title: Improved prolidase activity assay allowed enzyme kinetic characterization and faster prolidase deficiency diagnosis.
abstract: BACKGROUND
  Prolidase is a metallo-exopeptidase hydrolyzing X-Pro and X-Hyp dipeptides. Its absence or reduced level is typical in prolidase deficiency (PD) patients, and altered prolidase activity was reported in various diseases. Therefore, standardized and accurate measurement of prolidase activity is essential for PD diagnosis, as well as to elucidate the pathophysiology of other disorders.
  
  
  METHODS
  Human recombinant prolidase was used to optimize a spectrophotometric enzyme activity assay. Kinetic parameters and Mn(2+) affinity were evaluated. The method was validated on blood and fibroblasts from PD patients.
  
  
  RESULTS
  An activation step consisting in prolidase incubation with 1 mmol/l MnCl(2) and 0.75 mmol/l reduced glutathione at 50°C for 20 min was necessary to obtain the maximum activity and to accurately determine, for the recombinant enzyme, V(max) (489 U/mg), K(m) (5.4 mM) and Mn(2+) affinity (54 mM(-1)). The method applied to PD diagnosis revealed an intra-assay CV=8% for blood and 9% for fibroblasts lysates. The inter-assay CV was 21% for blood and 20% for cell lysates.
  
  
  CONCLUSIONS
  We optimized a faster spectrophotometric method to measure the activity when the enzyme is fully activated, this is crucial to allow a reliable evaluation of prolidase activity from different sources.
authors.affiliation: Department of Biochemistry, University of Pavia, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Besio
authors.forename: Enrico
authors.initials: E
authors.lastname: Monzani
authors.forename: Roberta
authors.initials: R
authors.lastname: Gioia
authors.forename: Stefania
authors.initials: S
authors.lastname: Nicolis
authors.forename: Antonio
authors.initials: A
authors.lastname: Rossi
authors.forename: Luigi
authors.initials: L
authors.lastname: Casella
authors.forename: Antonella
authors.initials: A
authors.lastname: Forlino
mesh_terms: D004150:Dipeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D056732:Prolidase Deficiency / Q000175:diagnosis*; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D011994:Recombinant Proteins; D004150:Dipeptidases; C022449:proline dipeptidase