Uptake of reconstituted Na,K-ATPase vesicles by isolated lymphocytes measured by FACS, confocal microscopy and spectrofluorometry.

Abstract:

:Na,K-ATPase (EC 3.6.1.37, Na,K-ATPase) is a fundamental vital membrane transport and receptor system which, after biosynthesis, is exported to the plasma membrane in inside-out vesicles. Na,K-ATPase can be extracted form the natural membrane and inserted into artificially formed phosphatidylcholine vesicles (liposomes). The ultrastructure of the reconstituted vesicles has been fully described. In the present work, the Na,K-ATPase-vesicles were labeled with fluorescent tracers either in their water or membrane phase, incubated with freshly isolated human lymphocytes, and the resulting cellular fluorescence measured with fluorescence activated cell sorting (FACS), confocal microscopy and spectrofluorometry. The FACS data show that all lymphocytes take up Na,K-ATPase-vesicles in a dose- and temperature-dependent fashion. Three-dimensional analysis of the fluorescence by confocal microscopy reveals that the fluorescence is contained within the cells. Quantitative determination by spectrofluorometry indicates that depending on the vesicle/cell ratio, a single lymphocyte takes up 650 to 36,500 vesicles within 30 min at 37 degrees C together with up to about 200,000 renal Na,K-ATPase molecules.

journal_name

Cell Biochem Biophys

authors

Anner BM,Volet B

doi

10.1007/BF02738123

subject

Has Abstract

pub_date

1999-01-01 00:00:00

pages

437-54

issue

3

eissn

1085-9195

issn

1559-0283

journal_volume

30

pub_type

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