Dipeptidyl peptidase IV hydrolyzes Xaa-Pro-Yaa- peptides to Xaa-Pro and Yaa-, while amino peptidase-P cleaves on the amino side of Pro. These two enzymes are found in the microvillous fraction of rabbit kidney. Catalysis is nonspecific with respect to Xaa and Yaa, except the Yaa position cannot be occupied by Pro or Hyp. Harada et al. have published several papers on assaying cleavage of proline-containing peptides found in structural proteins.
Gly-Pro-Ala was used as a substrate for dipeptidyl peptidase IV and was separated from Gly-Pro on a Zorbax ODS (15 cm X 4.6 mm) column by means of a mobile phase containing 10 mM KH2P04(pH 2.5) and 1.0 mM 1-octanesulfonate as an ion-pairing agent. The substrate used for amino peptidase-P was Gly-Pro-Hyp, which was separated from Pro-Hyp by means of a mobile phase containing a 15:85 (v/v) mixture of (10 mM KH2P04) (pH 2.5) and 1-octanesulfonate) and acetonitrile. The eluate was monitored at 210 nm.
The reaction mixture contained in a final volume of 500 /xL 0.03 mmol Tris-HCl (pH 8.0), 0.3 ¿imol of peptide substrate, and the appropriate amount of microvillous fraction, which was prepared from rabbit kidney. In the aminopeptidase-P assay, 1.0 /xmol of manganese chloride was also included. Reactions were run for 30 minutes at 37°C and were stopped by adding 400 nL of 10% perchloric acid. After centrifugation, 20 /xL aliquots were injected. The amount of product formed was linear with amount of enzyme added out to 250 /xg and 1000 /xg of microvillus protein for dipeptidyl peptidase IV and amino peptidase-P, respectively.
Figure 9.35 shows a typical chromatogram and Figure 9.36 the dependence of protein concentration and product formation.
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