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Protein synthesis in lactating guinea-pig mammary tissue perfused in vitro. I. Radiolabelling of membrane and secretory proteins.

Abstract：

:A method for the in vitro perfusion of isolated guinea-pig mammary tissue is described that allows the radiolabelling of secretory and membrane proteins. Glands were depleted of methionine, labelled with [35S]methionine for 5 min and perfused with medium containing an excess of unlabelled methionine for varying times. The structural integrity of the alveoli in the perfused glands appeared well maintained. Epithelial polarity was preserved and junctional complexes were evident. About 20% of the methionine provided in the medium was extracted by glands of 10 g wet weight under the labelling conditions employed. With chase periods from 15 to 40 min, 50-70% of the methionine was incorporated into trichloroacetic-acid (TCA)-precipitable material. The principal radiolabelled proteins recovered from the tissue fractions had Mrs and isoelectric points similar to the major secretory proteins (i.e. caseins and alpha-lactalbumin) of guinea-pig milk. Autoradiography of tissue sections at the resolution of the light microscope showed that secretory proteins were transported from sites of synthesis within secretory cells to the alveolar lumina after 45 min. These highly labelled secretory proteins could be almost completely removed from microsomal fractions by treatment with sodium carbonate solutions. Proteins with Mrs from 30 000 to 200 000 were detected in the washed membranes by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. These labelled membrane-associated proteins persisted in the microsomal membrane fraction after chase periods from 7.5 to 40 min.

journal_name

Exp Cell Res

journal_title

Experimental cell research

authors

Mather IH,Jarasch ED,Bruder G,Heid HW,Mepham TB

doi

10.1016/0014-4827(84)90369-0

subject

Has Abstract

pub_date

1984-03-01 00:00:00

pages

208-23

issue

1

eissn

0014-4827

issn

1090-2422

pii

0014-4827(84)90369-0

journal_volume

151

pub_type

杂志文章

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