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. 1976 Jan;33(1):15–26. doi: 10.1038/bjc.1976.3

Plasma membrane associated enzymes of mammary tumours as the biochemical indicators of metastasizing capacity. Analyses of enriched plasma membrane preparations.

S K Chatterjee, U Kim, K Bielat
PMCID: PMC2024917  PMID: 175819

Abstract

Plasma membranes from 6 spontaneously metastasizing and 4 non-metastasizing rat mammary carcinomata were isolated by discontinuous sucrose density gradient centrifugation of microsomal pellets. The starting microsomal fraction contained 40-50% plasma membranes as determined by the levels of 5'-nucleotidase activity, with a negligible amount of nuclear (1%), mitochondrial (5%) and lysomal (7%) contamination. Five distinct fractions (F1-F5) were banded at densities 1 X 09, 1 X 13, 1 X 15, 1 X 17 and 1 X 21 at 25 degrees C, in addition to a pellet (F6) obtained by centrifuging at 76,000 g for 17 h. The fractions F1 through F5, all contained various concentrations of membranous structures, while the pellet (F6) contained only amorphous materials as evidenced by electron microscopy. The F3 fraction at the gradient 1 X 15 had the highest specific as well as total activity of the plasma membrane marker enzyme, with aggregates of the least contaminated plasma membranes in vesicular forms. This fraction also had the lowest specific activity for glucose-6-phosphatase (smooth ER marker) and for beta-D-glucuronidase (lysomal marker), and therefore was considered to be the "cleanest" plasma membrane fraction. When the activity of 4 additional plasma membrane marker enzymes, i.e., alkaline phosphatase, phosphodiesterase I, nucleotide pyrophosphatase and alkaline ribonuclease was determined in the same F3 fraction, their levels were significantly lower in every metastasizing tumour than in the non-metastasizing ones, with the enzyme activity decreasing in direct proportion to the metastasizing capacity. On the other hand, the marker enzymes were high in all non-metastasizing tumours, with the activity seemingly increasing with the immunogenicity of tumour cells. There was no significant difference between the 2 groups of mammary tumours in the levels of sialic acid, hexosamine, phospholipid or cholesterol in the plasma membranes. Thus, the level of plasma membrane marker enzymes is considered an accurate indicator for metastasizing capacity in the rat mammary tumour system.

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Selected References

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

  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. Beck C., Tappel A. L. An automated multiple enzyme monitor for column chromatography. Anal Biochem. 1967 Nov;21(2):208–218. doi: 10.1016/0003-2697(67)90182-0. [DOI] [PubMed] [Google Scholar]
  3. Bosmann H. B., Hagopian A., Eylar E. H. Cellular membranes: the isolation and characterization of the plasma and smooth membranes of HeLa cells. Arch Biochem Biophys. 1968 Oct;128(1):51–69. doi: 10.1016/0003-9861(68)90008-8. [DOI] [PubMed] [Google Scholar]
  4. DePierre J. W., Karnovsky M. L. Plasma membranes of mammalian cells: a review of methods for their characterization and isolation. J Cell Biol. 1973 Feb;56(2):275–303. doi: 10.1083/jcb.56.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Demus H. Subcellular fractionation of human lymphocytes. Isolation of two plasma membrane fractions and comparison of the protein components of the various lymphocytic organelles. Biochim Biophys Acta. 1973 Jan 2;291(1):93–106. doi: 10.1016/0005-2736(73)90064-3. [DOI] [PubMed] [Google Scholar]
  6. GAREN A., LEVINTHAL C. A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta. 1960 Mar 11;38:470–483. doi: 10.1016/0006-3002(60)91282-8. [DOI] [PubMed] [Google Scholar]
  7. Gatt R., Berman E. R. A rapid procedure for the estimation of amino sugars on a micro scale. Anal Biochem. 1966 Apr;15(1):167–171. doi: 10.1016/0003-2697(66)90262-4. [DOI] [PubMed] [Google Scholar]
  8. Kim U., Baumler A., Carruthers C., Bielat K. Immunological escape mechanism in spontaneously metastasizing mammary tumors. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1012–1016. doi: 10.1073/pnas.72.3.1012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. MASTER R. W. POSSIBLE SYNTHESIS OF POLYRIBONUCLEOTIDES OF KNOWN BASE-TRIPLET SEQUENCES. Nature. 1965 Apr 3;206:93–93. doi: 10.1038/206093b0. [DOI] [PubMed] [Google Scholar]
  11. Michell R. H., Hawthorne J. N. The site of diphosphoinositide synthesis in rat liver. Biochem Biophys Res Commun. 1965 Nov 22;21(4):333–338. doi: 10.1016/0006-291x(65)90198-1. [DOI] [PubMed] [Google Scholar]
  12. PALADE G. E. A study of fixation for electron microscopy. J Exp Med. 1952 Mar;95(3):285–298. doi: 10.1084/jem.95.3.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. PORTEOUS J. W., CLARK B. THE ISOLATION AND CHARACTERIZATION OF SUBCELLULAR COMPONENTS OF THE EPITHELIAL CELLS OF RABBIT SMALL INTESTINE. Biochem J. 1965 Jul;96:159–171. doi: 10.1042/bj0960159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Prospero T. D., Burge M. L., Norris K. A., Hinton R. H., Reid E. Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes. Biochem J. 1973 Mar;132(3):449–458. doi: 10.1042/bj1320449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. RAZZELL W. E. Tissue and intracellular distribution of two phosphodiesterases. J Biol Chem. 1961 Nov;236:3028–3030. [PubMed] [Google Scholar]
  17. ROE J. H. The determination of sugar in blood and spinal fluid with anthrone reagent. J Biol Chem. 1955 Jan;212(1):335–343. [PubMed] [Google Scholar]
  18. SCHLISELFELD L. H., VANEYS J., TOUSTER O. THE PURIFICATION AND PROPERTIES OF A NUCLEOTIDE PYROPHOSPHATASE OF RAT LIVER NUCLEI. J Biol Chem. 1965 Feb;240:811–818. [PubMed] [Google Scholar]
  19. SWANSON M. A. Phosphatases of liver. I. Glucose-6-phosphatase. J Biol Chem. 1950 Jun;184(2):647–659. [PubMed] [Google Scholar]
  20. Spurr A. R. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res. 1969 Jan;26(1):31–43. doi: 10.1016/s0022-5320(69)90033-1. [DOI] [PubMed] [Google Scholar]
  21. Touster O., Aronson N. N., Jr, Dulaney J. T., Hendrickson H. Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase I as marker enzymes. J Cell Biol. 1970 Dec;47(3):604–618. doi: 10.1083/jcb.47.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]

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