A new variant of the gamma subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells.
The gamma subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, gamma runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the gamma chains of rat kidney Na, K-ATPase shows that gamma(a) (upper) has a mass of 7184.0 +/- 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while gamma(b) (lower) has a mass of 7337.9 +/- 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of gamma(a), TELSANH, are replaced by Ac-MDRWYL in gamma(b), but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either gamma(a) or gamma(b) of the Na,K-ATPase, respectively. gamma(a) or gamma(b) cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to gamma(a) or gamma(b) of renal Na, K-ATPase. Additional minor bands seen after transfection, namely gamma(a)' in human embryonic kidney and gamma(b)' in HeLa, are presumably cell-specific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na, K-ATPase contains two variants of the gamma subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.
Kuster
B
B
Protein Interaction Laboratory, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Shainskaya
A
A
Pu
H X
HX
Goldshleger
R
R
Blostein
R
R
Mann
M
M
Karlish
S J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011918:Rats, Inbred SHR; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D000254:Sodium-Potassium-Exchanging ATPase