Rapid isolation of plasma membranes in high yield from cultured fibroblasts.

The Biochemical journal ·Vol. 168 ·No. 2 ·1977-11-15 ·Pages 187-94

Thom D, Powell AJ, Lloyd CW, Rees DA

Abstract

1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2--lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.

MeSH Terms

Borohydrides Cell Fractionation/methods Cell Line Cell Membrane/analysis,enzymology,ultrastructure Centrifugation, Density Gradient Fibroblasts/ultrastructure Periodic Acid Tritium

Chemicals

Borohydrides Tritium Periodic Acid

Authors & Affiliations

4 authors, click to expand affiliations / ORCID

Thom D

Powell A J

Lloyd C W

Rees D A

References (27)

27 references, click to expand

The isolation and characterization of the plasma membrane of cultured chinese hamster ovary cells.
Can J Biochem. 1975 Aug;53(8):914-20 PMID: 126793
A new method for the direct determination of serum cholesterol.
J Lab Clin Med. 1953 Mar;41(3):486-92 PMID: 13035283
Studies on sialic acids.
Acta Soc Med Ups. 1956 Jun 30;61(1-2):1-25 PMID: 13339502
A sialomucopeptide liberated by trypsin from the human erythrocyte.
Nature. 1960 Dec 17;188:1011-2 PMID: 13695244
ISOLATION OF A MUCOPEPTIDE FROM THE SURFACE OF EHRLICH ASCITES TUMOUR CELLS.
Nature. 1964 Oct 3;204:53-4 PMID: 14240112
THE ISOLATION AND CHARACTERIZATION OF SUBCELLULAR COMPONENTS OF THE EPITHELIAL CELLS OF RABBIT SMALL INTESTINE.
Biochem J. 1965 Jul;96:159-71 PMID: 14343125
Protein measurement with the Folin phenol reagent.
J Biol Chem. 1951 Nov;193(1):265-75 PMID: 14907713
Membrane receptors as general markers for plasma membrane isolation procedures. The use of 125-I-labeled wheat germ agglutinin, insulin, and cholera toxin.
J Biol Chem. 1975 Jan 25;250(2):488-500 PMID: 163229
Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots.
Lipids. 1966 Jan;1(1):85-6 PMID: 17805690
Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells.
Biochim Biophys Acta. 1976 Jun 17;436(2):335-52 PMID: 179596
The isolation and characterization of plasma membranes from normal and leukaemic cells of mice.
Biochim Biophys Acta. 1973 Sep 27;323(1):55-68 PMID: 4148030
Isolation of plasma membrane fragments from BHK21 cells.
Acta Pathol Microbiol Scand B Microbiol Immunol. 1970;78(2):176-82 PMID: 4249781
The relation of structural and catalytic protein in the mitochondrial electron transfer chain.
Arch Biochem Biophys. 1966 Jul;115(1):215-25 PMID: 4290771
The isolation and characterization of the plasma membrane from chick embryo fibroblasts.
Biochim Biophys Acta. 1970;196(2):125-40 PMID: 4313177
Studies on the chemical and enzymatic modification of glycoproteins. A general method for the tritiation of sialic acid-containing glycoproteins.
J Biol Chem. 1971 Mar 25;246(6):1889-94 PMID: 4323238
Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane.
Biochemistry. 1971 Jun 22;10(13):2606-17 PMID: 4326772
The lipids of the plasma membranes and endoplasmic reticulum from cultured baby hamster kidney cells (BHK21).
Biochim Biophys Acta. 1972 Jan 17;255(1):66-78 PMID: 4334687
Membrane site modified on induction of the transformation of lymphocytes by periodate.
Proc Natl Acad Sci U S A. 1972 Nov;69(11):3207-10 PMID: 4343958
Modification of sialyl residues of sialoglycoprotein(s) of the human erythrocyte surface.
J Biol Chem. 1973 Dec 10;248(23):8247-53 PMID: 4356622
On the mechanism of the increased aggregation by neuraminidase of 16C malignant rat dermal fibroblasts in vitro.
J Cell Sci. 1974 Aug;15(3):575-90 PMID: 4426924
A low-viscosity epoxy resin embedding medium for electron microscopy.
J Ultrastruct Res. 1969 Jan;26(1):31-43 PMID: 4887011
A new method for the determination of serum nucleotidase.
Z Klin Chem Klin Biochem. 1968 Sep;6(5):441-6 PMID: 4972843
Isolation and characterization of lymphocyte plasma membranes.
Biochim Biophys Acta. 1972 May 9;266(2):494-504 PMID: 5038272
Structural studies on human erythrocyte glycoproteins. Alkali-labile oligosaccharides.
J Biol Chem. 1969 Nov 10;244(21):5943-6 PMID: 5350948
Isolation of meth A cell surface membranes possessing tumor-specific transplantation antigen activity.
Cancer Res. 1970 Dec;30(12):2832-7 PMID: 5494569
Studies on glycopeptides released by trypsin from intact human erythrocytes.
Biochemistry. 1967 Jul;6(7):2195-202 PMID: 6049454
Isolation of membranes from normal and thrombin-treated gel-filtered platelets using a lectin marker.
Biochim Biophys Acta. 1976 Apr 5;426(4):688-96 PMID: 816377

Article Info

Journal

The Biochemical journal

Abbr.

Biochem J

ISSN

0264-6021

DOI

10.1042/bj1680187

Published

1977-11-15

Pages

187-94

Language

English

Region

England

NLM ID

2984726R

PMCID

PMC1183751

Subset

External Links

PubMed source DOI: 10.1042/bj1680187 PMC Full Text View journal details

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Rapid isolation of plasma membranes in high yield from cultured fibroblasts.

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