Neutral aminopeptidase: a potential marker enzyme of the amphibian urinary bladder epithelial cell apical membrane.
Antidiuretic hormone induces, in the apical plasma membrane of amphibian urinary bladder epithelial cells, the exocytotic insertion of intramembranous particle aggregates that probably contain water channels. Purification of the apical membrane is a way to characterize the aggregates. The isolation of such purified membranous fractions involves the use of specific exogenous or endogenous markers. One of them could be the neutral aminopeptidase (AP), whose activity was detected in urinary bladder. Enrichment in AP activity was observed in plasma membrane preparations compared to cell homogenates (X2.7). However, a large part of the enzyme activity was also recovered in the soluble fraction of the preparation, suggesting large proteolysis of the protein. The enzyme presents a low optimal pH (6.4) and a high specificity for proline-p-nitroanilide as compared to the AP present in kidneys and intestines. To localize the protein in the amphibian bladder epithelium, an immunological approach was necessary due to the low activity of the enzyme in this tissue. The low enzymatic activity also prevented the purification of sufficient amounts of the urinary bladder AP as antigen, and we prepared antibodies against purified AP from frog or toad kidneys where the activity is 60 times higher than in the bladder. The serum specificity was verified by spot immunodetection, Western blot, inhibition capacity of antibodies, and immunoadsorption on a solid support with the renal enzyme. The sera were found to be able to react with native as well as denatured forms of the kidney enzyme. Antibodies cross-reacted with several peptides of low molecular weight (40-60 kDa) from urinary bladder plasma membrane proteins (Western blot).(ABSTRACT TRUNCATED AT 250 WORDS)
Tacnet
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Unité de Recherches sur la Pathologie Rénale et Vasculaire, INSERM U 28, Hôpital Broussais, Paris, France.
Chevalier
J
J
Coudrier
E
E
Berthonaud
V
V
Ripoche
P
P
D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002024:Bufo marinus; D018826:CD13 Antigens; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D004848:Epithelium / Q000201:enzymology / Q000648:ultrastructure; D007422:Intestines / Q000201:enzymology / Q000648:ultrastructure; D007668:Kidney / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification; D008854:Microscopy, Electron; D008871:Microvilli / Q000201:enzymology / Q000648:ultrastructure; D008970:Molecular Weight; D009928:Organ Specificity; D011893:Rana esculenta; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D001743:Urinary Bladder / Q000201:enzymology / Q000648:ultrastructure*
D015415:Biomarkers; D008565:Membrane Proteins; D000626:Aminopeptidases; D018826:CD13 Antigens