Identification of the beta-subunit for nongastric H-K-ATPase in rat anterior prostate.
The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase alpha-subunit (alpha(ng)) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to alpha(1)-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase beta-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of beta-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three beta-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is beta(1) > beta(3) >> beta(2). Immunohistochemical experiments demonstrate that beta(1) is present predominantly in apical membranes, coinciding with alpha(ng), whereas beta(3) is localized in the basolateral compartment, coinciding with alpha(1). This is the first direct demonstration of the alpha(ng)-beta(1) colocalization in situ indicating that, in rat AP, alpha(ng) associates only with beta(1). The existence of alpha(ng-)beta(1) complex has been confirmed by immunoprecipitation experiments. These results indicate that beta(1)-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.
Pestov
Nikolay B
NB
Department of Pharmacology, Medical College of Ohio, Toledo, 43614, USA.
Korneenko
Tatyana V
TV
Radkov
Rossen
R
Zhao
Hao
H
Shakhparonov
Mikhail I
MI
Modyanov
Nikolai N
NN
D000818:Animals; D002451:Cell Compartmentation / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007150:Immunohistochemistry; D008297:Male; D011467:Prostate / Q000166:cytology / Q000201:enzymology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020033:Protein Isoforms; D021122:Protein Subunits; D012333:RNA, Messenger; D017506:H(+)-K(+)-Exchanging ATPase