Uterine smooth muscle: Troponin

Uterine

Smooth

ABSTRACT

Similarity in the mechanism of contraction in smooth and skeletal muscle is based on the presence of contractile proteins, similar or identical in structure, properties and biological activity, in both types of muscle. Troponin, which together with tropomyosin confers calcium sensitivity to actomyosin, is shown to be present in bovine uterine smooth muscle. Uterine troponin was purified by isoelectric precipitation and ammonium sulfate fractionation. The uterine troponin preparations were shown to be free of tropomyosin in acrylamide gel electrophoresis. Electrophoresis in sodium dodecyl sulfate showed a minimum of six components. The intrinsic viscosity of uterine troponin was greatly increased upon addition of uterine tropomyosin indicating complex formation of the two proteins. The biological activity of uterine t,roponin was similar to that of skeletal troponin. The uterine troponin-tropomyosin complex inhibited uterine and skeletal actomyosin ATPase in the presence of EGTA. The proteins actin, myosin, and tropomyosin are recognized as structural elements of the myofibril of skeletal and heart muscle. More recently the protein troponin has been discovered (1). Combined wit,h tropomyosin, troponin is thought to confer calcium sensitivity to the actomyosin ATPase2 (2), calcium being required to activate the contractile proteins (3). In mammalian smooth muscle the relationships of the contractile proteins are not well defined. We previously isolated and characterized actin and tropomyosin from the myometrium (4, 5). Native tropomyosin and oc-actinin were found in chicken gizzard (6). This communication demonstrates the presence of troponin in the myometrium and describes some of its properties. Uterine horns from freshly killed nonpregnant cows or heifers were obtained at the slaughter 1 This investigation was supported by a U.S. Public Health Service grant (HD-00010) from the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md. 2 Abbreviations used: ATPase, adenosine triphosphatase; DTT, dithiothreitol; EGTA, ethyleneglycol bis (p)-aminoethyl ether N,2V-tetraacetic acid.

Muscle:

Troponin’

house, immediately placed on ice and taken to the laboratory. The muscle tissue was dissected free of fat and blood vessels and cut into pieces, l-2 in. square. Five to ten horns (400-800 g of tissue) were homogenized in a Waring Blendor for x min at high speed in 1 vol of deionized water containing 0.5 mM dithiothreitol. The homogenate was centrifuged at 9OOOgfor 20 min. Further preparation of the crude muscle extract was according to Bailey (7) ; dithiothreitol (0.5 mM) was added to all solutions (8) throughout the purification procedures. Bovine skeletal troponin and tropomyosin were prepared by the same methods as the uterine proteins. The 1 M KC1 extract was acidified to pH 4.6, the precipitate was set aside for preparation of tropomyosin, the supernatant fraction used to prepare troponin (9). The purification of SH-troponin was carried out essentially following the procedure of Yasui, Fuchs, and Briggs (10) for skeletal troponin, except that the pH was lowered to 4.0 for removal of contaminating tropomyosin. Final dialysis was against 0.05 M ammonium bicarbonateXl.5 mM DTT. The protein was freeze-dried. SH-tropomyosin was prepared from the precipitate starting with a series of isoelectric precipitations (8), followed by ammonium sulfate precipitation (11). The fraction between 53 and 60% ammonium sulfate saturation was collected, dissolved in a small amount of water, and dialyzed against three changes of 0.09 M KCIg.01 M HCl (5) for 48 hr or longer. Any precipitate formed was spun down, the supernatant fraction was dialyzed against water and freeze-dried. All solutions contained 0.5 mM DTT. Disc electrophoresis was performed according to Davis (12) using a spacer gel and a sample gel. The buffer system of Williams and Reisfeld (13) was chosen with the 6% acrylamide gel at pH 7.5, but the diethylbarbituric acid buffer was adjusted to pH 7.4. Urea (4 M) was present in all gels and in the protein solutions (10). Electrophoresis was carried out for 45 min at 5 mA per tube. The gels were stained for 30 min in 0.5$& naphthalene black in 7% acetic acid, and destained electrophoretically with 7% acetic acid at 5 mA per tube for approximately 1 hr. Elect,rophoresis in sodium dodecyl sulfate was carried out following Weber and &born (14). Samples of troponin, tropomyosin, and appropriate standards were run in lOrr/, gels. 353

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Viscosity measurements were carried out in an Ostwald viscometer at 20 f 0.02” in 20 mM TrisHCI buffer, pH 8.5. The proteins were dialyzed in this buffer for 24 hr. Three preparations were used: Uterine SH-troponin, uterine SH-tropomyosin, and a mixture of the two at a ratio of troponin: tropomyosin of 1.5: 1. Actomyosin from rabbit myofibrils was desensitized (15). Calcium sensitivity was estimated as the effect of varying ratios of troponin to tropomyosin on the Mg-activated ATPase activity in (A) the presence and (B) the absence of EGTA: $& inhibition = (B - A)/B X 100. The assay medium contained 0.06 M KCl, 2.5 mM MgC12, 2.5 rn~ ATP, 25 mM Tris buffer, pH 7.6. Uterine actomyosin was prepared by low ionic strength extraction in the presence of ATP according to Huys (16), except that Tris buffer was used in place of phosphate. The resulting gel was washed three times with deionized water and suspended in 0.6 M KCl. Assays were carried out in 0.6 M KCl, 10 mM MgC12, 1 mM ATP, and 10 mM histidine buffer, pH 7.0. The reactions were terminated after 5 min by addition of trichloroacetic acid and inorganic phosphate was determined (17). Protein concentrations were determined by the method of Lowry et al. (18) using accurately weighed samples of the respective proteins as standards. Correction for moisture was applied from drying at 80” in vacua over night. Protein concentrations are thus given on a dry weight basis. Tryptophan was determined spectrophotometritally in 0.1 N NaOH (19). Spectral data were taken in a Beckman DU-2 spectrophotometer. ‘Ultra pure” urea and “special enzyme grade” ammonium sulfate, both low in heavy metals (Mann Research Laboratories), were used throughout. The average yields obtained for uterine SHtroponin, skeletal SH-troponin, and uterine SHtropomyosin are 22, 53, and 62 mg/lOO g wet weight, respectively, with a troponin yield of gs that obtained for tropomyosin. Disc electrophoresis in urea clearly showed the SH-troponin preparations migrating faster than the tropomyosin preparations (Fig. 1). Moreover, the troponin preparations were free of tropomyosin. Like skeletal SH-tropomyosin (lo), uterine SH-tropomyosin appeared as a double band, and there was no troponin in the preparations (Fig. 1). The main band of uterine SH-troponin moved faster in the electrophoresis than that of the skeletal SH-troponin, and, indeed, the uterine troponin appeared cleaner than the skeletal troponin preparation. There was a variable amount of diffuse material at the top of the gel column in the SHtroponin electrophoresis. A thin fast-moving band was observed migrating to the bottom of the gel column with the indicator dye. Schaub and Perry

FIG. 1. Polyacrylamide gel electrophoresis. (A) 4 M urea, pH 7.4, uterine troponin, 200 pgg; (B) skeletal troponin, 200 pg; (C) uterine tropomyosin, 50 pg. (D) Sodium dodecyl sulfate, pH 7.0, uterine troponin, 50 pg; (E) uterine tropomyosin, 50 pg. (20) showed that partial dissociation of skeletal troponin occurred in the presence of urea and identified the fast-migrating material as a transformation product, present in all preparations carried out in the absence of calcium or in troponin prepared from ethanolor ether-treated muscle powder extracted with 1 M KC1 as used by us. The diffuse band close to the origin could be the inhibitory factor, or troponin B identified in skeletal troponin (20,21). Without urea the main band was slightly retarded and more retarded in the presence of 10% glycerol. Addition of DTT did not change the electrophoretic pattern. Electrophoresis in sodium dodecyl sulfate, run in our laboratory, generally showed at least six bands as were seen in skeletal troponin (22). Molecular weights calculated from electrophoresis in sodium dodecyl sulfate solution (14) were approximately 14,500, 26,000, 43,090, and 56,ooO for the major components, 66,000 and 70,006 for two high molecular-weight contaminants, and 49,060 for the occasional seventh band (Fig. 1). Uterine tropomyosin was computed to have a molecular weight of 37,000. Figure 2 shows the absorption spectra for the troponins and the tropomyosin, with the uterine and skeletal troponins similar to one another, but

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COMMUNICATIONS different from SH-tropomyosin. Notable is the relatively higher absorption of the uterine troponin, especially in the X 260 region. We computed 18.2 and 13.7 moles tryptophan per 100,009 g dry protein for uterine and skeletal troponin, respectively, while there was none in uterine tropomyosin.

Reduced viscosities of the SH-proteins are plotted as a function of protein concentration in Fig. 3. The intrinsic viscosities, calculat)ed by the method of least squares, were 0.296 and 5.51 dl/g for uterine SH-troponin and SH-tropomyosin respectively. Combining uterine SH-troponin and SH-tropomyosin in a ratio of 1.5:1 raised the re-

. -I -1 \ I \\ I ----4.

240

I 280 320 360 4100 WAVELENGTH, ii (mpL)

FIG. 2. Absorption spectra in 0.1 N NaOH. (A) Uterine troponin, tal troponin, 0.21 mg/ml; (C) uterine tropomyosin, 0.70 mg/ml.

0.22 mg/ml;

(B) skele-

7

PROTEIN CONCENTRATION, g/lOOml FIG. 3. Reduced viscosities as a function of protein concentration. (A, and (O), troponin with tropomyosin added; (B, 0 ), tropomyosin and (O), with troponin added; ratio troponin: tropomyosin 1.5: 1.

l

), Troponin tropomyosin

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3.5

3.0 2.5 I .5 2.0 I .o RATIO : TROPON IN / TROPOMYOSI N

3.5

4.0

FIG. 4. Inhibition of the Mgz+-activated ATPase activity by t,roponin and tropomyosin in the presence of 1 mM EGTA; 120 rg troponin-tropomyosin complex per milligram actomyosin. Desensitized skeletal actomyosin with (0) uterine troponin and uterine tropomyosin; (0) uterine troponin and skeletal tropomyosin; uterine actomyosin with (X) uterine troponin and uterine tropomyosin. duced viscosity (see Fig. 3). Extrapolation yielded an intrinsic viscosity of 6.26 for the mixture. As a criterion of biological activity the inhibition of the Mg2+-act.ivated ATPase of desensitized actomyosin in the presence of EGTA was chosen. The ATPase activity of desensitized skeletal or uterine actomyosin was not inhibited by EGTA alone, nor was there inhibition in the presence of EGTA with either uterine troponin alone or uterine or skeletal tropomyosin alone. In the presence of uterine troponin combined with either uterine or skeletal tropomyosin, EGTA inhibited the ATPase, i.e., restored calcium sensitivity. The effect of troponin and tropomyosin in various combining ratios is shown in Fig. 4. For the uterine proteins the inhibition was greatest at a ratio of 1.5:l whether skeletal or uterine actomyosin was used. The inhibitory effect was decreased when skeletal tropomyosin replaced uterine tropomyosin in the ATPase assay. The demonstration of troponin in the myometrium is of importance inasmuch as it is indicative of a mechanism of contraction, similar in smooth and in skeletal muscle. The low yields of uterine troponin and tropomyosin conform with the low content of contractile proteins in uterus smooth muscle compared to skeletal muscle as observed with actin (4) and myosin (23). Our yield of skeletal troponin is in the range obtained by Yasui, Fuchs, and Briggs 00) The electrophoretic pattern in urea demonstrates the absence of tropomyosin from the troponin preparations, but suggests the presence of more than one component, as were found in skeletal troponin (20, 22). Four major and three minor

components were identified in uterine troponin, with molecular weights similar to those of skeletal troponin (22). The molecular weight of uterine tropomyosin calculated to be 37,000 is in close agreement with 36,000 of skeletal tropomyosin 04). Interaction of troponin and tropomyosin is demonstrated by the elevation of the reduced viscosity upon adding the two proteins to one another, an elevation considerably higher than would be expected for two noninteracting proteins. Desensitized rabbit skeletal actomyosin was used to test for inhibit,ion of ATPase activity, because this system is well characterized (20). Maximum inhibition by the uterine troponin-tropomyosin complex in the presence of EGTA was approximately half of that reported for skeletal troponin-tropomyosin (20). However, troponin3 and tropomyosin (5) are considerably different in smooth and skeletal muscle. Hence, it was surprising to see that they affect the inhibition of skeletal actomyosin at all, and that the combination of uterine troponin with skeletal tropomyosin had any effect. Uterine actomyosin is highly unstable and known for its low ATPase activity (23). In agreement with this we could not demonstrate EGTA sensitivity in all our preparations and found the EGTA sensitivity relatively low. This EGTA sensitivity was confined to a narrow range of troponin: tropomyosin ratios and appears more specific than the inhibition of skeletal actomyosin with uterine troponin and tropomyosin. We may conclude that there is a protein in 3 Carsten,

M. E., unpublished.

COMMUNICATIONS uterine smooth muscle which fulfills a function similar to that of skeletal muscle troponin. The importance of troponin lies in its role in calcium sensitization of actomyosin and calcium binding in contraction. We demonstrated in the uterus a calcium-binding sarcoplasmic reticulum, capable of storing calcium in relaxation (24). Thus the components of the contractile system are present in uterus smooth muscle as they are in skeletal muscle. They are available for rhythmic contraction and relaxation of the uterus. ACKNOWLEDGMENTS Credit is due my staff-Kenneth Freshman, Ronald Wong, and Stephen Stram-for technical assistance at various stages of the investigation. REFERENCES 1. EBASHI, S., AND EBASHI, F., J. Biochem. 66, 604 (1964). 2. EBASHI, S., AND KODAMA, A., J. Biochem. 60, 733 (1966). 3. WEBER, A., AND HERZ, R., J. Biol. Chem. 238, 599 (1963). 4. CARSTEN, M. E., Biochemistry 4, 1049 (1965). 5. CARSTEN, M. E., Biochemistry ‘7,960 (1968). 6. EBASHI, S., IWAKURA, H., NAKAJIMB, H., NAKAMURA, R., AND 001, Y., Biochem. 2. 346, 201 (1966). 7. BAILEY,B., Biochem. J. 43,271 (1948). 8. MUELLER, H., Biochem. 2.346,300 (1966). 9. EBASHI, S., AND KODAMA, A., J. Biochem. 68, 107 (1965). 10. YASUI, B., FUCHS, F., AND BRIGGS, F. N., J. Biol. Chem. 243, 736 (1968). 11. HARTSHOR,NE, D. J., AND MUELLER, H., Biochim. Biophys. Acta 176,301 (1969). 12. DAVIS, B. J., Ann. iV. Y. Acad. Sci. 121, 404 (1964).

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13. WILLIAMS, D. E., AND REISFELD, R. A., Ann. N. Y. Acad. Sci. 121,373 (1964). 14. WEIIER, K., -END OSRORN, M., J. Biol. Chem. 244, 4406 (1969). 15. SCHAUB, M. C., HARTSHORNE, D. J., AND PERRY, S. V., Biochem. J. 104, 263 (1967). 16. HUYS, J., Bull. Sot. Roy. Beige Gynecol. Obstet. 33, 429 (1963). 17. ROCKSTEIN, M., AND HERRON, P. W., Anal. Chem. 23, 1500 (1951). 18. LOWRY, W. H., ROSEBROUGH, N. J., FARR, A. L., .~ND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 19. BEAVEN, G. H., SND HOLIDAY, E. R., Advan. Protein Chem. 7, 319 (1952). 20. SCHAUB, M. C., AND PERRY, 9. V., Biochem. J. 116, 993 (1969). 21. HARTSHORNE, D. J., SND MUELLER, H., Biothem. Biophys. Res. Commun. 31,647 (1968). 22. HARTSHORNE, D. J., AND PYUN, H. Y., Biochim. Biophys. Acta 229,698 (1971). 23. NEEDHAM, 1). M., AND SHOENBERG, C. F., in “Cellular Biology of the Uterus” (R. M. Wynn, ed.), 291 pp. Appleton-CenturyCrofts, New York, 1967. 24. CARSTEN, M. E., J. Gen. Physiol. 63,414 (1969). MARY E. CARSTEN’ Department of Obstetrics and Gynecology, Research Laboratory, University of California at Los Angeles, School of Medicine, LOS Angeles, California 90024 Received August 18, 1971 4 Recipient of a U. S. Public Health Service Research Career Development Award (K3-GM4647) from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Maryland.