Stimulation of ouabain binding to Na,K-ATPase in 40% dimethyl sulfoxide by a factor from Na,K-ATPase preparations.
In 40% dimethyl sulfoxide (Me2SO) high-affinity ouabain (O) binding to Na,K-ATPase (E) is promoted by Mg2+ in the absence of inorganic phosphate (Pi) (Fontes et al., Biochim. Biophys. Acta 1104, 215-225, 1995). Furthermore, in Me2SO the EO complex reacts very slowly with Pi and this ouabain binding can therefore be measured by the degree of inhibition of rapid phosphoenzyme formation. Here we found that, unexpectedly, the ouabain binding decreased with the enzyme concentration in the Me2SO assay medium. We extracted the enzyme preparation with Me2SO or chloroform/methanol and demonstrated that the extracted (depleted) enzyme bound ouabain poorly. Addition of such extracts to assays with low enzyme concentration or depleted enzyme fully restored the high-affinity ouabain binding. Dialysis experiments indicated that the active principle had a molecular mass between 3.5 and 12 kDa. It was highly resistant to proteolysis. It was suggested that the active principle could either be a low-molecular-weight, proteolysis-resistant-peptide (e.g., a proteolipid) or a factor with a nonproteinaceous nature. A polyclonal antibody raised against the C-terminal 10 amino acids of the rat kidney gamma-subunit was able to recognize this low-molecular-weight peptide present in the extracts. The previously depleted enzyme displayed lower amounts of the gamma-proteolipid in comparison to the native untreated enzyme, as demonstrated by immunoreaction with the antibody.
Fontes
C F
CF
Departamento de Bioquímica, ICB, CCS, Universidade Federal do Rio de Janeiro, Brasil.
Lopes
F E
FE
Scofano
H M
HM
Barrabin
H
H
Norby
J G
JG
D000818:Animals; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D015153:Blotting, Western; D003956:Dialysis; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism; D006868:Hydrolysis; D007679:Kidney Medulla; D008970:Molecular Weight; D010042:Ouabain / Q000737:chemistry* / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D010766:Phosphorylation; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D010710:Phosphates; D010743:Phospholipids; D011510:Proteolipids; D010042:Ouabain; D010450:Endopeptidases; D000254:Sodium-Potassium-Exchanging ATPase; D004121:Dimethyl Sulfoxide