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. 1965 May 1;48(5):737–752. doi: 10.1085/jgp.48.5.737

Isolation of Relaxing Particles from Rat Skeletal Muscles in Zonal Centrifuges

H Schuel 1, L Lorand 1, R Schuel 1, N G Anderson 1
PMCID: PMC2213757  PMID: 14324985

Abstract

Supernatants of rat skeletal muscle homogenates were fractionated by differential centrifugation and by zonal centrifugation in sucrose density gradients. Cytochrome oxidase was employed as an enzymatic marker for locating mitochondria. The subcellular fractions were also assayed for their ability to prevent the ATP-induced contraction of myofibrils. Both the mitochondrial and microsomal fractions obtained by differential fractionation were found to be rich in such relaxing activity, and the microsomal fraction was appreciably contaminated by mitochondria. In contrast to this, when fractionation was carried out by means of zonal centrifugation (4200 RPM x 205 min. to 40,000 RPM x 60 min.), relaxing activity was found to be associated only with particles having the sedimentation characteristics of microsomes (s 20,w estimated to be between 370 and 1880S). Relaxing activity was not detected in the regions of the gradient containing either the starting sample zone (soluble phase) or the mitochondrial peak. The microsomal relaxing particles showed negligible cytochrome oxidase activity.

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Selected References

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  1. ANDERSON N. G., BARRINGER H. P., BABELAY E. F., FISHER W. D. THE B-IV ZONAL ULTRACENTRIFUGE. Life Sci. 1964 Jul;3:667–671. doi: 10.1016/0024-3205(64)90016-5. [DOI] [PubMed] [Google Scholar]
  2. BAIRD G. D., PERRY S. V. The inhibitory action of relaxing-factor preparation on the myofibrillar adenosine triphosphatase. Biochem J. 1960 Nov;77:262–271. doi: 10.1042/bj0770262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BENDALL J. R. Muscle-relaxing factors. Nature. 1958 Apr 26;181(4617):1188–1190. doi: 10.1038/1811188a0. [DOI] [PubMed] [Google Scholar]
  4. BRIGGS F. N., KALDOR G., GERGELY J. Participation of a dialyzable cofactor in the relaxing factor system of muscle. I. Studies with single glycerinated fibres. Biochim Biophys Acta. 1959 Jul;34:211–218. doi: 10.1016/0006-3002(59)90249-5. [DOI] [PubMed] [Google Scholar]
  5. Baltscheffsky M. Calcium uptake and relaxing activity in a fractionated rabbit muscle homogenate. Biochem Biophys Res Commun. 1964;14:296–301. doi: 10.1016/0006-291x(64)90453-x. [DOI] [PubMed] [Google Scholar]
  6. COOPERSTEIN S. J., LAZAROW A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem. 1951 Apr;189(2):665–670. [PubMed] [Google Scholar]
  7. COSTANTIN L. L., FRANZINI-ARMSTRONG C., PODOLSKY R. J. LOCALIZATION OF CALCIUM-ACCUMULATING STRUCTURES IN STRIATED MUSCLE FIBERS. Science. 1965 Jan 8;147(3654):158–160. doi: 10.1126/science.147.3654.158. [DOI] [PubMed] [Google Scholar]
  8. DE DUVE C., WATTIAUX R., BAUDHUIN P. Distribution of enzymes between subcellular fractions in animal tissues. Adv Enzymol Relat Subj Biochem. 1962;24:291–358. doi: 10.1002/9780470124888.ch6. [DOI] [PubMed] [Google Scholar]
  9. EBASHI S. Calcium binding activity of vesicular relaxing factor. J Chir (Paris) 1961 Sep;82:236–244. doi: 10.1093/oxfordjournals.jbchem.a127439. [DOI] [PubMed] [Google Scholar]
  10. FUCHS F., BRIGGS F. N. The nature of the muscle-relaxing factor. II. Some physicochemical properties. J Gen Physiol. 1963 May;46:893–904. doi: 10.1085/jgp.46.5.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. GAMBLE J. L., Jr, LEHNINGER A. L. Activity of respiratory enzymes and adenosine-triphosphatase in fragments of mitochondria. J Biol Chem. 1956 Dec;223(2):921–933. [PubMed] [Google Scholar]
  12. GORNALL A. G., BARDAWILL C. J., DAVID M. M. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949 Feb;177(2):751–766. [PubMed] [Google Scholar]
  13. HASSELBACH W., MAKINOSE M. [The calcium pump of the "relaxing granules" of muscle and its dependence on ATP-splitting]. Biochem Z. 1961;333:518–528. [PubMed] [Google Scholar]
  14. HESS H. H., POPE A. Ultramicrospectrophotometric determination of cytochrome oxidase for quantitative histochemistry. J Biol Chem. 1953 Sep;204(1):295–306. [PubMed] [Google Scholar]
  15. HOGEBOOM G. H., SCHNEIDER W. C., STRIEBICH M. J. Localization and integration of cellular function. Cancer Res. 1953 Sep;13(9):617–632. [PubMed] [Google Scholar]
  16. KUMAGAI H., EBASHI S., TAKEDA F. Essential relaxing factor in muscle other than myokinase and creatine phosphokinase. Nature. 1955 Jul 23;176(4473):166–166. doi: 10.1038/176166a0. [DOI] [PubMed] [Google Scholar]
  17. LORAND L. RELAXING PARTICLES OF SKELETAL MUSCLE. Fed Proc. 1964 Sep-Oct;23:905–908. [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. MARSH B. B. The effects of adenosine triphosphate on the fibre volume of a muscle homogenate. Biochim Biophys Acta. 1952 Sep;9(3):247–260. doi: 10.1016/0006-3002(52)90159-5. [DOI] [PubMed] [Google Scholar]
  20. MARTONOSI A., FERETOS R. SARCOPLASMIC RETICULUM. I. THE UPTAKE OF CA++ BY SARCOPLASMIC RETICULUM FRAGMENTS. J Biol Chem. 1964 Feb;239:648–658. [PubMed] [Google Scholar]
  21. MOLNAR J., LORAND L. Transphosphorylating enzymes and muscle relaxation. Nature. 1959 Apr 11;183(4667):1032–1034. doi: 10.1038/1831032a0. [DOI] [PubMed] [Google Scholar]
  22. MOLNAR J., LORNAD L. A phosphoryl group acceptor attached to the microsomal fraction of muscle. Arch Biochem Biophys. 1962 Sep;98:356–363. doi: 10.1016/0003-9861(62)90198-4. [DOI] [PubMed] [Google Scholar]
  23. MUSCATELLO U., ANDERSSON-CEDERGREN E., AZZONE G. F., von der DECKEN The sarcotubular system of frog skeletal muscle. A morphological and biochemical study. J Biophys Biochem Cytol. 1961 Aug;10(4):201–218. doi: 10.1083/jcb.10.4.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. NAGAI T., MAKINOSE M., HASSEL BACH W. [The physiological relaxing factor produced by the muscle granules]. Biochim Biophys Acta. 1960 Sep 23;43:223–238. doi: 10.1016/0006-3002(60)90433-9. [DOI] [PubMed] [Google Scholar]
  25. NAGAI T., UCHIDA K., YASUDA M. Some further properties of the muscle relaxing-factor system and the separation of the effective substance. Biochim Biophys Acta. 1962 Jan 29;56:205–215. doi: 10.1016/0006-3002(62)90557-7. [DOI] [PubMed] [Google Scholar]
  26. PORTZEHL H. Die Bindung des Erschlaffungsfaktors von Marsh an die Muskelgrana. Biochim Biophys Acta. 1957 Nov;26(2):373–377. doi: 10.1016/0006-3002(57)90019-7. [DOI] [PubMed] [Google Scholar]
  27. SCHUEL H., ANDERSON N. G. STUDIES ON ISOLATED CELL COMPONENTS. XVI. THE DISTRIBUTION OF ACID PHENYL PHOSPHATASE ACTIVITIES IN RAT LIVER BREI FRACTIONATED IN THE ZONAL ULTRACENTRIFUGE. J Cell Biol. 1964 Jun;21:309–323. doi: 10.1083/jcb.21.3.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. SCHUEL H., TIPTON S. R., ANDERSON N. G. STUDIES ON ISOLATED CELL COMPONENTS. XVII. THE DISTRIBUTION OF CYTOCHROME OXIDASE ACTIVITY IN RAT LIVER BREI FRACTIONATED IN THE ZONAL ULTRACENTRIFUGE. J Cell Biol. 1964 Aug;22:317–326. doi: 10.1083/jcb.22.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. SKEGGS L. T., Jr An automatic method for colorimetric analysis. Am J Clin Pathol. 1957 Sep;28(3):311–322. doi: 10.1093/ajcp/28.3_ts.311. [DOI] [PubMed] [Google Scholar]
  30. SMITH L. Spectrophotometric assay of cytochrome c oxidase. Methods Biochem Anal. 1955;2:427–434. doi: 10.1002/9780470110188.ch13. [DOI] [PubMed] [Google Scholar]
  31. STAM A. C., Jr, HONIG C. R. The preparation and characterization of a cardiac relaxing substance. Biochim Biophys Acta. 1962 Jul 2;60:259–264. doi: 10.1016/0006-3002(62)90401-8. [DOI] [PubMed] [Google Scholar]
  32. WEBER A., HERZ R., REISS I. On the mechanism of the relaxing effect of fragmented sarcoplasmic reticulum. J Gen Physiol. 1963 Mar;46:679–702. doi: 10.1085/jgp.46.4.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. WEBER A., HERZ R., REISS I. THE REGULATION OF MYOFIBRILLAR ACTIVITY BY CALCIUM. Proc R Soc Lond B Biol Sci. 1964 Oct 27;160:489–501. doi: 10.1098/rspb.1964.0063. [DOI] [PubMed] [Google Scholar]
  34. WEBER A., HERZ R. The binding of calcium to actomyosin systems in relation to their biological activity. J Biol Chem. 1963 Feb;238:599–605. [PubMed] [Google Scholar]
  35. WEBER A., WINICUR S. The role of calcium in the superprecipitation of actomyosin. J Biol Chem. 1961 Dec;236:3198–3202. [PubMed] [Google Scholar]

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