Interference of substrate quenching with the kinetics of placental peptidases.
The study was undertaken to clarify the putative presence and influence of substrate inhibition and substrate quenching on the kinetic analysis of placental peptidases using naphthylamide substrates in a fluorometric assay. Using the appropriate naphthylamide substrates, we analysed microsomal alanine aminopeptidase (EC 3.4.11.2) and angiotensinase A (EC 3.4.11.7) in the presence or absence of inhibitors (1,10'-phenanthroline, EDTA) or activators (calcium) in the human and rat placenta. All substrates were shown to quench the fluorescence of the enzymatically released unspecific moiety. Since Michaelis-Menten-derived kinetic equations do not include quenching phenomena, we deduced a mathematical model integrating enzyme kinetics and quenching kinetics, thereby allowing simultaneous analysis of both processes from the same raw data. Curves derived from this expanded kinetic model fitted significantly better to velocity data than those derived from Michaelis-Menten-equation. According to the presented data, we assume that substrate inhibition for both enzymes, as previously proposed in the literature, in contrast may be due to formerly unconsidered substrate quenching. The presented kinetic model could serve as a useful tool in the study of putative physiological substrates of the investigated placental peptidases, characterizing the physiologic substrates by their competitive interaction with the synthetic naphthylamides. Additionally, this model may not only be important in fluorometric analysis of peptidases, but also of other enzymes which are analysed fluorometrically with synthetic substrates.
Frank
H G
HG
Institut für Anatomie, Freie Universität Berlin.
Graf
R
R
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D018826:CD13 Antigens; D002118:Calcium / Q000494:pharmacology; D005260:Female; D043384:Glutamyl Aminopeptidase; D006801:Humans; D007700:Kinetics; D010618:Phenanthrolines / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D051381:Rats; D017208:Rats, Wistar
D010618:Phenanthrolines; D000626:Aminopeptidases; D018826:CD13 Antigens; D043384:Glutamyl Aminopeptidase; D002118:Calcium