Dipeptidyl peptidase IV (DPP-IV) from pig kidney cleaves analogs of bovine growth hormone-releasing factor (bGRF) modified at position 2 with Ser, Thr or Val. Extended DPP-IV substrate specificity?
The literature reported DPP-IV substrate specificity includes oligopeptides with a penultimate Pro, Hyp or Ala residue. Bovine GRF is a substrate for DPP-IV and is rapidly degraded by the enzyme via removal of its N-terminal Tyr-Ala. Incubation of selected GRF analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series with a porcine-kidney-derived DPP-IV in PBS (pH 7.4) resulted in cleavage at the X2-Asp3 bond. The extent of enzymatic hydrolysis varied with X2 as reflected in the following relative cleavage rates: Ala2 (100%), Ser2 (4%), Thr2 (2.5%), Val2 (0.53%), Ile2 (0%). These cleavages were sequestered when similar experiments were performed in the presence of the DPP-IV-specific inhibitor N-epsilon-(p-NO2-benzyloxycarbonyl)-Lys-Pro-OH. A side reaction, buffer-induced deamidation of Asn8, contributed less than 5% of the total substrate degradation. Although our finding qualitatively extends the DPP-IV substrate specificity to also include N-terminal X-Ser, X-Thr and X-Val sequences, quantitatively, relatively fast cleavages of the GRFs with Ala2 make the latter preferred substrates for DPP-IV. The data presented here indicates that the observed GRF(3-29) fragment formation upon incubation of Ser2- and Thr2-substituted bGRF analogs in bovine plasma could have been DPP-IV-related.
Martin
R A
RA
Upjohn Company, Kalamazoo, MI 49001.
Cleary
D L
DL
Guido
D M
DM
Zurcher-Neely
H A
HA
Kubiak
T M
TM
D000818:Animals; D002021:Buffers; D002417:Cattle; D018819:Dipeptidyl Peptidase 4; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism*; D006207:Half-Life; D007668:Kidney / Q000201:enzymology*; D012694:Serine; D013379:Substrate Specificity; D013552:Swine; D013912:Threonine; D014633:Valine
D002021:Buffers; D013912:Threonine; D012694:Serine; D013007:Growth Hormone-Releasing Hormone; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4; D014633:Valine