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PMID: 6310568 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Monoclonal antibodies to rat Na+,K+-ATPase block enzymatic activity.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 80 ·No. 17 ·1983-09-00 ·Pages 5281-5

Schenk DB, Leffert HL

Abstract

A panel of nine mouse monoclonal antibodies has been prepared against purified preparations of rat kidney Na+,K+-ATPase (EC 3.6.1.3). Selection for specific antibody was based upon the ability of crude hybridoma fluids to inhibit Na+-ATPase activity (using luciferase-linked ATPase assays) and upon antibody binding to both the purified kidney membrane enzyme and to glutaraldehyde-fixed hepatocytes by using standard enzyme-linked immunoadsorbent assays. After immunoaffinity purification, two of the antibodies (both of the IgG1 subclass) fully inhibit kidney and liver membrane Na+,K+-ATPase activity with Ki (apparent) values of 30 nM ("9-A5") and 600 nM ("9-B1"). Immunoblots demonstrate directly that three different 125I-labeled antibodies (6-4, 9-A5, and 9-B1) bind predominantly to a 94,000 Mr protein that comigrates in NaDodSO4/polyacrylamide gels with the fluorescein isothiocyanate-labeled alpha subunit of the Na+,K+-ATPase. Indirect immunofluorescence studies with these antibodies on paraformaldehyde-fixed liver slices reveal staining patterns congruent with bile canalicular membrane domains. These results together suggest that the antibodies exert inhibitory effects by recognizing alpha subunits of both liver and kidney Na+ pumps in their native conformations.

MeSH Terms
Animals Antibodies, Monoclonal Antigen-Antibody Complex Enzyme-Linked Immunosorbent Assay Fluorescent Antibody Technique Hybridomas/immunology Kidney Medulla/enzymology Liver/enzymology Male Mice Mice, Inbred BALB C Molecular Weight Rats Rats, Inbred Strains Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors,immunology
Chemicals
Antibodies, Monoclonal Antigen-Antibody Complex Sodium-Potassium-Exchanging ATPase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Schenk D B
Leffert H L
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29 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.80.17.5281
Published
1983-09-00
Pages
5281-5
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC384237
Subset
IM
Grants
NIADDK NIH HHS · AM28215 · United States
NIADDK NIH HHS · AM28392 · United States
NIGMS NIH HHS · GM 07752 · United States
External Links
PubMed source DOI: 10.1073/pnas.80.17.5281 PMC Full Text View journal details
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