Synthesis of myoglobin by muscle polysomes

BIOCHIMICA ET BIOPHYSICA ACTA

523

BBA 96358

SYNTHESIS OF MYOGLOBIN BY MUSCLE POLYSOMES

LAV~rRENCE J. K A G E N AND S H O S H A N A L I N D E R

Department o/Medicine, Columbia University College o/Physicians and Surgeons, and the Edward Daniels Faulkner Arthritis Clinic o/the Presbyterian Hospital, New York, N . Y . (U.S.A .) (Received J u l y i o t h , 1969)

SUMMARY

I. Polysome preparations derived from muscle, but not from liver, of the duck brought about incorporation of uniformly 14C-labeled lysine into antibody-detectable myoglobin in vitro. This synthesis of myoglobin accounted for approximately 5-9 ~/o of the total soluble protein synthesis demonstrable in this system. 2. Tetrameric, pentameric and hexameric polysomes seemed to be the active components in myoglobin synthesis. 3. The efficiency of myoglobin synthesis per unit of polysomes in the standard cell-free system was the same in preparations obtained from immature and adult ducks, although approx. 8 times as much polysomal material could be isolated from immature muscle. 4. Myoglobin (0.06-0.08 raM) produced sligbt, though consistent, stimulation of myoglobin synthesis. Concentrations of myoglobin above o.15 mM inhibited myoglobin synthesis in vitro. There was no detectable effect of these concentrations of added myoglobin on total protein synthesis measured as a whole, however. 5. The combination of reduced polysome content, and reduced efficiency may account for the reduced synthetic ability of adult muscle, for myoglobin, compared to immature muscle.

INTRODUCTION

Myoglobin, the single polypeptide chain, Oz-binding heme protein, is synthesized in striated muscle tissuek The myoglobin content of immature and embryonic muscle is markedly less than that of the adult, and its content in muscle rises with age ~-4. Minces and homogenates of embryonic muscle have been found, however, to be more active in myoglobin synthesis than those of adult tissue 1. The reasons for this increased synthetic ability of embryonic muscle are not known. Recently techniques for improved preparation of stable, "nuclease-free" polyribosomes have been reported by EARL AND MORGAN5 from heart muscle. Using their system, we wish here to present studies on myoglobin synthesis by polysomes derived from immature and mature duck muscle. A comparison of polysome efficiency with age, the approximate size of polysomes needed for synthesis and the effect of the environmental concentration of myoglobin on synthesis will be discussed. Biochim. Biophys. Acta, 195 (1969) 523-53 °

524

L.J. KAGEN,S. LINDER

MATERIALS AND METHODS Polysomal and ribosomal preparations were made from skeletal muscle of adult (1-year-old) and newly hatched (i-4-day-old) ducks, as described by EARL AND MORGAN5. The A2no nm/A28on m ratio of these preparations was 1.7-1.8. The system in vitro for the study of radioactive amino acid incorporation into protein was described in ref. 5. The cell-free incorporation system contained o.02 M Tris buffer (pH 7.6); o.I M KC1; 0.04 M NaC1; I I mM magnesium acetate; I mM EDTA; 6 mM 2-mercaptoethanol; 5 mM ATP; uniformly 14C-labeled lysine 0.25 #C/ml; 5-1o mg/ml of cell sap (supernatant fluid obtained after centrifugation of crude muscle homogenate at IOO ooo ×g for 2 h); 3-1o A26on m units of the polyribosome preparation. The total volume was I.O ml. The technique of assay of supernatant fluids from the incubation system for myoglobin synthesis is based on specific precipitation with antimyoglobin serum, and is described in ref. I. The antiserum was added to the supernatant fluids in several times the required amount for antibody excess. This allowed the same volume of serum to be used in each of the experimental samples. In these experiments, 0. 4 ml of antiserum was added to 0.2 ml of supernatant fluid. Precipitates which formed after 17-18 h at 2 °, were washed 4 times by the addition of i.o-ml vol. of o.15 M NaC1, followed by centrifugation. These were then dissolved in 0.2 ml i M NaOH, digested and counted in the presence of liquid scintillator in a Tri-Carb scintillation counter (Packard Inst. Co., Downer's Grove, Ill.), as described. Duck myoglobin was prepared as in ref. 4. The preparation of antimyoglobin serum in rabbits was performed as in ref. 4. Uniformly 14C-labeled lysine (0.32 #g/ml) was obtained from the New England Nuclear corp. (Boston, Mass.). Its specific activity was 241 mC/mmole. Standardization of sucrose gradient ultracentriiugal polysome profiles was made by comparison with polysome preparations from reticulocytes kindly supplied by Dr. P. Marks. Gradients were of 15-3o % sucrose, and were centrifuged at 37 ooo rev./min, 5 °, in SW-56 rotor (Beckman Instruments, Palo Alto, Calif.), for 75 min. Droplets of a ribosome suspension were placed on carbonized formvar-coated electron microscope grids, inverted on to 20 % phosphate-buffered formalin (pH 7.4), and fixed for 20 rain. Grids were transferred to a drop of 0.005 % uranyl acetate, partially dried and negatively stained with 2 ~o potassium phosphotungstate. Specimens were examined with a Hitachi HU-IIC electron microscope operating at 75 kV. RESULTS Skeletal muscle polyribosomal characteristics

Polysomal preparations obtained from duck gastrocnemius muscle were incubated with "cell sap", crude transferase preparations, amino acids and energy sources 5 in the presence of uniformly l~C-labeled lysine (0.25/,C/ml). Fig. i illustrates incorporation of radioactivity from lysine into antibody detectable myoglobin, by this system using two different amounts of polysome-containing material. Incorporation was usually maximal after 5-1o rain and ceased after 20 rain. Table I demonstrates that lysine incorporation into myoglobin occurred with muscle polyBiochim. Biophys. Acta, 195 (1969) 523-53°

525

MYOGLOBIN SYNTHESIS BY MUSCLE POLYSOMES 4O0 / 0 A 2 6 0 Units

300

200 ._C

E 0 tO0

.,d' O

"~'3 Aeeo u.;,~" i

5

t

i

I0 15 TIME IN MINUTES

21 0

216

Fig. I. Incorporation of radioactivity from u n i f o r m l y z4C-labeled lysine into a n t i b o d y detectable myoglobin. Two p o l y s o m e preparations (io and 3 A ~80nra units) were used in the s y s t e m in vitro.

TABLE I I N C O R P O R A T I O N O F U N I F O R M L Y 1 4 C - L A B E L E D L Y . S I N E BY" R I B O S O M A L P R E P A R A T I O N S AND TOTAL SOLUBLE PROTEIN

INTO MY"OGLOBIN

I n c u b a t i o n time, 15 min. E a c h experimental value represents the average of 4 replicate experiments.

Source o/ribosomes Skeletal muscle 7.3 A200 nm units 3.0 A260nm units

Myoglobin antibody ppt. (counts /min )

Total soluble protein Tricholoroacetic acid ppt. (counts /min )

2504-18

8

50024-282 13464-213

04- 2 34- 2

14364-127 I9o34-136

I I 7 4,

Liver units 5.0 A260nm units

7.8 zJ~60n m

somes, and not with liver polysomes, in an otherwise identical system. Myoglobin synthesis accounted for 5-9 % of the uniformly z4C-labeled lysine uptake into trichloroacetic acid-precipitable, total soluble proteins. Fig. 2 demonstrates the appearance of these polysome preparations, active in myoglobin synthesis, in the electron microscope. Before examination, they had been frozen at --7 °0 and thawed once. The photograph is intended to show the abundance of ribosomes, polysomes and the relative absence of membranes, or nonribosomal, particulate materials. Active polysome preparations were subjected to centrifugation in density gradients of sucrose after a brief (2 min) incubation at 37 °, in the incorporation system in vitro containing uniformly z4C-labeled lysine. As shown (Fig. 3) these preparations, then, contained single ribosomes (at the 8o-S mark) amounting to 3o-4o % of the total ribosomal material. The rest was polydispersed in several more dense groups found lower in the gradient. Anti-myoglobin serum (0.4 ml) was added to each of Biochim. Biophys. Acta, 195 (1969) 523-53o

526

L . J . KAGEN, S. LINDER

Fig. 2. Electron micrograph of polysome preparation. This preparation had been frozen and thawed before examination. Many ribosomes display the characteristic division into larger and s m a l l e r s u b u n i t s . Single r i b o s o m e s a n d r i b o s o m a l c l u s t e r s (polyribosomes) containing two or m o r e ribos o m e s are seen (arrows). × 15 ° ooo.

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Fig. 3. Sucrose density gradient ultracentrifugal pattern of polysome preparation after brief (2 rain) incubation with uniformly 14C-labeled lysine in the system in vitro. Sedimentation is f r o m right to left. A r r o w m a r k s the position of single r i b o s o m e s (8o S).

12 pooled fractions of the gradient after centrifugation, in order to demonstrate those regions which contained radioactive or newly synthesized myoglobin antigenic determinants. Carrier myoglobin (iooffg) was added to each of the fractions to ensure formation of adequate antigen-antibody precipitates. Radioactivity, detectable with the antiserum to myoglobin (Fig. 3), was present in two areas of the gradient. At the top, radioactivity appeared in free newly synthesized myoglobin unassociated with ribosomes. Fractions (lO-14) in the midportion of the gradient representing zones containing polysomes of probable tetrameric, pentameric and hexameric size also contained radioactivity which precipitated in the presence of antiBiochim. Biophys. Acta, 195 (1969) 52:3-53 °

527

MYOGLOBIN SYNTHESIS BY MUSCLE POLYSOMES

myoglobin serum. The newly formed myoglobin determinants were associated with these intermediate size ribosomal aggregates. Addition of normal rabbit serum as a control failed to precipitate more than 2-6 counts/min of radioactivity from such gradient preparations.

E//ect o/age on polyribosomal e[/iciency Polysome preparations, obtained from gastrocnemius muscles of adult and immature ducks were tested in vitro in the incubation system for myoglobin synthetic ability (Table II). In the usual experiment, using 3-1o A280 nm units of polyribosomes

TABLE

II

EFFICIENCY OF POLYSOMAL PREPARATIONS DERIVED FROM GASTROCNEMIUS MUSCLE OF DUCKS OF DIFFERENT AGES COMPARED TO EFFICIENCY OF WHOLE TISSUE HOMOGENATES IN MVOGLOBIN SYNTHESIS Each experimental

value represents the average of 6 replicate experiments.

Age

Counts/rain in myoglobin per polysome A zs0 nm unit

Counts/rain in myoglobin per mg muscle tissue homogenate

1-year adult 4-day duckling 1-day duckling

3 7 -4- 4 424-3 46~4

3± I io~: I" 18+2"

* Yield of polysomal material from these two preparations from the preparation of adult gastrocnemius muscle.

was approx. 8 times as great as

or 2o-3 ° mg of crude muscle homogenate fluid, between lOO-5OO counts/min were incorporated into myoglobin in all cases, except with crude homogenates of adult muscle tissue (where only about 1/3-1/6 as much radioactivity was detected in myoglobin as with extracts of immature muscle tissue). This was a small but consistently obtained value. Although crude homogenate preparations of immature muscle were 3-6 times as active in myoglobin synthesis as those of adults, isolated polysomal preparations in otherwise similar incubation systems did not demonstrate this increased efficiency. There were no apparent differences in the efficiency (defined as counts/min incorporated into myoglobin per A26o n m unit) of polysomes with age. However, approx. 8 times as many polysomes could be prepared from immature muscle as could be isolated from adult muscle.

E//ect o/ added myoglobin Fig. 4 demonstrates the effect of varying concentrations of myoglobin in the incubation medium. There was a slight but consistent stimulation of myoglobin synthesis in vitro at myoglobin concentrations of o.06--0.08 mM (o.11-o.14 mg/ml). At higher concentrations, however, this effect was not present; rather there was inhibition of lysine incorporation into myoglobin, particularly at concentrations above o.15 mM (2. 7 mg/ml). The time-course of this effect is shown in Fig. 5. Here, myoglobin at a concentration of 2.5 mg/ml had no effect while at 12 mg/ml (approx. 0.6 mM) there was approx. 80 ~/o inhibition. The control preparations in these experiBiochim. Biophys. Acta, 195 ( 1 9 6 9 ) 5 2 3 - 5 3 °

.oo

528

L. J. KAGEN, S. LINDER

120 100

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03

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20

30

40

TIME IN MINUTES

Fig. 4. E f f e c t o f added m y o g l o b i n on m y o g l o b i n synthesis b y muscle polysomes. I n c o r p o r a t i o n of

radioactivity by systems in vitro containing added myoglobin plotted as a percent of control (initial) incorporation, without added myoglobin. Fig. 5- Effect of added myoglobin on incorporation of radioactivity from uniformly 14C-labeled lysine into antibody detectable myoglobin. Three concentrations of myoglobin ( A, 0.45 mg/ml; @, 2.5 mg/ml; O, IZ mg/ml) were used. 140-

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Fig. 6. Effect of added myoglobin on total protein synthesis b y muscle polysomes. Incorporation of radioactivity by systems in vitro containing added myoglobin plotted as a percent of control (initial) incorporation, without added myoglobin.

ments always contained some myoglobin in the cell sap fraction, in this case (o.45 mg/ml) 0.025 raM. Fig. 6 shows the lack of effect of these myoglobin concentrations on total protein synthesis (i.e., incorporation of radioactivity into trichloroacetic acid precipitates). DISCUSSION

Myoglobin is piesent in greater concentration in adult than in immature muscle. The data in Table In, based upon our previous findings, summarize these differences TABLE I I I CONCENTRATION

OF MYOGLOBIN,

DETERMINED

IMMUNOLOGICALLY

IN DUCK

MUSCLE

Data based upon studies reported in ref. 4. Age

Adult z days post-hatching

Cardiac

Femorotibial

mg/g muscle

mM

mg/g muscle

mM

4.6 2. I

o.26 o. I2

3.5 o.7

o.2o 0.04

Biochim. Biophys. Acta, 195 (1969) 5z3-53 o

MYOGLOBIN SYNTHESIS BY MUSCLE POLYSOMES

529

in terms of concentration in entire homogenates prepared from muscle, without making any attempt to discriminate between possible concentration differences in various parts of the muscle cell. This relationship with age holds true for other animals including man 1,2. Immature muscle, although deficient in myoglobin content, is however, quite active in myoglobin synthesis (3~5 times as active as adult muscle when crude homogenates are compared). The technique of assessment of myoglobin synthesis, which measured radioactivity incorporated from uniformly 14C-labeled lysine into antibody-precipitable myoglobin, probably allows for the recognition of globin chains as well as complete myoglobin molecules. Details of this have been reported elsewhere 1. In the present studies, it has been possible to study myoglobin synthesis by muscle polysome preparations using methods of isolation and incubation proposed by EARL AND MORGAN5. Density gradient centrifugation experiments indicated that newly synthesized radioactive antigenic determinants of myoglobin could be recognized by specific antiserum in the lightest zones, unassociated with ribosomes, representing free myoglobin polypeptide chains as well as in zones of intermediate density, associated with polysomes in the region where tetrameric, pentameric and hexameric polysomes would be expected. The polysome size needed to bring about synthesis of the myoglobin polypeptide chain (mol. wt approx. 17 800) would then be similar to that required for the single polypeptide chain of hemoglobin ( mol. wt. approx. 17 ooo). In the latter instance, the pentameric polysome may be the most prevalent active unit e. Recently antibody to myosin has been reported to cause precipitation of polysomes synthesizing this nascent protein 1°. Approx. 5-9 ~/o of the total soluble protein synthesis could be detected as myoglobin synthesis. These findings are the same as reported in experiments using crude muscle minces and homogenates 1. The activity of polysomes derived from immature muscle was approximately the same as that of polysomes obtained from adult muscle. More polysomes (8 X), however, were isolated from immature muscle. Similar findings of increased polyribosome concentration in young mammalian muscle have recently been reported 9. The reasons for the increased myoglobin synthetic ability of immature muscle may, therefore, be based, at least in part, upon two factors. First, increased numbers of polysomes present in immature rapidly growing muscle, and second, inhibitory effects of myoglobin upon synthetic rate. This latter factor, as indicated by the present studies could well be operative in adult muscle. Although the myoglobin concentration of the entire muscle cell may be estimated, its concentration in the environment of sites of protein synthesis, the polysomes, is not known. Localization of myoglobin within the muscle has not been possible on a quantitative basis; however, regions near the contractile proteins and sarcolemma seem qualitatively richest in this protein 7,s. If the average adult concentrations (3.5 rag/g) of myoglobin were present at, or near, active ribosomal sites, 25 % inhibition of maximal myoglobin synthesis would be anticipated. The reason for this inhibitory effect is not, at present, known. The combination, therefore, of reduced polysome content and reduced efficiency of synthesis and perhaps other unknown factors might account for the reduced synthetic ability of adult muscle, replete with myoglobin, compared to ilnmature muscle, still relatively deficient in content of myoglobin. Biochim. Biophys. Acta, 195 (1969) 523-530

530

L . J . KAGEN, S. LINDER

ACKNOWLEDGEMENTS T h e a u t h o r s a r e m o s t g r a t e f u l f o r t h e k i n d a s s i s t a n c e of D r . R i c h a r d A. R i f k i n d of t h e D e p a r t m e n t of M e d i c i n e w h o p e r f o r m e d t h e e l e c t r o n m i c r o s c o p y o n t h e p o l y ribosome preparations. This study was supported in part by Grant 5 R0I AMII659, N a t i o n a l I n s t i t u t e s of H e a l t h , P u b l i c H e a l t h S e r v i c e .

REFERENCES I 2 3 4 5 6 7 8 9 IO

L. J. KAGEN, S. LINDER AND R. GUREVICH, Am. J. Physiol., 217 (1969) 591. G. BI6RCK, Acta Med. Scan& suppl., 226 (1949) I. L. J. KAGEN AND C. L. CHRISTIAN, Am. J. Physiol., 211 (1966) 656. L. J. KAGEN AND S. LINDER, Proc. Soc. Exptl. Biol. Med., 128 (1968) 438. D. C. N. EARL AND H. E. MORGAN, Arch. Biochem. Biophys., 128 (1968) 460. J. R. WARNER, A. RICH AND C. E. HALL, Science, 138 (1962) 1399. L. J. KAGEN AND R. GUREVlCH, J. Histochem. Cytochem., 15 (1967) 436. S. C-OLDFISCHER, J. Cell. Biol., 34 (1967) 398. U. SRIVASTAVA,Arch. Biochem. Biophys., 13o (1969) 129. E. R. ALLEN AND C. F. TERRENCE, Proc. Natl. Acad. Sci. U.S., 6o (I968) 12o9.

Biochim. Biophys. Acta, 195 (1969) 523-53 °