Abstract
This chapter presents the procedure for purification and assay of prolylcarboxypeptidase (PCP). PCP is purified to homogeneity from human kidney, steps involve: extraction; DEAE-cellulose chromatography; CM-cellulose chromatography; SP-Sephadex chromatography; CM-cellulose chromatography; hydroxyapatite chromatography; and Sephadex G-I00 Chromatography. The molecular weight of human kidney PCP determined by gel filtration on Sephadex G-100 is 115,000. The C-terminal end of the naturally occurring substrates for PCP, angiotensins II and III, and it has been used to assay PCP activity during the purification of the enzyme from human kidney. The phenylalanine released from the synthetic substrate is measured with an automatic amino acid analyzer. Because this method requires the use of an amino acid analyzer, it introduces substantial delay in assaying fractions for PCP activity. To overcome this limitation, a more rapid and simpler procedure has been developed. This procedure employs 14C (or 3H)-labeled Cbz-Pro-Ala as substrate. The substrate represents the C-terminal end of the angiotensin II antagonist saralasin and is cleaved at a rate 4.2 times greater than Cbz- Pro-Phe. The amount of [14C]Ala or [3H]Ala released by PCP is measured in a scintillation counter.
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