Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1965 Feb;94(2):494–501. doi: 10.1042/bj0940494

Protein synthesis in mitochondria. 4. Preparation and properties of mitochondria from Krebs II mouse ascites-tumour cells*

K B Freeman 1
PMCID: PMC1206534  PMID: 14348211

Abstract

1. Methods of disrupting Krebs II mouse ascites-tumour cells have been studied. After washing the cells free of ions with sucrose solutions, rapid disruption was obtained in sucrose by use of an Ultra-Turrax disintegrator or a Dounce homogenizer. 2. Disruption of cells after osmotic shock led to the loss of proteins, especially cytochrome c, from the mitochondria. Such losses did not occur when cells were disrupted by shear in 0·3 m-sucrose. 3. The distribution of protein, RNA, DNA, malate dehydrogenase, cytochrome c, cytochrome oxidase and succin-oxidase was measured in the various cell fractions after separation by differential centrifuging. 4. The mitochondrial fraction sedimented at 9500g was further fractionated by equilibrium sedimentation in a sucrose gradient. The distribution of protein and enzyme activity in the gradient indicated that the 9500g pellet contains other material besides mitochondria. 5. Krebs-cell mitochondria contain up to five times as much RNA as do liver mitochondria. 6. After purification by equilibrium centrifugation Krebs-cell mitochondria still contain traces of DNA.

Full text

PDF
501

Selected References

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

  1. ANDERSON N. G., WILBUR K. M. Studies on isolated cell components. IV. The effect of various solutions on the isolated rat liver nucleus. J Gen Physiol. 1952 May;35(5):781–796. doi: 10.1085/jgp.35.5.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BEAUFAY H., BENDALL D. S., BAUDHUIN P., DE DUVE C. Tissue fractionation studies. 12. Intracellular distribution of some dehydrogenases, alkaline deoxyribonuclease and iron in rat-liver tissue. Biochem J. 1959 Dec;73:623–628. doi: 10.1042/bj0730623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. CHANCE B., HESS B. Metabolic control mechanisms. I. Electron transfer in the mammalian cell. J Biol Chem. 1959 Sep;234:2404–2412. [PubMed] [Google Scholar]
  5. CHARLWOOD P. A. Applications of radioactively labeled marker proteins in density gradient ultracentrifugation. Anal Biochem. 1963 Mar;5:226–245. doi: 10.1016/0003-2697(63)90120-9. [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. DE DUVE C., PRESSMAN B. C., GIANETTO R., WATTIAUX R., APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. doi: 10.1042/bj0600604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. HAYANO K., TSUBONE T., AZUMA I., YAMAMURA Y. The prevention of the orotic acid fatty liver by the administration of 5-aminoimidazolecarboxamide. J Biochem. 1962 Nov;52:379–380. doi: 10.1093/oxfordjournals.jbchem.a127632. [DOI] [PubMed] [Google Scholar]
  9. KERR I. M., MARTIN E. M., HAMILTON M. G., WORK T. S. The initiation of virus protein synthesis in Krebs ascites tumor cells infected with EMC virus. Cold Spring Harb Symp Quant Biol. 1962;27:259–269. doi: 10.1101/sqb.1962.027.001.025. [DOI] [PubMed] [Google Scholar]
  10. LITTLEFIELD J. W., KELLER E. B., GROSS J., ZAMECNIK P. C. Studies on cytoplasmic ribonucleoprotein particles from the liver of the rat. J Biol Chem. 1955 Nov;217(1):111–123. [PubMed] [Google Scholar]
  11. 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]
  12. MARTIN E. M., MALEC J., COOTE J. L., WORK T. S. Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 3. Methods for the disruption of Krebs II mouse-ascites-tumour cells. Biochem J. 1961 Sep;80:606–611. doi: 10.1042/bj0800606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. MARTIN E. M., MALEC J., SVED S., WORK T. S. Studies on protein and nucleic acid metabolism in virus-infected mammalian cells. 1. Encephalomyocarditis virus in Krebs II mouse-ascites-tumour cells. Biochem J. 1961 Sep;80:585–597. doi: 10.1042/bj0800585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. PETERS T., Jr The biosynthesis of rat serum albumin. II. Intracellular phenomena in the secretion of newly formed albumin. J Biol Chem. 1962 Apr;237:1186–1189. [PubMed] [Google Scholar]
  15. ROODYN D. B. Protein synthesis in mitochondria. 3. The controlled disruption and subfractionation of mitochondria labelled in vitro with radioactive valine. Biochem J. 1962 Oct;85:177–189. doi: 10.1042/bj0850177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. ROODYN D. B., REIS P. J., WORK T. S. Protein synthesis in mitochondria. Requirements for the incorporation of radioactive amino acids into mitochondrial protein. Biochem J. 1961 Jul;80:9–21. doi: 10.1042/bj0800009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. ROODYN D. B., SUTTIE J. W., WORK T. S. Protein synthesis in mitochondria. 2. Rate of incorporation in vitro of radioactive amino acids into soluble proteins in the mitochondrial fraction, including catalase, malic dehydrogenase and cytochrome c. Biochem J. 1962 Apr;83:29–40. doi: 10.1042/bj0830029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. SAUER L. A., MARTIN A. P., STOTZ E. Cytochemical fractionation of the Lettre-Ehrlich ascites tumor. Cancer Res. 1960 Feb;20:251–256. [PubMed] [Google Scholar]
  19. SAUER L. A., MARTIN A. P., STOTZ E. Oxidative phosphorylation in ascites tumor mitochondria. Cancer Res. 1962 Jun;22:632–636. [PubMed] [Google Scholar]
  20. SCHWEET R., BISHOP J., MORRIS A. Protein synthesis with particular reference to hemoglobin synthesis--a review. Lab Invest. 1961 Nov-Dec;10:992–1011. [PubMed] [Google Scholar]
  21. SIEKEVITZ P., PALADE G. E. A cytochemical study on the pancreas of the guinea pig. 5. In vivo incorporation of leucine-1-C14 into the chymotrypsinogen of various cell fractions. J Biophys Biochem Cytol. 1960 Jul;7:619–630. doi: 10.1083/jcb.7.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. SIEKEVITZ P., WATSON M. L. Cytochemical studies of mitochondria. II. Enzymes associated with a mitochondrial membrane fraction. J Biophys Biochem Cytol. 1956 Nov 25;2(6):653–669. doi: 10.1083/jcb.2.6.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. SIMKIN J. L., WORK T. S. Protein synthesis in guinea-pig liver; incorporation of radioactive amino acids into proteins of the microsome fraction in vivo. Biochem J. 1957 Feb;65(2):307–315. doi: 10.1042/bj0650307. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES