A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane.

The Biochemical journal ·Vol. 154 ·No. 1 ·1976-01-15 ·Pages 11-21

Luzio JP, Newby AC, Hales CN

Abstract

1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5'-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 mum-adrenaline.

MeSH Terms

Adenylyl Cyclases/analysis Adipose Tissue/analysis Adsorption Animals Antibodies Cell Fractionation/methods Cell Membrane/enzymology Epididymis/analysis Immunoglobulin G Immunologic Techniques Male Nucleotidases/analysis Rats

Chemicals

Antibodies Immunoglobulin G Nucleotidases Adenylyl Cyclases

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Luzio J P

Newby A C

Hales C N

References (27)

27 references, click to expand

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Article Info

Journal

The Biochemical journal

Abbr.

Biochem J

ISSN

0264-6021

DOI

10.1042/bj1540011

Published

1976-01-15

Pages

11-21

Language

English

Region

England

NLM ID

2984726R

PMCID

PMC1172670

Subset

External Links

PubMed source DOI: 10.1042/bj1540011 PMC Full Text View journal details

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A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane.

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