Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.
Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.
Kitlar
T
T
Max Planck Institut für Molekulare Physiologie, Abteilung Epithelphysiologie, Dortmund, Germany.
Döring
F
F
Diedrich
D F
DF
Frank
R
R
Wallmeier
H
H
Kinne
R K
RK
Deutscher
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D002374:Catalase / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D007668:Kidney / Q000648:ultrastructure; D008099:Liver / Q000201:enzymology; D008871:Microvilli / Q000737:chemistry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors*; D009249:NADP / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010695:Phlorhizin / Q000378:metabolism*; D017385:Sequence Homology; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D009249:NADP; D010695:Phlorhizin; D002374:Catalase; D014357:Trypsin