A K+ competitive, conformational probe of the H,K-ATPase.
The H,K-ATPase was noncovalently labelled with a fluorescent quinoline derivative, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo [3,2-c]quinoline, (MDPQ). MDPQ competitively inhibited the K+ stimulated ATP hydrolysis with a Ki of 0.22 microM but did not inhibit the MgATP-dependent phosphoenzyme to an extent greater than 10% of control. Inhibitor binding to the H,K-ATPase enhanced MDPQ fluorescence. This fluorescence was quenched by lumenal K+ with a K0.5 of 1.8 mM. MDPQ binding to the H,K-ATPase shifted the fluorescence Ex/Em maxima from 342/478 nm to 342/453 nm. Phosphorylation of the H,K-ATPase by MgATP further enhanced fluorescence with a difference spectra [MgATP-(MgATP+KCl)] emission peak at 446 nm. Trypsin dependent proteolysis of the H,K-ATPase stabilized within the E2K conformation eliminated the phosphoenzyme response, but enhanced the K+ specific dephosphoenzyme response. These observations show that MDPQ is a fluorescent, competitive inhibitor of the H,K-ATPase that interacts with a lumenal cation binding site. Under specific conditions, both the cation and MDPQ binding sites remain intact within trypsin produced cleavage peptides of the H,K-ATPase.
Rabon
E C
EC
UCLA/VA 90077.
Sachs
G
G
Leach
C A
CA
Keeling
D
D
D000634:Aminoquinolines / Q000494:pharmacology*; D005453:Fluorescence; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D010455:Peptides / Q000737:chemistry; D011188:Potassium / Q000494:pharmacology*; D011487:Protein Conformation; D054328:Proton Pump Inhibitors; D014357:Trypsin
D000634:Aminoquinolines; D010455:Peptides; D054328:Proton Pump Inhibitors; C069657:1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo(3,2-c)quinoline; D014357:Trypsin; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium