Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1965 Aug 1;26(2):641–656. doi: 10.1083/jcb.26.2.641

DENSITY GRADIENT SEPARATION OF SARCOTUBULAR VESICLES AND OTHER PARTICULATE CONSTITUENTS OF RABBIT MUSCLE

K Seraydarian 1, W F H M Mommaerts 1
PMCID: PMC2106741  PMID: 4222283

Abstract

Separation of particulate matter in rabbit muscle extracts by differential centrifugation leads, in first approximation, to the isolation of fraction I (15,000 to 41,000 g) and fraction II (41,000 to 150,000 g). The former consists mainly of sarcotubular material, actively transporting calcium ions, and displaying relaxation factor activity. The latter is heterogeneous, shows little calcium accumulation, and contains factors both inhibiting and activating myofibrillar ATPase. Fraction I is resolved by density gradient centrifugation into 2 main subfractions. The lighter one represents sarcotubular material in the best state of preservation, with biochemical activities stable for weeks in the cold. The heavier one may consist of the same material in a less well preserved form. Upon aging, it develops an activating activity toward myofibrillar ATPase, when the relaxing effect has declined. Fraction II is resolved by density gradient centrifugation into 3 or more fractions, with some variability. Relaxing activity in terms of inhibition of myofibrillar ATPase predominates among the lighter subfractions, increase of ATPase among the heavier. The intrinsic ATPase of fraction II is activated by calcium ions, but there is little or no bulk accumulation of calcium oxalate. Nevertheless, its limited calcium uptake seems to suffice to explain its relaxing activity. The particulate material contains mucopolysaccharide and lipid. Most of the lipid in fraction I is phospholipid; in fraction II this is less than half, if calculated as lecithin. The unfractionated material contains an adenylcyclase. There is no acetylcholine esterase.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. BOAS N. F. Method for the determination of hexosamines in tissues. J Biol Chem. 1953 Oct;204(2):553–563. [PubMed] [Google Scholar]
  3. 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]
  4. CARSTEN M. E., MOMMAERTS W. F. THE ACCUMULATION OF CALCIUM IONS BY SARCOTUBULAR VESICLES. J Gen Physiol. 1964 Nov;48:183–197. doi: 10.1085/jgp.48.2.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DAVIES R. E. A MOLECULAR THEORY OF MUSCLE CONTRACTION: CALCIUM-DEPENDENT CONTRACTIONS WITH HYDROGEN BOND FORMATION PLUS ATP-DEPENDENT EXTENSIONS OF PART OF THE MYOSIN-ACTIN CROSS-BRIDGES. Nature. 1963 Sep 14;199:1068–1074. doi: 10.1038/1991068a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. LORAND L. RELAXING PARTICLES OF SKELETAL MUSCLE. Fed Proc. 1964 Sep-Oct;23:905–908. [PubMed] [Google Scholar]
  8. 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]
  9. MARINETTI G. V., KOCHEN J., ERBLAND J., STOTZ E. The lipide composition of a purified cytochrome preparation of pig heart. J Biol Chem. 1957 Dec;229(2):1027–1035. [PubMed] [Google Scholar]
  10. 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]
  11. MARTONOSI A. ROLE OF PHOSPHOLIPIDS IN ATPASE ACTIVITY AND CA TRANSPORT OF FRAGMENTED SARCOPLASMIC RETICULUM. Fed Proc. 1964 Sep-Oct;23:913–921. [PubMed] [Google Scholar]
  12. MOMMAERTS W. F., KHAIRALLAH P. A., DICKENS M. F. Acetylcholinesterase in the conductive tissue of the heart. Circ Res. 1953 Sep;1(5):460–465. doi: 10.1161/01.res.1.5.460. [DOI] [PubMed] [Google Scholar]
  13. MOMMAERTS W. F., SERAYDARIAN K., UCHIDA K. ON THE RELAXING SUBSTANCE OF MUSCLE. Biochem Biophys Res Commun. 1963 Sep 10;13:58–60. doi: 10.1016/0006-291x(63)90162-1. [DOI] [PubMed] [Google Scholar]
  14. MOMMAERTS W. F. THE MUSCLE CELL AND ITS FUNCTIONAL ARCHITECTURE. Am J Med. 1963 Nov;35:606–610. doi: 10.1016/0002-9343(63)90132-3. [DOI] [PubMed] [Google Scholar]
  15. PERRY S. V. The bound nucleotide of the isolated myofibril. Biochem J. 1952 Jul;51(4):495–499. doi: 10.1042/bj0510495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. RABINOWITZ M., DESALLES L., MEISLER J., LORAND L. DISTRIBUTION OF ADENYL-CYCLASE ACTIVITY IN RABBIT SKELETAL-MUSCLE FRACTIONS. Biochim Biophys Acta. 1965 Jan 4;97:29–36. doi: 10.1016/0304-4165(65)90266-7. [DOI] [PubMed] [Google Scholar]
  17. RALL T. W., SUTHERLAND E. W. Formation of a cyclic adenine ribonucleotide by tissue particles. J Biol Chem. 1958 Jun;232(2):1065–1076. [PubMed] [Google Scholar]
  18. SPIRO R. G. GLYCOPROTEINS: STRUCTURE, METABOLISM AND BIOLOGY. N Engl J Med. 1963 Sep 19;269:616–CONCL. doi: 10.1056/NEJM196309192691206. [DOI] [PubMed] [Google Scholar]
  19. SUTHERLAND E. W., RALL T. W. Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles. J Biol Chem. 1958 Jun;232(2):1077–1091. [PubMed] [Google Scholar]
  20. SUTHERLAND E. W., RALL T. W., MENON T. Adenyl cylase. I. Distribution, preparation, and properties. J Biol Chem. 1962 Apr;237:1220–1227. [PubMed] [Google Scholar]
  21. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
  22. 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]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES