Rat hepatic (Na/sup +/, K/sup +/)-ATPase:. cap alpha. =subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping

Journal Article · Tue Jul 01 00:00:00 EDT 1986 · Biochemistry; (United States)

OSTI ID:6582973

Hubert, J J; Schenk, D B; Skelly, H; Leffert, H L

The catalytic ..cap alpha..-subunit of rat hepatic (Na/sup +/, K/sup +/)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody (9-A5) covalently linked to Sepharose 4B that specifically block phosphorylation of the sodium pump's ..cap alpha..-subunit from (..gamma..-/sup 32/P)ATP. The hepatic subunit is virtually identical with purified rat, dog, and human renal ..cap alpha..-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (M/sub r/ 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal ..beta..-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic ..beta..-subunits (M/sub r/ 50K and 55K) eluted from 9-A5-Spharose. Additional studies of ouabain-sensitive /sup 86/Rb/sup +/ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two know structurally conserved forms of catalytic subunit (the renallike ..cap alpha.. form) and, if at all, structurally divergent forms of the sodium pump's ..beta..-subunit.

• Research Organization: Univ. of California, San Diego, La Jolla (USA)
• OSTI ID: 6582973
• Journal Information: Biochemistry; (United States), Vol. 25:13
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ATP-ASE
ANTIGEN-ANTIBODY REACTIONS
LIQUID COLUMN CHROMATOGRAPHY
STRUCTURAL CHEMICAL ANALYSIS
RATS
MICROSOMES
LABELLED COMPOUNDS
MONOCLONAL ANTIBODIES
PHOSPHORUS 32
TRACER TECHNIQUES
ACID ANHYDRASES
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ENZYMES
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
NUCLEI
ODD-ODD NUCLEI
ORGANOIDS
PHOSPHOHYDROLASES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RODENTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques