Changes in myosin caused by hypothyroidism

518

ht. J. Biochrm.l 1972, 3, 518-524.

CHANGES A. Department

IN MYOSIN L.

JACOBSON*,

of Chemistry,

CAUSED B.

(Publishers)

Ltd.]

BY HYPOTHYROIDISMS

HUMPHREY,

The University

[Scientechnica

of Calgary,

AND

V. GREY

Calgary,

Alberta,

Canada

(Received I 6 June, I 972) ABSTRACT The effects of hormone control on the muscle protein myosin have been studied. Hypothyroidism was induced in rabbits by ingestion of n-propyhhiouracil and myosin was isolated from the back muscle. The yield of myosin was very much lower from the hypothyroid animals. 2. The enzymatic activity of the myosin from the hypothyroid animals was lower than that from the control animals. 3. The helical content of the myosin from the hypothyroid animals was significantly greater than that from the controls. 4. Some differences in the amino-acid composition of the proteins were also observed. 5. However, sedimentation studies and disc gel electrophoresis indicated that the myosins from the control and from the hypothyroid animals have similar size, charge, and subunits. I.

VARIOUS studies have shown that the biosynthesis of muscle proteins can be altered by The changes in the hormonal balance. enzymatic activity of the contractile proteins appears to be a very sensitive indicator of selective small changes in the proteins at or near the active site. Hjalmarson, ?Vhitfield, and Morgan (1970) reported that the calcium-activated ATPase of rat cardiac myosin was decreased by hypophysectomy, while the potassium activated ATPase was unchanged. Thyroxine administration in the rat increased the cardiac myosin ATPase (Goodkind, Damback, and Luchi, 1969) and stimulated protein biosynthesis in the rat heart (Michels, Cason, and Sokoloff, 1963). Thyrum, Kritcher, and Luchi (1970) reported that in cardiac myosin from the guineapig both the amino-acid composition and the helical content, as well as the ATPase activity, were altered in hyperthyroid animals. Muscular dystrophy and weakness are common secondary effects of hypothyroidism (Deb& and Semelaigne, 1935). The water content of the muscle tissues is increased and the tissue becomes spongy. Localized areas of necrosis in the muscle-fibre have been * To whom correspondence should be addressed.

observed by Norris and Parner (1966). In these areas the myofibrillar organization is disrupted and the cross-striation decreased. Nuclear and mitochondrial morphological changes also have been reported. Ingestion of the goitrogen n-propylthiouracil produces a thyroxine deficiency by blockage of the conversion of monoiodotyrosine to di-iodot~osine (Richards and Morphological changes in Ingbar, t 959). the muscle, similar to those reported in hypothyroidism with localized areas of necrosis, have been reported in rabbits treated with the goitrogen n-propylthiouracil (Jacobson and Stapleton, 1969). However, most of the muscle tissue appeared normal. The firm normal texture of the muscle was lost and the tissues appeared ‘ spongy ’ or ’ mushy ‘. It appears likely that the morphological changes in the muscle tissue after n-propylthiouracil ingestion are due to changes in the thyroxine concentration, since the onset of the changes in the muscle and magnitude of the changes directly parallel the characteristic histological changes in the thyroid indicative of hypothyroidism. Changes in the enzymatic activity of actomyosin were also reported after n-propyIthiouracil treatment.

HYPOTHYROIDISM

In this work the properties of myosin isolated from h~~th~rroid animals have been studied. EXPERIMENTAL In this study a 0.03 per cent solution of nprop~lthiouracll was used as the drinking water for hew Zealand white rabbits for 3-4 months. Control animals of the same age were paired with the of-propylthiouracil-t!~ated %nimals- and maintained under identical conditions. Roth tvnes of animals were sacrified at the same time and parallel preparations of myosin from the back muscle were made immediately after sacrifice. Thyroid biopsy was used to confirm hypothyroidism. Myosin was prepared by the methad of Tonomura, Appel, and Morales (1966). The myosin solutions were sequentially chromatographed with cellulose phosphate followed by DEAE-Sephadex. The cellulose phosphate chromatography has been reported by Harris and Suelter (1967) to remove traces of AMP deaminase, which may be coprecipitated during the p~i6cation procedure. The DEAF-Sephadex purification has been reported by Bar& Love, and Herrmann (x966) to remove nucleic acid or nucleotide contaminants, which can also be coprecipitated or bound to the protein. The chromatography technique and results were similar to the previous reports. The specific activitv of the myosin was increased and the nucleotide absorbance at 260 nm. decreased relative to the absorbance at 280 nm. Protein concentrations were measured by the micro-Kjeldahj technique. Double~istilled water was used throughout. All solutions were passed through Chelex JOO resin (Bio Rad Laboratories) to remove trace heavv metal contaminants. ATPase activity was measured by the modified Fiske SubbaRow method proposed by Lecocq and Inesi (1966). The agreement with the standard Fiske SubbaRow method was within .zo’~ pmole phosphate per g. protein per second. A control and a sample from the n-propvlthiouraciftreated animal were always measured’in the same experimental determination. Sedimentation measurement were made at G.000 r.p.m. at lo’ C. in a Spinco Model E ultracenrrifu,ge. Optical rotation measurements were made at 233 nm. in fixed-end-plate temperature-jacketed cells (5.0 mm. path length) at 20” C, in a DurrumJasco spectropolarimeter. Amino-acid analysis was with a Beckman Model 12 I amino-acid analyser. Digestion was for 24 hours in 6 ;11 HCI. The myosins from the control and from the hypothyroid animals were run sequentially with standard samples preceding and following the experimental determination.

EFFECTS

5’9

ON MYOSIN

The technique and conditions used in the sodium dod~ylsuiphate (SDS) polyac~lam~de gel electrophoresis were similar to those of Sarkar, Sreter, and Gergeley ($971). Samples were prepared by incubating protein in buffer with I per Eent SDS(with I percent n-mercaptoethanol, 2IdE) for I hour at 37” C., and then left overnight at 25 C. Gels were 6-25 per cent acrylamide, with bisacrvlamide 8 ner cent of total acrvlamide. The gels were also 0% per cent SDS and 0.8 per cent 2ME. The reservoir buffer was Tris/glycine, pH 8.3; the gel buffer was Tris/HCl, pH 8.6. Samples were applied as solutions in 40 per cent sucrose in reservoir buffer, and layered on the gel at I mA. per tube for 10 minutes. The current was then increased to 4 mA. per tube. For Agarose gel chromatography, I ‘25 per cent gels were prepared in Tris/HCl at pII 8.6, After the tubes were filled, very hot water was layered on top to provide a flat gel surface. Application and running conditions were similar to the SDS electrophoresis. In both cases the pH was lower than that of previous workers to avoid base degradation of the proteins. RESC’LTS

In all cases ingestion of the goitrogen n-propylthiouracil produced muscle atrophy. The total weight of muscle tissue obtained from the back muscle of the rabbits treated with ~-propylthiouracil varied from I I to 30 per cent below the control. However, a more dramatic comparison is the 50 per cent or greater decrease in the amount of myosin extracted per g. of muscle tissue. The percentage yields are shown in Table I. As a qualitative observation, there appeared to be a correlation between the ‘spongy ’ nature of the tissue and the decrease in the amount of myosin which could be extracted by normal procedures. The very porous ’ spongy ’ nature of muscle tissues from the hypothyroid animalsprobabIyindicatesahigherpercentage of water in these tissues, so that the total weight of the back muscle probably is not the best basis for comparison for myosin content. However, the changes are so large that it appears Iikely that there is significantly less myosin in the muscie of hypothyroid animals. The potassium-activated ATPases at PH 8-o and 7.4 are also shown in Table I. The enzymatic activity of the myosin from the control animals was consistently si~i~~antl~ higher than the enzymatic activity of the myosin from the hypothyroid animals,

Zrzt.3. Biochcm.

JXOBSON AND OTHERS

520

In Table f the specific rotations of the myosins are given at 233 nm., the trough in the Cotton effect due to the a helical structure. The specific rotations of the myosin samples from the n-propylthiouracil-treated animals are significantly larger, indicating a significantly higher helical content for these

greater weight is given to the higher protein concentration determinations. llleasurements are more accurate in this area, since at lower protein cdncentrations the peaks are more diffuse. Electrophoresis of the intact chromatographed myosin on Agarose showed only one

Tubk Z.-PROPERTIES OF THE MYOSINS ISOLATED FROM CONTROL AND R-PROPYLTHIOUUCIL-TRE.XTEU RABBITS

ANIMAL PAIR

PERCENTAGE YIELD OF MYOSIN PER g. OF MUSCLE TISSUE

l-

PTU 5

I

0.23

0’09

2 3 4 5

0.29 0.4s 0.32 0’47

0.08 0.17 0.07 0.23

_-

T

pH 8.0

__ Control

SPECIFIC ROTATION

ACTIVITY (Irmoles Pi per g. per second)*

PH 7.4:

Control

PTU $

Control

2.8

I ,6

2’4

3.1 3-o

2’4 2.2 2’1

2’2 2.3 -

3‘0 * ‘2

I.9zk.5

2.3*.x

I.2

;:;

(-[cd

x 10-Y:

!JH 7’4t PTU !j

Control

PTU 5

1’7

9.8 7.5

10.6 10.4

I ‘6

’ ‘4

;::

-

;:: IO.3

7’5

8.0 5 I ‘0

t.6i.2

10’”

3-

A-.-

3

* 0.2 mg. per ml. myosin 0.5 M KCI, 0.02 M Tris, I m&f ATP. t o+I mN EDTA added. f 5 mm. fixed-end-plate cell temperature-jacketed at 20.’ C. S,My&n

from n-propylthiouracil-treated

animals.

myosin samples. If a value of I 15 is used as the mean residue weight to convert the specific rotation to the mean residue rotation, and if -16,000 is used to represent the mean residue rotation for IOO per cent helix (Beychok, 1968)) the myosin from the hypothyroid animals is on average I o per cent more helical than the control samples. The amino-acid analysis of the myosin samples is given in Table II. There is a statistically significant difference in the Thr, Glu, and Leu contents of the myosin from the hypothyroid animals. There is a possibility that there is some difference in the Lys content, but this is not statistically significant. Sedimentation coefficients for several myosin preparations are shown in Fig. I as a function of protein concentration. There are no significant differences in the sedimentation behaviour of the myosins from the control and the #-propylthiouracil-treated animals, if

Tabte Il.--AMINO-AGIL I”LUALYSES OF THE X&osws OF CONTROL AND HYPOTHYROID RABBITS AMINO-ACID

ASP Thr Se1 GIU

Pro Gly Ala Vd Met lie Leu T-C PGe LYS His Arg

CONTROL*

84&r 40&3 4o*4 146 k6

16&5 45i2 80*3 36k5 24&2 4*+4 z”,: 27 zk3 9257 27 *3 42k2

85i1 4gi1t 38i1 159+3+ ‘7Ifr5 43*3 78~~2 +3+5 221-3 4oc4 84 _~6+ 18=r 2852 85i-10 24~4 48--,ro

I * Average of Ei different myc n samples from

6 animals. f Significant difference samples and control.

between

h~~oth~ro~d

I

j22

JACOBSON

19693 . However, the mechanism of the ATPase of actomyosin is known to be complex, and actomyosin ATPase activity is not equivalent to myosin ATPase activity. 1VhiIe dissociation of the actin-myosin complex by ATP has been observed, it has also beenshown that the steady-state rate of hydrolysis of myosin is greatly increased in the presence of actin (Eisenberg and Moos, 1968). Recent evidence (Lynn and Taylor, 1970; Taylor, Lynn, and Mall, 1970) suggests that the activation of myosin ATPase by actin is due to an increased dissociation of the enzymeproduct complex and that this occurs in conjunction with the dissociation of the actinmyosin complex. Differences in actin binding and dissociation co&d account for the differences in behaviour between myosin and actomyosin from the control and hypothyroid animals. Decreased yields of myosin and actomyosin from dystrophic muscle appear to be a common factor despite the many different types of myopathy. In muscular dystrophic chicks a 75 per cent decrease in the yield of myosin has been reported by Morey and coworkers (Morey, Tarczy-Hornock, Richards, and Brown, 1967). The greater reduction in the amount of myosin extractable from whole dystrophic muscle, compared to the amount of actomyosin extractable, also appears to be a common occurrence in dystrophic muscle. Such relative decreases were noted in denervation atropy by Fisher (1950) and in vitamin E deficiency by Corsi, Gallucci, and BarHowever, in the case of gellini ( r 959). vitamin E deficiency Corsi and coworkers ( xg5g) observed that a normal amount of myosin may be extracted from isolated myofibrils while a reduced yield was observed with whole-muscle extracts. With the muscle from vitamin-E-deficient rabbits Corsi and others (1959) suggested that a larger proportion of myosin was bound with actin than in the case of normal animals. A similar effect may be operative with muscle from hypothyroid animals and supports the suggestion that the differences in enzymatic activity between the myosins and the actomyosins from normal and hypothyroid animals might be due to differences in actin binding.

AND

OTHERS

ht.

3.

Biochem.

It is possible that differences in the potassium-activated ATPase of actomyosin were masked because of the presence of small amounts of protein contaminants in the actomyosin solutions. Rampton, Pearson, Walker, and Kapsalis ( 197 I) have recently shown that a number of disc gel electrophoresis bands, which are present in the WeberEdsall extraction media, are still present in the actomyosin after standard preparative procedures. This suggests that the protein purity in all preparations of natural actomyosin may be suspect. There is some sample variation in the [02s3] values reported here. This type of variation was also observed by Tonomura, Sekiga, Imamura, and Tokiwa (1963). The increase in helical content reported here for myosin from hypothyroid animals is greater than sample-variation effects and is of the same order of magnitude as the increase previously reported by Thyrum and others o or cardiac myosin from hyperthyroid ~~~~~1~ The similarities in the sedimentation coefficients indicate that there are probably no major differences in the gross size and shape of the myosin molecules. The values of the sedimentation constants extrapolated to zero protein concentration are similar to values of Laki, Spicer, and Carrol (1951). Since normal myosin is already a very highly helical molecule, a change in helical content from 50 to 60 per cent may not be large enough to be measurable in the tertiar! structure as indicated by sedimentation. Since myosin is a large molecule, electrophoretic studies are usually limited to the digestion products. With diiute Agarose the degradation of the molecule is not necessary and the iigarose electrophoresis indicates that there is no significant difference in charge or shape between the myosins and that only one major component is present. There are no previous reports of Agarose efectrophoresis of myosin to which the results might be compared. The degradation of the purified myosin to four components in the SDSpolyacrylamide and the loss of light components after chromatography are similar to reports of Sarkar and others (1g71), and the

19721 3

HYPOTHYROIDISM

similarities between the myosin samples indicate that the major subunit structures of the myosins are the same. It appears that the enzymatic activity of the myosins and actomyosins is the most sensitive method of determining changes in the structure caused by hormone control. This is not surprising, since only a very small change in or near the active site is necessary for a large change in activity. It is not known whether the biosynthesis of cardiac and skeletal myosin is affected in the same manner by hormone control. However, it is interesting to note that excess thyroxine produced a more active cardiac myosin while deficiency produced a less active skeletal myosin. In both cases differences in the amino-acid composition of the myosin have been observed. However, the changes are not parallel. In cardiac myosin from hypcrthyroid animals, Asp, Glu, Lys, Ser, and Thr changes were observed by Thyrum and others (1970). In the skeletal myosin from the hypothyroid animals the changes were in Thr, Glu, and Leu. The amino-acid analysis of the myosin from the control animals is in good agreement with previous values reported by Kominz, Hough, Symonds, and Laki ( 1954) and by Thyrum and others (I 970). Hjalmarson and others (1970) reported that the calcium-activated cardiac myosin ATPase in rats is decreased by hypophysectomy, while the I(+-activated ATPase was unchanged. Hypothyroidism is a secondary effect of hypophysectomy, and thyroxine administration restored the Ca2+-activated ATPase in these animals. The changes reported here for rabbit skeletal myosin K+activated ATPase induced by n-propylthiouracil administration were not paralleled by changes in the cardiac myosin from hypophysectomized rats. However, a direct comparison may not be valid for several reasons. There may be additional hormonal changes caused by hypophysectomy, species differences are possible, and there may be differences in effects on cardiac and skeletal protein. In summary, some differences, notably in enzymatic activity, helical content, and amino-acid comoosition. have been shown n

EFFECTS ON MYOSIN

523

between rabbit skeletal myosin from normal animals and animals in which hypothyroidism has been induced. However, the general size, shape, and subunit structure of the myosin has not been altered by change in hormonal levels. The amount of myosin extractable is drastically reduced by the fz-propylthiouracil ingestion. The differences in amino-acid composition between normal myosin and the myosin from hypothyroid animals indicates that thyroid hormone levels affect the biosynthesis of the muscle proteins. The changes in skeletal muscle, which occur in conjunction with hypothyroidism and the skeletal muscle weakness, may be due in large part to the decreased amounts of the contractile proteins and possibly to a lesser extent to the changes in organization of the muscle tissue (since such changes are of a localized nature only). However, differences in the structure and activity of muscle proteins may also play a part in the clinical changes reported in muscle. ACKNOWLEDGEMENTS

The authors gratefully acknowledge financial support from the National Research Council for this work. All experimental animals were cared for in accord with the principles of Care of Experimental Animals-A Guide for Canada from the Canadian Council on Animal Care. REFEREl’ICES E. F., Lovz, D. S., and HERRMANN. H. ( I 966)) ’ Investigation of. myosin heterogeneity

BARIL,

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diethyl-

Gem.,

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(1959L

vitamin

S. (IgSB), dichroism

‘ ‘:

Rotatory

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Rev.

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and

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; hlyosin

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JACOBSONAND OTHERS

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Key Word Index: Myosin, hypothyroidism, contractile proteins, muscle proteins, hormone control, propylthiouracil.