Dipeptidyl peptidase 9 (DPP9) from bovine testes: identification and characterization as the short form by mass spectrometry.
The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.
Dubois
Véronique
V
Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, B-2610 Antwerp, Belgium.
Lambeir
Anne-Marie
AM
Vandamme
Stefaan
S
Matheeussen
Veerle
V
Guisez
Yves
Y
Scharpé
Simon
S
De Meester
Ingrid
I
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D013737:Testis / Q000201:enzymology*; D014357:Trypsin
D010446:Peptide Fragments; C471241:DPP9 protein, human; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D014357:Trypsin