+ 10 000 for the control inhibitor, and 120 000 _+ 5 000 for the dystrophic inhibitor (Fig. 4A). The active fraction obtained from the sucrose gradients was analyzed by gel electrophoresis and found to be homogeneous (Fig. 4B). Enzymatic digestion of inhibitory material Inhibitory material isolated from dystrophic muscle was previously reported to be sensitive to pronase digestion, but relatively insensitive to tryptic digestion and stable to heat (Petryshyn and Nicholls 1978). The inhibitory materials from control and dystrophic muscle were compared for sensitivity towards these agents. The possibility that the control or dystrophic inhibitory material was either DNA or RNA such as previously described for muscle and other cells (Adelman and Lovett 1974; Heywood et al. 1974; Lee-Huang et al. 1977) was discounted by the fact that treatment with micrococcal nuclease or with ribonuclease A did not reduce inhibitor activity. Control or dystrophic inhibitory material obtained from the ion exchange step and treated with trypsin, chymotrypsin or papain did not result in decreased inhibitory activity. With pronase, however, the inhibitory activities in control and dystrophic material were both reduced to a level of approximately 10~ inhibition in the reticulocyte assay and these decreases were statistically significant. The effect of heat and 2-mercaptoethanol on inhibitory activity Control and dystrophic inhibitory protein were heated to 95°C for 4 rain and/or pretreated with 5 °/ (v/v) 2-mercaptoethanol prior to assay. Both proteins were relatively insensitive to heat treatment, or to the presence of 2-mercaptoethanol alone. A combination of these treatments however, markedly reduced the activity in both cases, suggesting the presence of relatively inaccessible disulphide bonds essential for maintaining the activity of the protein (Table 2).
320
AI
I
20 k
--
~Y-Globulin
I,o in%",,o°'r f
"//
'c
BOVlDe$~rurnolburlTl}l
J Ovolburnin I
I /O
I 15
SUCROSE
Fig. 4. A : Molecular weight estimation by sucrose gradient centrifugation. 5 mg of ovalbumin, bovine serum albumin and ~,-globulin were layered as 1~ solution in buffer C in separate tubes containing 4.5 ml linear gradients of 10-20~ sucrose (w/v) in buffer C, and the gradients were centrifuged a s described in Fig. 3.0.3 ml fractions were collected, and the protein concentrations were determined in each fraction. The sucrose density of the fraction containing the highest amount of protein in each case were determined by refractometry. Molecular weight estimates for both control and dystrophic inhibitors were estimated in the tubes at which maximum inhibitory activity was observed. Molecular weight markers are as shown in Fig. 3. The horizontal bars indicate SEM for 4 experiments. B: Electrophoretic analysis of sucrose density gradient fractions from 13-13.5 % (w/v) sucrose. 50 #g protein from control (C) and dystrophic (D) preparations was subjected to electrophoresis on 8 ~ (w/v) acrylamide gels by the method of Laemmli (t970). Developed gels were stained with Coomassie brilliant blue and destained by diffusion in 7.5~ (v/v) acetic acid (bromphenol blue marker dye, B).
321 TABLE 2 THE EFFECT OF 2-MERCAPTOETHANOL AND HEAT ON INHIBITOR ACTIVITY l0 #g of control or dystrophic inhibitory protein obtained from the ion exchange step was treated either with 5% (v/v) 2-mercaptoethanol or heated for 4 min at 95 °C, or both treated and heated, prior to assay for inhibition of protein synthesis. Values are mean +_ SEM for 3 separate experiments. Treatment
% Inhibition
2-Mercaptoethanol
Heat
+ +
+ +
Control
21.95 27.40 19.20 2.20
-
+ + + +
Dystrophic
1.34 2.27 0.16 1.05
54.64 45.73 48.65 1.57
___4.74 + 4.38 + 2.72 + 0.77
SDS polyacrylamide gel electrophoresis Purified inhibitory protein labelled with 125I was subjected to electrophoresis on 8 ~ (w/v) polyacrylamide gels in the presence of 0.1 ~o (w/v) sodium dodecyl sulphate by the method of Laemmli (1970). Control and dystrophic labelled protein were resolved into 2 major protein components (Fig. 5). Pretreatment of labelled
f i
i
'
I
I
'
B
/o
G
I
A
o
I0--
I
r~
R
9
:b \
i:
//! 5
°'/,/'d L)'
5-
°'.o, •
0
'
0
o~
Q~l~e~',D
'
I
a
Ol 5
MOBIL I T Y IN GEL
,
I
/0
o
~b
~ 0
I
0
°'°~o.'O
I
~0
IlO
MOSILITY IN GEL
Fig. 5. Resolution of 125I-labelled (A) dystrophic and (B) control purified inhibitory material on SDS polyacrylamide gels. 10000 cpm of 125I-labelled protein inhibitory material was subjected to electrophoresis on 8 ~ (w/v) acrylamide gels by the method of Laemmli (1970) either with or without pretreatment in the presence of 5 ~ (v/v) 2-mercaptoethanol a n d heat for 2 min at 95 °C. The developed gels were fixed for 30 min in 1 0 ~ (w/v) trichloroacetic acid and sliced into 0.5 cm segments. The gel slices were leached in 1 ml Protosol for 8-12 h prior to the addition of 8 ml of toluene based scintillation fluid for counting. Molecular weight markers are as shown in Fig. 3. • -~, dystrophic muscle without pretreatment; • ~ , dystrophic muscle with pretreatment; 0 O, control muscle without pretreatment; 0 ~ 3 , control muscle with pretreatment.
322 .......
r
I
I
-
~
• ....
1
20
k I0 'R
5
~ 2 0
O
u
n
0.°,°;7,2.
l
f
I
I
I
[
I
0.2
04
0.6
0.,9
I0
MOBILITY IN 8~ SOS GEL Fig. 6. Molecular weight estimation by SDS polyacrylamide gel electrophoresis. 50#g of marker proteins of molecular weights as described in Fig. 3. i.e. ovalbumin, bovine serum albumin. 7-globulin, and aldolase subunit (M r 37 000) were subjected to electrophoresis on 8% (w/v) acrylamide gels. Developed gels were stained with Coomassie brilliant blue and destained by diffusion in 7.5% (v/v) acetic acid. The molecular weights of 125I-labelled dystrophic and control inhibitory materials were determined according to Weber and Osborn 0969). The horizontal bars indicate SEM for 4 experiments.
material with 5~o (v/v) 2-mercaptoethanol and heating for 2 min at 95 °C prior to electrophoresis resulted in an increase in the tow molecular weight component, suggesting the effect of pretreatment with 2-mercaptoethanol and heat is to dissociate the native inhibitor into subunits. Molecular weight estimation of these components (Fig. 6) using molecular weight marker proteins according to Weber and Osborn (1969) suggested molecular weights of 120000 + 10000 and 125 000 + 5 000 for the heavy component of control and dystrophic preparations, respectively. Estimated molecular weights of 74000 + 8000 and 71 000 + 4000 were obtained for the light component of control and dystrophic preparations, respectively. The absence of a discrete activity peak in the region of the 70 000 dalton component on the sucrose gradient (Fig. 3) suggests that this component is relatively inactive.
Determination of Stokes radii The Stokes radii of ~25I-labelled inhibitory proteins, together with their subunits, were determined by interpolation according to the method of Demassieux and La Chance (1974) on a Biogel P150 column. The estimated Stokes radii for the native protein from control and dystrophic preparations were 670 + 25 nm and 640 + 30 nm respectively, while those for the subunits were 320 _+ 22 nm and 330 + 18 nm, respectively (Fig. 7). Assuming a molecular w e i ~ t average of 120000 and 72 000 for the native protein and its subunits (as determined by centrifugation and SDS polyacrylamide gel electrophoresis), another physical characteristic, the frictional ratio (fifo) can be calculated: a
f/fo = (3W4 N)1/3 M~,3
323 I
~ 60 o
[
I
I
i
\ f~control inhibitor ~dystropht¢ tnhibitor ~poforritin ~ . _ olobulin
~k~d°l°s° i4 0 ~ o~ 2O
~ovine serumolburn dystrophicsu~unit--~-- contro,subuni, ~volbumin I 0
i
I OE
K~
L
I 0.4
Fig. 7. Determination of Stokes radii. A 2.5 x 55 cm column of Biogel P200 previously equilibrated with buffer B was calibrated as described by Demassieux and LaChance (1974) using proteins with the following Stokes radii : ovalbumin 273 n m ; bovine serum albumin, 349 n m ; aldolase, 450 n m ; y-globulin, 520 n m and apoferritin, 610 n m (Tremblay et al. 1981). For each protein, a distribution coefficient was calculated from the equation: Ve - Vo K d = - Vi where Vo is the void volume, estimated by blue dextran 2000 to be 39 ml; Vi is the inner volume of the gel pores, estimated by glycylglycine to be 121 ml, and Ve is the elution volume of the proteins studied. The Stokes radii of control and dystrophic inhibitors were estimated with 1.0 x 104 cpm of 1251-labelled inhibitory material with 0 . 2 5 ~ (w/v) bovine serum albumin contained in buffer B as eluant. The horizontal bars indicate SEM for 3 experiments.
where a is the Stokes radius, M is the molecular weight, and N is Avogadro's number. A partial specific volume of 0.74 ml/g was assumed for the inhibitors. The frictional ratios calculated for control and dystrophic native proteins are 2.04 and 1.95, respectively. The frictional ratios for control and dystrophic subunits are 1.16 and 1.19, respectively. The relatively high frictional ratios for the native proteins indicate extremely elliptical molecules. The subunits, however, appear to be relatively globular (f/fo for globular proteins is 1). DISCUSSION
Previously it was found that in the 70-day-old dystrophic mouse, the cytosol of the hindlimbs showed a pronounced decrease in the capacity to support protein synthesis, and this decrease was due to a pronase sensitive inhibitor which appeared to be a 70 000 dalton protein (Petryshyn and Nicholls 1978). The results of the experiments described here, using muscle from homozygous control mice, support the view that reduced amounts of the same inhibitory activity are present in control mice that are present in dystrophic mice. The muscle from dystrophic mice (dy/dy) contains approximately 6 times more inhibitory activity per unit weight. Since the hindleg muscle weight at 70 days of age is about half of that of the
324 controls owing to a loss of myofibrils, there is approximately 3 times more inhibitory activity per dystrophic animal. The specific activity of the inhibitory material following purification, however, was similar in the control and dystrophic preparations. Furthermore, the inhibitory material from the control muscle was essentially identical to that from the dystrophic muscle in a number of physical and chemical parameters. The present experiments have extended our previous results by taking advantage of a more sensitive reticulocyte lysate assay system and show that there is an active high molecular weight form of the inhibitor (approximately 120000) which can be denatured to yield the less active subunits of approximately molecular weight 70 000 which were described previously. The capacity of the machinery for protein synthesis as well as the translatability of various mRNA species appear to be altered differently in different tissues and in the various types of inherited muscular dystrophies. Dystrophic muscle that does not exhibit wasting appears to have an increase in protein synthesis. For example, the hypertrophied heart muscle of the dystrophic mouse had an increased cytosolic activity (Petryshyn et al. 1977). In dystrophic hamsters which do not exhibit wasting an increased incorporation of labelled amino acids into muscle protein has been found in whole cell studies (Goldspink and Goldspink 1971; Li 1980; Nicholls et al. 1980) and in cell-free preparations (Saleem and Nicholls 1979; Nicholls et al. 1980). On the other hand, in Duchenne dystrophy it was found that the muscle cytosol fraction, like that of the mouse leg muscle, exhibited a decreased capacity for amino acid incorporation (Ionasescu et al. 1971). Moreover, fibroblasts cultured from such patients exhibited a decreased labelling of the intraceltular collagen and a lowered synthesis of non-collagen proteins (Ionasescu et al. 1977). Muscle cells cultured from the patients exhibited a decrease in total protein synthesis but normal myosin synthesis (Ionasescu et al. 1979). More importantly, the rate of muscle protein synthesis in vivo in these patients is markedly reduced (Rennie et al. 1982). The inhibitory protein that is described in the present results is capable of decreasing both muscle and reticulocyte mRNA translation. Although no direct action on mRNA binding was detected, elongation was inhibited (Petryshyn and Nicholls 1978). Lodish (1976) has shown that changes either in the elongation steps or in the initiation steps of protein synthesis have a direct role in determining the amount of specific proteins that are synthesized. Thus it is possible that the inhibitory protein plays a role in wasting of the hindleg muscle in the dystrophic mouse. The possibility that the inhibitor that we have described is the result of an abnormal gene product produced only in dystrophic and heterozygous mice is precluded by our present results. Indeed, no evidence for a qualitative change of transcription has been detected in several tissues from dystrophic mice compared to normal mice nor even during normal muscle development (Ordahl and Caplan 1976; Grouse et al. 1978). It is not clear whether the inhibitor is more abundant in the dystrophic hindleg preparations because of changes in synthesis and/or degradation, or e.g. changes in an 'anti-inhibitor' which is missing in the dystrophic preparation.
325 REFERENCES Adelman, T.G. and J.S. Lovett (1974) Evidence for a ribosome-associated translation inhibitor during differentiation of Blastocladiella emersonii, Biochim. biophys. Acta, 335: 236-245. Demassieux, S. and J.P. LaChance (1974) Molecular filtration on Sepharose 6B, J. Chromat., 89: 251258. Girgis, G. R. and D. M. Nicholls (1971) Control of the increased protein synthesis in kidney of nephrotic rats by supernatant factors, Biochim. biophys. ,4cta, 247: 335--347. Girgis, G. R. and D.M. Nicholls (1973) Hypophysectomy and growth hormone alter the aminoacyltRNA binding activity of renal cortical supernatant, Endocrinology, 93 : 436~44. Goldspink, D.F. and G. Goldspink (1971) Age-related changes in protein turnover and ribonucleic acid of the diaphragm muscle of normal and dystrophic hamsters, Biochem. J., 162: 191-194. Greenwood, F.C. and W.M. Hunter (1963) The preparation of 131I-labelled human growth hormone of high specific radioactivity, Biochem. J., 89:114-123. Grouse, L.D., P.G. Nelson, G.S. Omenn and B.K. Schrier (1978) Measurements of gene expression in tissues of normal and dystrophic mice, Exp. Neurol., 59: 47~478. Hayashi, Y., H.O. Suzuki and T. Totsuka (1975) Protein and RNA synthesis in the skeletal muscle of hereditary dystrophic mouse, J. Biochem. (Tokyo), 77: 761-768. Heywood~ S. M., D. S. Kennedy and H. J. Bester (1974) Separation of specific initiation factors involved in the translation of myosin and myoglobin messenger RNAs and the isolation of a new RNA involved in translation, Proc. Nat. Acad. Sci. (USA), 71: 2428-2431. Hunt, T., G. Vanderhoff and I.M. London (1972) Control of globin synthesis: the role of heme, J. molec. Biol., 66: 471-481. Ionasescu, V., H. Zellweger and T. W. Conway (1971) Ribosomal protein synthesis in Duchenne muscular dystrophy, Arch. Biochem. Biophys., 144: 51-58. Ionasescu, V., C. Lara-Braud, H. Zellweger, R. Ionasescu and L. Burmeister (1977) Fibroblast cultures in Duchenne muscular dystrophy: alterations in synthesis and secretion of collagen and noncollagen proteins, Acta neurol, scand., 55: 407417. lonasescu, I.V., L.Z. Stern, R. lonasescu and P. Rubenstein (1979) Stimulatory effects of drugs for protein synthesis on muscle cell cultures in Duchenne dystrophy, Ann. Neurol., 5: 107-I10. Kitchin, S.E. and D.C. Watts (1973) Comparison of the turnover patterns of total and individual muscle proteins in normal mice and those with hereditary muscular dystrophy, Biochem. J., 136: 1017-1028. Kuliszewski, M.J. and D.M. Nicholls (1980) Peptide elongation in rat kidney after cadmium administration, Biochem. J., 190: 791-797. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227: 680-685. Lee-Huang, S., J. M. Sierra, R. Naranjo, W. Filipowicz and S. Ochoa (1977) Eucaryotic oligonucleotides affecting mRNA translation, Arch. Biochem. Biophys., 180: 276-287. Li, J. B. (1980) Protein synthesis and degradation in skeletal muscle of normal and dystrophic hamsters, Amer. J. Physiol., 239 (Endocrinol. Metab. 2): E401~06. Lodish, H.F. (1976) Translational control of protein synthesis, Ann. Rev. Biochem., 45: 39-72. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. biol. Chem., 193: 265-275. Nicholls, D.M., M. Chan and G.R. Girgis (1975) Aminoacyl-tRNA binding activity in regenerating kidney following contralateral nephrectomy or folic acid, Develop. Biol., 47 : 1 11. Nicholls, D. M., J. Carey and W. Sendecki (1977) Growth of liver accompanied by an increased binding of aminoacyl-transfer ribonucleic acids, Biochem. J., 166: 462-471. Nicholls, D.M., R.C. Creasy, M.W. Chin-See, J.A. Carlisle, A.B. Lange and M. Saleem (1980) Incorporation of amino acids into soluble and membrane protein fractions of dystrophic hamsters, Biochem. J., 190: 341-348. Ordahl, C.P. and A.I. Caplan (1976) Transcriptional diversity in myogenesis, Develop. Biol., 54: 61 72. Petryshyn, R. and D.M. Nicholls (1976) Protein synthesis in dystrophic muscle. Activity of the pH 5 supernatant fraction of muscle in dystrophic mice, Biochim. biophys. Acta, 435: 391~,04. Petryshyn, R. and D.M. Nicholls (1978) Studies of a factor from dystrophic mouse muscle inhibitory towards protein synthesis, Biochem. J., 176 : 907-917. Petryshyn, R.A., R.C. Creasy and D.M. Nicholls (1977) Protein synthesis in the heart of genetic dystrophic mice, Biochem. Med., 18: 139-151.
326 Rennie, M. J., R. H. T. Edwards, D. J. Millward, S. L. Wolman, D. Halliday and D. E. Matthews (t982l Effects of Duchenne muscular dystrophy on muscle protein synthesis, Nature (Lond.), 296: 165- 167. Saleem, M. and D. M. Nicholls (1979) Protein synthesis in muscles from normal and dystrophic hamster~. Biochem. J., 180: 51--58. Spargo, E., O.E. Pratt and P.M. Daniel (1979) The effects of murine muscular dystrophy on the metabolic and homeostatic functions of the skeletal muscles, J. neurol. Sci., 43 : 277 290. Srivastava, U. (1968) Biochemical changes in progressive muscular dystrophy. VII. Studies on the biosynthesis of protein and RNA in various cellular fractions of the muscle of normal and dystrophic mice, Canad. J. Biochem., 46: 35-41. Srivastava, U. (1972) Biochemical changes in progressive muscular dystrophy, Canad. J. Biochem., 50: 409415. Tombs, M.P., F. Souter and N.F. MacLagan (1959) The spectrophotometric determination of protein at 210 nm, Biochem. J., 73:167 17l. Tremblay, J., G. Thibault, J. Gutkowska, R. Boucher and J. Genest (1981) Purification and partial characterization of a plasma inhibitor of tonin, Canad. J. Biochem., 59: 256-261. Weber, K. and M. Osborn (1969) The reliability of molecular weight determination by sodium dodecylsulphate-polyacrylamide gel electrophoresis, J. biol. Chem., 244: 4406~412. Young, E. T. and D. M. Nicholls (1978) Liver enzyme induction by 1,1,1-trichloro-2,2-(p-chlorophenyl) ethane (DDT) is accompanied by an increase in the specific activity of elongation factor 1, Biochem. J., 172 : 479-486.
311
Elsevier Biomedical Press
THE PURIFICATION AND CHARACTERIZATION OF AN INHIBITOR OF PROTEIN SYNTHESIS FROM THE MUSCLE OF DYSTROPHIC MICE AND HOMOZYGOUS CONTROL MICE
M O N R O E W. C H I N - S E E and D. M c E W E N N I C H O L L S *
Department of Biology, York University, 4700 Keele Street, Downsview, M3J IP3 Toronto (Canada) (Received 8 February, 1982) (Accepted 7 April, 1982)
SUMMARY
Homogenates of hindlimb muscle were obtained from homozygous ( + / + ) control mice and dystrophic (dy/dy) mice of the ReJ 129 strain and subjected to purification by ion exchange chromatography, gel filtration, centrifugation on sucrose gradients, and sodium dodecyl sulphate gel electrophoresis. A proteinaceous material inhibitory to protein synthesis that was detected previously in fractions from dystrophic muscle (Petryshyn and Nicholls 1978) was present at reduced levels of activity in muscle fractions from homozygous controls. Although the specific activity after the sucrose gradient step was similar in the 2 groups of animals, the activity per animal was 3 times higher in the dystrophic mice. Evidence is presented to support the view that the inhibitory material is identical in the 2 groups and also that the active protein, of an approximate molecular weight of 120 000, can be denatured by heat and 2-mercaptoethanol to the less active form that was described previously, of an approximate molecular weight of 70 000.
INTRODUCTION
Although the earliest studies of dystrophic mice carried out in vitro reported increases in protein synthesis, more detailed investigations questioned these reports and showed that, provided corrections are made for cell size and for DNA content, the synthesis of total proteins is not changed (Kitchin and Watts 1973; Hayashi et al. 1975). These studies can be affected by differences in the transport This investigation was supported by the Muscular Dystrophy Association of Canada and the Natural Sciences and Engineering Research Council of Canada, Grant 3489. M. W. Chin-See held a Predoctoral Fellowship of the Muscular Dystrophy Association of Canada. * To w h o m reprint requests should be addressed. 0022-510X/82/0000-0000/$02.75 © Elsevier Biomedical Press
312 of amino acids into the precursor pool and by differences in the amount of proteil~ and in the species of labelled protein that is measured in the cell. Thus Srivastaw~ (1972) studied labelling in cell-free preparations (polyribosomes) and reported a decrease in myosin labelling, no change in actin labelling and an increase in tropomyosin labelling. From an early stage until the disease is well-advanced (e.g. by 90 days of age) the animals fail to thrive and the wasting of the hindlimb is accompanied by a marked decrease in myofibril content and a marked decrease in the protein content of soluble cytosolic fractions (Srivastava 1968; Petryshyn and Nicholls 1976). Muscle wasting may be due to decreased protein synthesis or to increased protein breakdown, and the results of Spargo et al. (1979) suggest that both of these changes are taking place owing to the use of amino acids for energy metabolism rather than for protein synthesis. In cell-free studies of the machinery for protein synthesis in this laboratory it was found that the cytosolic fraction, which contains most of the elongation factor 1 and 2 activity (i.e. the pH 5 supernatant fraction), exhibited changes in activity during altered states of hormone balance and of growth (Girgis and Nicholls 1973; Nicholls et al. 1975; Petryshyn et al. 1977; Nicholls et al. 1977; Kuliszewski and Nicholls 1980). These changes were chiefly due to elongation factor 1 activity (Young and Nicholls 1978). In the case of dystrophic mice, this cytosolic fraction of hindlimb muscle, but not other tissues, showed a pronounced decrease in the capacity to support protein synthesis (Petryshyn and Nicholls 1976). However, the lower incorporation with dystrophic preparations was not due to altered activity of elongation factors, ribonuclease, proteolytic enzymes, GTP, or sulphydryl reagents, but it was attributable to the presence of activity that was inhibitory to protein synthesis. The inhibitory activity was partially purified by gel filtration and ion exchange chromatography and was found to be sensitive to pronase and insensitive to heat, trypsin, ribonuclease A, deoxyribonuclease I and phospholipase C (Petryshyn and Nicholls 1978). When muscle from the tittermate controls was studied no inhibitory activity could be detected in the pH 5 supernatant fraction nor after gel filtration but it could be detected after DEAE-cellulose chromatography. Since the littermate control group contained heterozygotes (dy/+ ) as well as homozygotes (+ / + ), it was not known whether the inhibitory activity in the littermate controls was due to the presence of the heterozygous dystrophic animals. A preliminary experiment showed that the pH 5 supernatant fraction from homozygous controls behaved the same in protein synthesis as the pH 5 supernatant fraction from heterozygous controls. However, even if the inhibitor were present in a more purified fraction (i.e. after DEAE-cellulose chromatography) in the homozygous control animals, it was not known whether the inhibitory activity was the same as that from dystrophic mice. The following experiments were carried out to resolve these questions.
313 MATERIALSAND METHODS ATP, GTP and creatine phosphate as their sodium salts, creatine phosphokinase (rabbit muscle type E.C. 2.7.3.2), acetyl phenylhydrazine, micrococcal nuclease, papain, pronase P, ribonuclease A, and trypsin were obtained from Sigma Chemical Co., St. Louis, MO, U.S.A. Hemin chloride and Biogel P150 (50 150 mesh) were obtained from Calbiochem, Los Angeles, CA, U.S.A. Chloramine-T trihydrate (N-chloro-p-sulfonamide, sodium salt) was from Aldrich Chemical Co., Milwaukee, WI, U.S.A. Diethylaminoethyl cellulose (microgranular DE 32) was purchased from Whatman. L-[U-laC]Leucine (339 Ci/Mol) and carrier-free [1251]sodium iodide (16.05 mCi//~g iodine) were obtained from Amersham, Oakville, Ont., Canada. All other chemicals were of reagent grade, and were obtained as described previously (Girgis and Nicholls 1971 ; Young and Nicholls 1978).
Animals Male dystrophic mice (dy/dy) and control animals (+ /+ ) of the strain ReJ 129 were obtained from Roscoe B. Jackson laboratory, Bar Harbor, ME, U.S.A. These animals were maintained with free access to food (fox cubes, Master Feeds Inc.) and water until they were 60 to 70 days old. New Zealand white rabbits (2.5 kg) were supplied by Rabbits Unlimited, Stouffville, Ont., Canada.
Preparation of reticulocyte lysates The procedure for the preparation of rabbit reticulocytes was similar to that of Hunt et al. (1972), except that the rabbits were injected subcutaneously for 5 consecutive days with 2.5 ml of a 1~ (w/v) acetylphenylhydrazine solution to induce anaemia and made further anaemic by collecting 20 ml blood from the marginal ear vein on the first, third and fifth days. Three days after the final injection, the blood was collected and the reticulocytes washed, lysed and stored at -70°C.
Assay for inhibitory activity using reticulocyte lysate The following components were contained in a final volume of 65 pl: 10 mM Tris-HC1 (pH 7.8 at 25 °C), 2 mM magnesium acetate, 76 mM KC1, 1.0 mM ATP, 0.2 mM GTP, 30/~M of each of 19 amino acids (cysteine was excluded according to Hunt et al. 1972) 5 mM creatine phosphate, 5 #g creatine phosphokinase, 25 pM hemin chloride, 25 #1 of freshly thawed reticulocyte lysate, and 15 #1 of buffer or extract to be tested for inhibitory activity. 0.125 pCi of [J4C]leucine was added together with unlabelled leucine to give a final concentration of 30 pM. The reaction mixture was incubated for 30 min at 30°C, and the reaction was terminated by applying 10/~1 aliquots of the reaction mixture to 15 mm squares of 3 MM filter paper (Whatman) and then soaking in ice-cold 10~o (w/v) trichloroacetic acid
314 containing 1~o (w/v) leucine. The filter squares were prepared tbr counting by heating for 5 min at 95°C in fresh 5~i trichloroacetic acid containing 1';i', carrier leucine and then consecutive washes with cold 5~/o trichloroacetic acid, ethanol, and acetone. After drying for 1-2 min, the filters were stirred at 30 °C in 1J~°"/,,(v/v) H202 for 2 min to decolorize and were washed with ethanol and acetone as before. The filters were dried and counted in 8 ml of toluene-based scintillation fluid. Counting efficiency was approximately 85°/0 and was corrected for quenching by the external standardization method. Percent inhibitory activity was expressed as follows: incorporation with buffer - incorporation with inhibitor x 100. This assay incorporation with buffer was approximately 20 times more sensitive than the rat liver ribosome assay used previously (Petryshyn and Nicholls 1978).
Determination of protein concentration Non-collagen protein concentration of the soluble protein fractions was measured by the method of Lowry et al. (1951) with bovine serum albumin as a standard. For samples free of nucleic acids, protein concentration was determined at A2t0 according to Tombs et al. (1959).
Preparation of pH 5 supernatant fraction All of the work was performed at 2-4 °C. Mice were killed by cervical dislocation, and the hindlimbs were quickly removed and placed on ice, and the muscles were removed, weighed and minced with a pair of chilled scissors. The muscle mince was homogenized in a glass homogenizer with 15-20 complete passes of a Teflon pestle in 3 vol. of buffer A consisting of 0.25 M sucrose, 50 mM Tris-HC1 (pH 7.8 at 25 °C), 80 mM potassium acetate, 6 mM magnesium acetate and 10 mM 2-mercaptoethanol. The preparation of the pH 5 supernatant fraction from the muscle was as described previously (Petryshyn and Nicholls 1976).
Radioiodination of purified inhibitor Iodination of control and dystrophic inhibitor with [1251]iodidewas performed essentially as described by Greenwood and Hunter (1963). Fractions from the sucrose gradient step of inhibitor purification that demonstrated peak activity (13-13.5~ (w/v) sucrose) from both control and dystrophic preparations were pooled and concentrated by lyophilization. The freeze-dried residues were subsequently redissolved in 50/~1 of double-distilled water, and the protein concentration of each sample determined by method of Lowry et al. (1951). The protein was iodinated in 1.5 ml Eppendorf tubes by mixing 1 mCi of [12SI]sodium iodide, 10 #1 of 0.5 M phosphate buffer (pH 7.5 at 25 °C), 2-5/~g of protein contained in 10 t~t of 0.05 M phosphate buffer (pH 7.5 at 25 °C) and 10 #1 of a 14.2 mM solution of chloramine-T
315 trihydrate in 0.05 M phosphate buffer (pH 7.5 at 25°C). The reaction was immediately terminated by adding 25 /~1 of a 131.5 mM solution of sodium metabisulphite in 0.05 M phosphate buffer (pH 7.5 at 25 °C). Residual [~25I]iodine was diluted by adding 50/~1 of a 60.3 mM solution of potassium iodide in 0.05 M phosphate buffer (pH 7.5 at 25 °C). The entire contents of the tube were transferred to a 1 x 10 cm Sephadex G-50 gel column previously equilibrated with 0.05 M phosphate buffer (pH 8.6 at 25 °C). The column also was pretreated by passing through 1 ml of 2% (w/v) bovine serum albumin in phosphate buffer (pH 8.6 at 25 °C) and washing with 20 ml of the same buffer. This pretreatment minimized loss of labelled protein due to non-specific adsorption. Elution was carried out at a rate of 0.5 ml/min with 0.5 M phosphate buffer (pH 8.6 at 25°C). Material eluting between 1.5 and 5.5 ml was collected directly into vials containing 50 mg of crystalline bovine serum albumin in 1 ml of elution buffer as carrier protein. This fraction was found to contain most of the labelled protein. RESULTS
Chromatograph), The pH 5 supernatant fractions from control and dystrophic muscles were subjected to DEAE-celtulose chromatography. Elution of bound proteins with buffer containing a salt gradient resulted in a profile showing prominent protein peaks. The profiles were similar for both control and dystrophic preparations (Fig. 1). When the individual fractions were assayed for inhibitory activity using the lysate system, 2 regions of inhibitory activity could be seen. One peak occurred at 0.30 M KC1 as previously reported for dystrophic mice (Petryshyn and Nicholls 1978), and another peak was seen at 0.05 M KC1. The present investigation describes studies using only the former peak. The elution of the inhibitory material occurred at the same ionic strength in the control preparation as it did in the dystrophic preparation. The ~ inhibition, however, was much lower in the control preparation than it was in the dystrophic preparation.
Gel filtration Protein fractions that contained the inhibitory activity were obtained following DEAE-cellulose chromatography and were subjected to Biogel P150 gel filtration. A single protein peak was seen to elute near the void volume. On assaying for inhibitory activity, peak activity in both control and dystrophic preparations were seen to coincide with the leading edge of the main protein peak (Fig. 2) which was 0.6 mg/ml for controls and 2.0 mg/ml for dystrophic. The ~o inhibition was markedly lower in the control preparations.
316 I
I
I
I
60
04"12'0 ° ~40 P-.
20
0 0
I0
I --
20 30 FRACTION NO, I
I
I
--0
4O
I
B
~4
20 o
4O
03
P.. 0~
/.0 20
0 0
FRAC~ON N O
Fig. 1. Chromatography of the pH 5 supernatant fraction obtained from (A) dystrophic and (B) control muscle. 300 mg of the dystrophic and control wildtype pH 5 supernatant fractions were chromat0graphed separately on a 2.5 x 9 cm column of DEAE cellulose equilibrated in buffer B [50 mM Tris-HCt (pH 7.8 at 25°C) containing 10~ glycerol]. Bound protein was eluted at a flow rate 0.4 ml/min, using a linear gradient of 0-1 M KC1 contained in 200 ml of buffer B. 5 ml fractions were collected; Inhibitory activity was assayed using 3 #g of protein from each fraction. O, dystrophic protein; ©, control protein ; A, dystrophic inhibitory activity; A, control inhibitory activity.
Sucrose gradient centrifugation When the inhibitory activity from the control and dystrophic fractions obtained by gel filtrations (fractions 5-8) was subjected to centrifugation on individual 10-20% (w/v) linear sucrose gradients, the peak inhibitory activities from both dystrophic and control material were seen to coincide at the same sucrose concentration (Fig. 3). The activity however was markedly reduced in the control preparation compared to the dystrophic preparations (i.e. approximately 20 U/fraction for control compared to 100 U for dystrophic). Table 1 summarizes the results obtained when the inhibitory activity was
317 A
I
BSA
I
I
1 10G
-20
•
56
Q.
5
I
B
I0 FRACTION NOI
BSA
I
15
I
1 I 0 0 --
-- 2 . 0
50
-- /.0 o~ Q.
0
0
0
5 FRACTION
I0 NO
15
Fig. 2. Gel filtration of the D E A E cellulose fractions eluting at 0.3 M KC1 obtained from (A) dystrophic and (B) control muscle. Protein fractions containing inhibitory activity that were eluted from the D E A E cellulose column by between 0.25 and 0.38 M KC1 were pooled and concentrated by ultrafiltration in an Amicon cell fitted with a PM-10 membrane. This protein was filtered on a 1 × 55 cm column of Biogel PI50 equilibrated with buffer B. The column was eluted with buffer B at a flow rate of 0.2 ml/min. The void volume was 4.5 ml and 2 ml fractions were collected. Bovine serum albumin (BSA) eluted in fraction 7. Inhibitory activity was assayed using 15/~1 aliquots from each fraction. Symbols as in Fig. 1.
purified by the procedures discussed above. Although the dystrophic material yielded more than 6 times as much total activity (units) as the control material, the specific activity was approximately equal after step 4 (about 600 U/mg protein). Despite the low inhibitory activity in the initial pH 5 supernatant fraction obtained from control mice, the percent yield of activity recovered from control sources compared to the activity recovered from dystrophic sources improved at each step of purification. Thus the final yield upon sucrose gradient centrifugation was 3 ~ for control preparations but only 1~o for dystrophic preparations. When the inhibitory activity was recalculated per pair of hindlimbs, the ratio of the units/dystrophic mouse to the units/control mouse fell from 8.5 at step 1 to 3.3 at step 4.
t
I 0
A
I---~
C
IOC
r
2
8C
i
k
~-- 6 c .,,a
~ 40
20
I0
15 % SUCROSE
20
Fig. 3. Sucrose gradient centrifugation of inhibitory material obtained after gel filtration from dystrophic and control muscle. Inhibitory preparations (1.3 mg protein) were obtained from fractions 5-8 of the Biogel P150 gel filtration step. The fractions were pooled and concentrated by ultrafiltration as described under Fig. 2. The control and dystrophic preparations (0.5 ml in buffer B) were layered on a 4.5 ml linear gradient of 10-20~ (w/v) sucrose in buffer C [50 mM Tris-HCl(pH 7.8 at 25°C), 80 mM potassium acetate~ 6 mM magnesium acetate and 10 mM 2-mercaptoethanol]. The gradient was centrifuged at 0°C for 16 h at 105000 x g in a Beckman SW 65 rotor ray 6.4 cm and 0.3 ml fractions were collected from the bottom of the tubes. The sucrose density of each fraction was determined with a refractometer at 24 °C. Protein synthesis inhibitory activity in each fraction was assayed using 15/~1 aliquots. The sucrose gradient was calibrated with the following molecular weight markers: ovatbumin, 43000 (O); bovine serum albumin, 68 000 (A); and 7-globulin, 155 000 (G). A, dystrophic inhibitory activity; A, control inhibitory activity. TABLE 1 PURIFICATION OF INHIBITORY ACTIVITY FROM NORMAL AND DYSTROPHIC MICE a Purification step
1. pH 5 supernatant
+/+ dy/dy
2. DEAE cellulose
+/+ dy/dy
3. Biogel P150
+/+ dy/dy
4.10 209o sucrose gradient
+/+ dy/dy
Soluble Total Specific protein b activityc activity (mg) (units) (units/mg)
Yield Activity Ratio activity (units/hindlimbs) (units/dystrophic hindtimbs)/ (units/control hindllmbs)
300 300
1500 26 300
5 88
I00 100
375 3180
8.5
5.0 16
132 2090
26 130
9 8
33 253
7.7
2.0 11
75 1000
39 91
5 4
19 122
6.4
45 286
663 550
3 1
11 35
3.3
0.068 0.52
The recovered activity values for control and dystrophic inhibitory material are representative of 3 separate experiments using pooled material obtained from 4 control and 8 dystrophic animals per experiment. b Soluble protein was estimated as described in Materials and Methods for the following: at the ion exchange step, pooled fractions eluting between 0.25 and 0.38 M KC1; at the gel filtration step, pooled fractions eluting between fractions number 5 and 8; at the equilibrium centrifugation step, fractions occurring between 12.50 and 13,50Wo(w/v) sucrose. c Total activity was estimated by assaying 5-15 #g of protein from pooled inhibitory material at various stages of purification. One unit (U) of activity is defined as the amount of protein needed to effect 50~o inhibition of protein synthesis after 30 min of incubation at 30°C in a standard lysate translation system.
319 This could be explained in several ways. For example, each purification step in the dystrophic preparation may be removing proteolytic enzymes which digest the inhibitory substance or may be removing unrelated inhibitory material present. Alternatively, the 'dystrophic' inhibitor may be more labile than the 'control' inhibitor, and hence, more 'dystrophic' inhibitor is inactivated at each purification step. Possibly there may be a masking protein (an 'anti-inhibitor') normally present in control muscle, and decreased in dystrophic muscle, but which is successively stripped away at each purification step.
Characterizatton of inhibitor Molecular weight determination of active inhibitory mater&l Estimations of the molecular weight of active inhibitory material from control and dystrophic muscle were made by comparing the sedimentation in sucrose density gradients with that of several marker proteins and the results suggested a molecular weight of 115 000 _+ 10 000 for the control inhibitor, and 120 000 _+ 5 000 for the dystrophic inhibitor (Fig. 4A). The active fraction obtained from the sucrose gradients was analyzed by gel electrophoresis and found to be homogeneous (Fig. 4B). Enzymatic digestion of inhibitory material Inhibitory material isolated from dystrophic muscle was previously reported to be sensitive to pronase digestion, but relatively insensitive to tryptic digestion and stable to heat (Petryshyn and Nicholls 1978). The inhibitory materials from control and dystrophic muscle were compared for sensitivity towards these agents. The possibility that the control or dystrophic inhibitory material was either DNA or RNA such as previously described for muscle and other cells (Adelman and Lovett 1974; Heywood et al. 1974; Lee-Huang et al. 1977) was discounted by the fact that treatment with micrococcal nuclease or with ribonuclease A did not reduce inhibitor activity. Control or dystrophic inhibitory material obtained from the ion exchange step and treated with trypsin, chymotrypsin or papain did not result in decreased inhibitory activity. With pronase, however, the inhibitory activities in control and dystrophic material were both reduced to a level of approximately 10~ inhibition in the reticulocyte assay and these decreases were statistically significant. The effect of heat and 2-mercaptoethanol on inhibitory activity Control and dystrophic inhibitory protein were heated to 95°C for 4 rain and/or pretreated with 5 °/ (v/v) 2-mercaptoethanol prior to assay. Both proteins were relatively insensitive to heat treatment, or to the presence of 2-mercaptoethanol alone. A combination of these treatments however, markedly reduced the activity in both cases, suggesting the presence of relatively inaccessible disulphide bonds essential for maintaining the activity of the protein (Table 2).
320
AI
I
20 k
--
~Y-Globulin
I,o in%",,o°'r f
"//
'c
BOVlDe$~rurnolburlTl}l
J Ovolburnin I
I /O
I 15
SUCROSE
Fig. 4. A : Molecular weight estimation by sucrose gradient centrifugation. 5 mg of ovalbumin, bovine serum albumin and ~,-globulin were layered as 1~ solution in buffer C in separate tubes containing 4.5 ml linear gradients of 10-20~ sucrose (w/v) in buffer C, and the gradients were centrifuged a s described in Fig. 3.0.3 ml fractions were collected, and the protein concentrations were determined in each fraction. The sucrose density of the fraction containing the highest amount of protein in each case were determined by refractometry. Molecular weight estimates for both control and dystrophic inhibitors were estimated in the tubes at which maximum inhibitory activity was observed. Molecular weight markers are as shown in Fig. 3. The horizontal bars indicate SEM for 4 experiments. B: Electrophoretic analysis of sucrose density gradient fractions from 13-13.5 % (w/v) sucrose. 50 #g protein from control (C) and dystrophic (D) preparations was subjected to electrophoresis on 8 ~ (w/v) acrylamide gels by the method of Laemmli (t970). Developed gels were stained with Coomassie brilliant blue and destained by diffusion in 7.5~ (v/v) acetic acid (bromphenol blue marker dye, B).
321 TABLE 2 THE EFFECT OF 2-MERCAPTOETHANOL AND HEAT ON INHIBITOR ACTIVITY l0 #g of control or dystrophic inhibitory protein obtained from the ion exchange step was treated either with 5% (v/v) 2-mercaptoethanol or heated for 4 min at 95 °C, or both treated and heated, prior to assay for inhibition of protein synthesis. Values are mean +_ SEM for 3 separate experiments. Treatment
% Inhibition
2-Mercaptoethanol
Heat
+ +
+ +
Control
21.95 27.40 19.20 2.20
-
+ + + +
Dystrophic
1.34 2.27 0.16 1.05
54.64 45.73 48.65 1.57
___4.74 + 4.38 + 2.72 + 0.77
SDS polyacrylamide gel electrophoresis Purified inhibitory protein labelled with 125I was subjected to electrophoresis on 8 ~ (w/v) polyacrylamide gels in the presence of 0.1 ~o (w/v) sodium dodecyl sulphate by the method of Laemmli (1970). Control and dystrophic labelled protein were resolved into 2 major protein components (Fig. 5). Pretreatment of labelled
f i
i
'
I
I
'
B
/o
G
I
A
o
I0--
I
r~
R
9
:b \
i:
//! 5
°'/,/'d L)'
5-
°'.o, •
0
'
0
o~
Q~l~e~',D
'
I
a
Ol 5
MOBIL I T Y IN GEL
,
I
/0
o
~b
~ 0
I
0
°'°~o.'O
I
~0
IlO
MOSILITY IN GEL
Fig. 5. Resolution of 125I-labelled (A) dystrophic and (B) control purified inhibitory material on SDS polyacrylamide gels. 10000 cpm of 125I-labelled protein inhibitory material was subjected to electrophoresis on 8 ~ (w/v) acrylamide gels by the method of Laemmli (1970) either with or without pretreatment in the presence of 5 ~ (v/v) 2-mercaptoethanol a n d heat for 2 min at 95 °C. The developed gels were fixed for 30 min in 1 0 ~ (w/v) trichloroacetic acid and sliced into 0.5 cm segments. The gel slices were leached in 1 ml Protosol for 8-12 h prior to the addition of 8 ml of toluene based scintillation fluid for counting. Molecular weight markers are as shown in Fig. 3. • -~, dystrophic muscle without pretreatment; • ~ , dystrophic muscle with pretreatment; 0 O, control muscle without pretreatment; 0 ~ 3 , control muscle with pretreatment.
322 .......
r
I
I
-
~
• ....
1
20
k I0 'R
5
~ 2 0
O
u
n
0.°,°;7,2.
l
f
I
I
I
[
I
0.2
04
0.6
0.,9
I0
MOBILITY IN 8~ SOS GEL Fig. 6. Molecular weight estimation by SDS polyacrylamide gel electrophoresis. 50#g of marker proteins of molecular weights as described in Fig. 3. i.e. ovalbumin, bovine serum albumin. 7-globulin, and aldolase subunit (M r 37 000) were subjected to electrophoresis on 8% (w/v) acrylamide gels. Developed gels were stained with Coomassie brilliant blue and destained by diffusion in 7.5% (v/v) acetic acid. The molecular weights of 125I-labelled dystrophic and control inhibitory materials were determined according to Weber and Osborn 0969). The horizontal bars indicate SEM for 4 experiments.
material with 5~o (v/v) 2-mercaptoethanol and heating for 2 min at 95 °C prior to electrophoresis resulted in an increase in the tow molecular weight component, suggesting the effect of pretreatment with 2-mercaptoethanol and heat is to dissociate the native inhibitor into subunits. Molecular weight estimation of these components (Fig. 6) using molecular weight marker proteins according to Weber and Osborn (1969) suggested molecular weights of 120000 + 10000 and 125 000 + 5 000 for the heavy component of control and dystrophic preparations, respectively. Estimated molecular weights of 74000 + 8000 and 71 000 + 4000 were obtained for the light component of control and dystrophic preparations, respectively. The absence of a discrete activity peak in the region of the 70 000 dalton component on the sucrose gradient (Fig. 3) suggests that this component is relatively inactive.
Determination of Stokes radii The Stokes radii of ~25I-labelled inhibitory proteins, together with their subunits, were determined by interpolation according to the method of Demassieux and La Chance (1974) on a Biogel P150 column. The estimated Stokes radii for the native protein from control and dystrophic preparations were 670 + 25 nm and 640 + 30 nm respectively, while those for the subunits were 320 _+ 22 nm and 330 + 18 nm, respectively (Fig. 7). Assuming a molecular w e i ~ t average of 120000 and 72 000 for the native protein and its subunits (as determined by centrifugation and SDS polyacrylamide gel electrophoresis), another physical characteristic, the frictional ratio (fifo) can be calculated: a
f/fo = (3W4 N)1/3 M~,3
323 I
~ 60 o
[
I
I
i
\ f~control inhibitor ~dystropht¢ tnhibitor ~poforritin ~ . _ olobulin
~k~d°l°s° i4 0 ~ o~ 2O
~ovine serumolburn dystrophicsu~unit--~-- contro,subuni, ~volbumin I 0
i
I OE
K~
L
I 0.4
Fig. 7. Determination of Stokes radii. A 2.5 x 55 cm column of Biogel P200 previously equilibrated with buffer B was calibrated as described by Demassieux and LaChance (1974) using proteins with the following Stokes radii : ovalbumin 273 n m ; bovine serum albumin, 349 n m ; aldolase, 450 n m ; y-globulin, 520 n m and apoferritin, 610 n m (Tremblay et al. 1981). For each protein, a distribution coefficient was calculated from the equation: Ve - Vo K d = - Vi where Vo is the void volume, estimated by blue dextran 2000 to be 39 ml; Vi is the inner volume of the gel pores, estimated by glycylglycine to be 121 ml, and Ve is the elution volume of the proteins studied. The Stokes radii of control and dystrophic inhibitors were estimated with 1.0 x 104 cpm of 1251-labelled inhibitory material with 0 . 2 5 ~ (w/v) bovine serum albumin contained in buffer B as eluant. The horizontal bars indicate SEM for 3 experiments.
where a is the Stokes radius, M is the molecular weight, and N is Avogadro's number. A partial specific volume of 0.74 ml/g was assumed for the inhibitors. The frictional ratios calculated for control and dystrophic native proteins are 2.04 and 1.95, respectively. The frictional ratios for control and dystrophic subunits are 1.16 and 1.19, respectively. The relatively high frictional ratios for the native proteins indicate extremely elliptical molecules. The subunits, however, appear to be relatively globular (f/fo for globular proteins is 1). DISCUSSION
Previously it was found that in the 70-day-old dystrophic mouse, the cytosol of the hindlimbs showed a pronounced decrease in the capacity to support protein synthesis, and this decrease was due to a pronase sensitive inhibitor which appeared to be a 70 000 dalton protein (Petryshyn and Nicholls 1978). The results of the experiments described here, using muscle from homozygous control mice, support the view that reduced amounts of the same inhibitory activity are present in control mice that are present in dystrophic mice. The muscle from dystrophic mice (dy/dy) contains approximately 6 times more inhibitory activity per unit weight. Since the hindleg muscle weight at 70 days of age is about half of that of the
324 controls owing to a loss of myofibrils, there is approximately 3 times more inhibitory activity per dystrophic animal. The specific activity of the inhibitory material following purification, however, was similar in the control and dystrophic preparations. Furthermore, the inhibitory material from the control muscle was essentially identical to that from the dystrophic muscle in a number of physical and chemical parameters. The present experiments have extended our previous results by taking advantage of a more sensitive reticulocyte lysate assay system and show that there is an active high molecular weight form of the inhibitor (approximately 120000) which can be denatured to yield the less active subunits of approximately molecular weight 70 000 which were described previously. The capacity of the machinery for protein synthesis as well as the translatability of various mRNA species appear to be altered differently in different tissues and in the various types of inherited muscular dystrophies. Dystrophic muscle that does not exhibit wasting appears to have an increase in protein synthesis. For example, the hypertrophied heart muscle of the dystrophic mouse had an increased cytosolic activity (Petryshyn et al. 1977). In dystrophic hamsters which do not exhibit wasting an increased incorporation of labelled amino acids into muscle protein has been found in whole cell studies (Goldspink and Goldspink 1971; Li 1980; Nicholls et al. 1980) and in cell-free preparations (Saleem and Nicholls 1979; Nicholls et al. 1980). On the other hand, in Duchenne dystrophy it was found that the muscle cytosol fraction, like that of the mouse leg muscle, exhibited a decreased capacity for amino acid incorporation (Ionasescu et al. 1971). Moreover, fibroblasts cultured from such patients exhibited a decreased labelling of the intraceltular collagen and a lowered synthesis of non-collagen proteins (Ionasescu et al. 1977). Muscle cells cultured from the patients exhibited a decrease in total protein synthesis but normal myosin synthesis (Ionasescu et al. 1979). More importantly, the rate of muscle protein synthesis in vivo in these patients is markedly reduced (Rennie et al. 1982). The inhibitory protein that is described in the present results is capable of decreasing both muscle and reticulocyte mRNA translation. Although no direct action on mRNA binding was detected, elongation was inhibited (Petryshyn and Nicholls 1978). Lodish (1976) has shown that changes either in the elongation steps or in the initiation steps of protein synthesis have a direct role in determining the amount of specific proteins that are synthesized. Thus it is possible that the inhibitory protein plays a role in wasting of the hindleg muscle in the dystrophic mouse. The possibility that the inhibitor that we have described is the result of an abnormal gene product produced only in dystrophic and heterozygous mice is precluded by our present results. Indeed, no evidence for a qualitative change of transcription has been detected in several tissues from dystrophic mice compared to normal mice nor even during normal muscle development (Ordahl and Caplan 1976; Grouse et al. 1978). It is not clear whether the inhibitor is more abundant in the dystrophic hindleg preparations because of changes in synthesis and/or degradation, or e.g. changes in an 'anti-inhibitor' which is missing in the dystrophic preparation.
325 REFERENCES Adelman, T.G. and J.S. Lovett (1974) Evidence for a ribosome-associated translation inhibitor during differentiation of Blastocladiella emersonii, Biochim. biophys. Acta, 335: 236-245. Demassieux, S. and J.P. LaChance (1974) Molecular filtration on Sepharose 6B, J. Chromat., 89: 251258. Girgis, G. R. and D. M. Nicholls (1971) Control of the increased protein synthesis in kidney of nephrotic rats by supernatant factors, Biochim. biophys. ,4cta, 247: 335--347. Girgis, G. R. and D.M. Nicholls (1973) Hypophysectomy and growth hormone alter the aminoacyltRNA binding activity of renal cortical supernatant, Endocrinology, 93 : 436~44. Goldspink, D.F. and G. Goldspink (1971) Age-related changes in protein turnover and ribonucleic acid of the diaphragm muscle of normal and dystrophic hamsters, Biochem. J., 162: 191-194. Greenwood, F.C. and W.M. Hunter (1963) The preparation of 131I-labelled human growth hormone of high specific radioactivity, Biochem. J., 89:114-123. Grouse, L.D., P.G. Nelson, G.S. Omenn and B.K. Schrier (1978) Measurements of gene expression in tissues of normal and dystrophic mice, Exp. Neurol., 59: 47~478. Hayashi, Y., H.O. Suzuki and T. Totsuka (1975) Protein and RNA synthesis in the skeletal muscle of hereditary dystrophic mouse, J. Biochem. (Tokyo), 77: 761-768. Heywood~ S. M., D. S. Kennedy and H. J. Bester (1974) Separation of specific initiation factors involved in the translation of myosin and myoglobin messenger RNAs and the isolation of a new RNA involved in translation, Proc. Nat. Acad. Sci. (USA), 71: 2428-2431. Hunt, T., G. Vanderhoff and I.M. London (1972) Control of globin synthesis: the role of heme, J. molec. Biol., 66: 471-481. Ionasescu, V., H. Zellweger and T. W. Conway (1971) Ribosomal protein synthesis in Duchenne muscular dystrophy, Arch. Biochem. Biophys., 144: 51-58. Ionasescu, V., C. Lara-Braud, H. Zellweger, R. Ionasescu and L. Burmeister (1977) Fibroblast cultures in Duchenne muscular dystrophy: alterations in synthesis and secretion of collagen and noncollagen proteins, Acta neurol, scand., 55: 407417. lonasescu, I.V., L.Z. Stern, R. lonasescu and P. Rubenstein (1979) Stimulatory effects of drugs for protein synthesis on muscle cell cultures in Duchenne dystrophy, Ann. Neurol., 5: 107-I10. Kitchin, S.E. and D.C. Watts (1973) Comparison of the turnover patterns of total and individual muscle proteins in normal mice and those with hereditary muscular dystrophy, Biochem. J., 136: 1017-1028. Kuliszewski, M.J. and D.M. Nicholls (1980) Peptide elongation in rat kidney after cadmium administration, Biochem. J., 190: 791-797. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227: 680-685. Lee-Huang, S., J. M. Sierra, R. Naranjo, W. Filipowicz and S. Ochoa (1977) Eucaryotic oligonucleotides affecting mRNA translation, Arch. Biochem. Biophys., 180: 276-287. Li, J. B. (1980) Protein synthesis and degradation in skeletal muscle of normal and dystrophic hamsters, Amer. J. Physiol., 239 (Endocrinol. Metab. 2): E401~06. Lodish, H.F. (1976) Translational control of protein synthesis, Ann. Rev. Biochem., 45: 39-72. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. biol. Chem., 193: 265-275. Nicholls, D.M., M. Chan and G.R. Girgis (1975) Aminoacyl-tRNA binding activity in regenerating kidney following contralateral nephrectomy or folic acid, Develop. Biol., 47 : 1 11. Nicholls, D. M., J. Carey and W. Sendecki (1977) Growth of liver accompanied by an increased binding of aminoacyl-transfer ribonucleic acids, Biochem. J., 166: 462-471. Nicholls, D.M., R.C. Creasy, M.W. Chin-See, J.A. Carlisle, A.B. Lange and M. Saleem (1980) Incorporation of amino acids into soluble and membrane protein fractions of dystrophic hamsters, Biochem. J., 190: 341-348. Ordahl, C.P. and A.I. Caplan (1976) Transcriptional diversity in myogenesis, Develop. Biol., 54: 61 72. Petryshyn, R. and D.M. Nicholls (1976) Protein synthesis in dystrophic muscle. Activity of the pH 5 supernatant fraction of muscle in dystrophic mice, Biochim. biophys. Acta, 435: 391~,04. Petryshyn, R. and D.M. Nicholls (1978) Studies of a factor from dystrophic mouse muscle inhibitory towards protein synthesis, Biochem. J., 176 : 907-917. Petryshyn, R.A., R.C. Creasy and D.M. Nicholls (1977) Protein synthesis in the heart of genetic dystrophic mice, Biochem. Med., 18: 139-151.
326 Rennie, M. J., R. H. T. Edwards, D. J. Millward, S. L. Wolman, D. Halliday and D. E. Matthews (t982l Effects of Duchenne muscular dystrophy on muscle protein synthesis, Nature (Lond.), 296: 165- 167. Saleem, M. and D. M. Nicholls (1979) Protein synthesis in muscles from normal and dystrophic hamster~. Biochem. J., 180: 51--58. Spargo, E., O.E. Pratt and P.M. Daniel (1979) The effects of murine muscular dystrophy on the metabolic and homeostatic functions of the skeletal muscles, J. neurol. Sci., 43 : 277 290. Srivastava, U. (1968) Biochemical changes in progressive muscular dystrophy. VII. Studies on the biosynthesis of protein and RNA in various cellular fractions of the muscle of normal and dystrophic mice, Canad. J. Biochem., 46: 35-41. Srivastava, U. (1972) Biochemical changes in progressive muscular dystrophy, Canad. J. Biochem., 50: 409415. Tombs, M.P., F. Souter and N.F. MacLagan (1959) The spectrophotometric determination of protein at 210 nm, Biochem. J., 73:167 17l. Tremblay, J., G. Thibault, J. Gutkowska, R. Boucher and J. Genest (1981) Purification and partial characterization of a plasma inhibitor of tonin, Canad. J. Biochem., 59: 256-261. Weber, K. and M. Osborn (1969) The reliability of molecular weight determination by sodium dodecylsulphate-polyacrylamide gel electrophoresis, J. biol. Chem., 244: 4406~412. Young, E. T. and D. M. Nicholls (1978) Liver enzyme induction by 1,1,1-trichloro-2,2-(p-chlorophenyl) ethane (DDT) is accompanied by an increase in the specific activity of elongation factor 1, Biochem. J., 172 : 479-486.