• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
J Cell Biol. Sep 1, 1970; 46(3): 576–591.
PMCID: PMC2107885

A NEW METHOD FOR THE PREPARATION OF RAT LIVER LYSOSOMES

Separation of Cell Organelles of Rat Liver by Carrier-Free Continuous Electrophoresis

Abstract

A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and β-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to β-glucuronidase. The ratio of β-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5–8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.

Full Text

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

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • DE DUVE C, PRESSMAN BC, 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. [PMC free article] [PubMed]
  • SAWANT PL, SHIBKO, KUMTA US, TAPPEL AL. ISOLATION OF RAT-LIVER LYSOSOMES AND THEIR GENERAL PROPERTIES. Biochim Biophys Acta. 1964 Apr 6;85:82–92. [PubMed]
  • BEAUFAY H, BENDALL DS, 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. [PMC free article] [PubMed]
  • BEAUFAY H, BENDALL DS, BAUDHUN P, WATTIAUX R, DE DUVE C. Tissue fractionation studies. 13. Analysis of mitochondrial fractions from rat liver by density-gradient centrifuging. Biochem J. 1959 Dec;73:628–637. [PMC free article] [PubMed]
  • Baudhuin P, Beaufay H, De Duve C. Combined biochemical and morphological study of particulate fractions from rat liver. Analysis of preparations enriched in lysosomes or in particles containing urate oxidase, D-amino acid oxidase, and catalase. J Cell Biol. 1965 Jul;26(1):219–243. [PMC free article] [PubMed]
  • Beaufay H, Jacques P, Baudhuin P, Sellinger OZ, Berthet J, De Duve C. Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients. Biochem J. 1964 Jul;92(1):184–205. [PMC free article] [PubMed]
  • Ragab H, Beck C, Dillard C, Tappel AL. Preparation of rat liver lysosomes. Biochim Biophys Acta. 1967 Nov 28;148(2):501–505. [PubMed]
  • SAWANT PL, DESAI ID, TAPPEL AL. FACTORS AFFECTING THE LYSOSOMAL MEMBRANE AND AVAILABILITY OF ENZYMES. Arch Biochem Biophys. 1964 May;105:247–253. [PubMed]
  • DAVENPORT JB. SEPARATION OF SUBCELLULAR PARTICLES BY DENSITY-GRADIENT ELECTROPHORESIS. Biochim Biophys Acta. 1964 Jul 29;88:177–190. [PubMed]
  • Plummer DT. The electrophoretic behaviour of mitochondria from kidney and liver of rats. Biochem J. 1965 Sep;96(3):729–732. [PMC free article] [PubMed]
  • HANNIG K. EINE NEUENTWICKLUNG DER TRAEGERFREIEN KONTINUIERLICHEN ELEKTROPHORESE. ZUR TRENNUNG HOCHMOLEKULARER UND GROBDISPERSER TEILCHEN. Hoppe Seylers Z Physiol Chem. 1964;338:211–227. [PubMed]
  • Hannig K, Krüsmann WF. Die Anwendung der trägerfreien kontinuierlichen Elektrophorese zur Auftrennung der weissen Blutzellen aus Humanblut. Hoppe Seylers Z Physiol Chem. 1968 Feb;349(2):161–170. [PubMed]
  • HANNIG K, WRBA H. ISOLIERUNG VON VITALEN TUMORZELLEN DURCH TRAEGERFREI ELEKTROPHORESE. Z Naturforsch B. 1964 Sep;19:860–860. [PubMed]
  • Schnaitman C, Erwin VG, Greenawalt JW. The submitochondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria. J Cell Biol. 1967 Mar;32(3):719–735. [PMC free article] [PubMed]
  • COOPERSTEIN SJ, LAZAROW A. A microspectrophotometric method for the determination of cytochrome oxidase. J Biol Chem. 1951 Apr;189(2):665–670. [PubMed]
  • HORIE S, MORRISON M. Cytochrome c oxidase components. I. Purification and properties. J Biol Chem. 1963 May;238:1855–1860. [PubMed]
  • KUN E, ACHMATOWICZ B. STUDIES WITH SPECIFIC ENZYME INHIBITORS. IX. SELECTIVE INHIBITORY EFFECTS OF SUBSTRATE ANALOGUES ON THE CATALYTIC ACTIVITY OF CRYSTALLINE GLUTAMATE DEHYDROGENASE. J Biol Chem. 1965 Jun;240:2619–2627. [PubMed]
  • BEERS RF, Jr, SIZER IW. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952 Mar;195(1):133–140. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Heidrich HG, Stahn R, Hannig K. The surface charge of rat liver mitochondria and their membranes. Clarification of some controversies concerning mitochondrial structure. J Cell Biol. 1970 Jul;46(1):137–150. [PMC free article] [PubMed]
  • WATTIAUX R, WATTIAUX-DECONINCK S, RUTGEERTS MJ, TULKENS P. INFLUENCE OF THE INJECTION OF A SUCROSE SOLUTION ON THE PROPERTIES OF RAT-LIVER LYSOSOMES. Nature. 1964 Aug 15;203:757–758. [PubMed]
  • Vaes G, Jacques P. Studies on bone enzymes. The assay of acid hydrolases and other enzymes in bone tissue. Biochem J. 1965 Nov;97(2):380–388. [PMC free article] [PubMed]
  • Rahman YE, Howe JF, Nance SL, Thomson JF. Studies on rat liver ribonucleases. II. Zonal centrifugation of acid ribonuclease; implications for the heterogeneity of lysosomes. Biochim Biophys Acta. 1967;146(2):484–492. [PubMed]
  • Sellinger OZ, Hiatt RA. Cerebral lysosomes. IV. The regional and intracellular distribution of arylsulfatase and evidence for two populations of lysosomes in rat brain. Brain Res. 1968 Feb;7(2):191–200. [PubMed]
  • Treadwell PE, Santos-Buch CA. Disruption of Kupffer cells during systemic anaphylaxis in the mouse. I. Properties and distribution of heat- and formaldehyde-stable liver acid p-nitrophenylphosphatase. Am J Pathol. 1967 Oct;51(4):483–503. [PMC free article] [PubMed]
  • Brown DH. Separation of microchonria, peroxisomes and lysosomes by zonal centrifugation in a Ficoll gradient. Biochim Biophys Acta. 1968 Jul 16;162(1):152–153. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...