Prolylcarboxypeptidase Activity Research Articles

Prolyl Carboxypeptidase Activity Is Present in Human Adipose Tissue and Is Elevated in Serum of Obese Men with Type 2 Diabetes

Prolyl carboxypeptidase (PRCP) is involved in metabolic disorders by hydrolyzing anorexigenic peptides. A link between serum PRCP activity and obesity has been reported, but its origin/source is still unclear. Previously proven correlations between human serum PRCP activity and the amount of adipose tissue may suggest that adipose tissue is an important source of circulating PRCP. We investigated PRCP activity in visceral, subcutaneous adipose tissue (VAT and SCAT), skeletal muscle tissue and serum of lean and obese men with or without type 2 diabetes (T2D). Correlations between PRCP activity, metabolic and biochemical parameters and immune cell populations were assessed. PRCP activity was the highest in VAT, compared to SCAT, and was very low in skeletal muscle tissue in the overall group. Serum PRCP activity was significantly higher in T2-diabetic obese men, compared to lean and obese non-diabetic men, and was positively correlated with glycemic control. A positive correlation was observed between serum PRCP activity and VAT immune cell populations, which might indicate that circulating PRCP activity is deriving rather from the immune fraction than from adipocytes. In conclusion, PRCP activity was observed in human adipose tissue for the first time and serum PRCP activity is correlated with T2D in obese men.

Proline-specific peptidase activities (DPP4, PRCP, FAP and PREP) in plasma of hospitalized COVID-19 patients

BackgroundCOVID-19 patients experience several features of dysregulated immune system observed in sepsis. We previously showed a dysregulation of several proline-selective peptidases such as dipeptidyl peptidase 4 (DPP4), fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP) and prolylcarboxypeptidase (PRCP) in sepsis. In this study, we investigated whether these peptidases are similarly dysregulated in hospitalized COVID-19 patients. MethodsFifty-six hospitalized COVID-19 patients and 32 healthy controls were included. Enzymatic activities of DPP4, FAP, PREP and PRCP were measured in samples collected shortly after hospital admission and in longitudinal follow-up samples. ResultsCompared to healthy controls, both DPP4 and FAP activities were significantly lower in COVID-19 patients at hospital admission and FAP activity further decreased significantly in the first week of hospitalization. While PRCP activity remained unchanged, PREP activity was significantly increased in COVID-19 patients at hospitalization and further increased during hospital stay and stayed elevated until the day of discharge. ConclusionThe changes in activities of proline-selective peptidases in plasma are very similar in COVID-19 and septic shock patients. The pronounced decrease in FAP activity deserves further investigation, both from a pathophysiological viewpoint and as its utility as a part of a biomarker panel.

Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells.

The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1β-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.

Prolylcarboxypeptidase as a Predictive Biomarker in Prostate Cancer

Prolylcarboxypeptidase (PRCP) selectively targets specific substrates. It cleaves angiotensin II to generate angiotensin (1-7) and inactivates angiotensin III. Prostate tissues from patients with prostate cancer (PCa) show elevated PRCP expression and moderate cytoplasmic staining. Elevated levels of PRCP have been detected in carcinomas of the kidney, breast, and prostate. However, the detailed biological functions and link with the prostate cancer cells remain unclear. To evaluate the role of prostate PRCP, we characterized and measured the expression pattern and activity of PRCP in 22Rv1, LNCaP, DU 145, and PC3 and compared with RWPE-1 cells, as control. We optimized multiple steps of the protocol, e.g. cell number, cell viability, S-phase test, Hoechst 33258 staining, and quantification of amplified cDNA yields and final qPCR quantification, to quantify gene expression of Prcp gene in the PCa cell lines. The expression levels of PRCP was significantly elevated in LNCaP cells and 22Rv1. Accordingly, PRCP protein expression were increased in LNCaP cells and 22Rv1. PRCP protein expression and activity were reduced by PRCP-siRNA. We conclude from these data that increased PRCP abundance may be a feature of castration-resistant prostate cancer. Our findings shed new insight on PRCP function, and suggest that PRCP may have the potential to become useful target for the diagnosis and treatment of PCa.

Prolylcarboxypeptidase Mitigates Myocardial Ischemia/Reperfusion Injury by Stabilizing Mitophagy.

The role of prolylcarboxypeptidase (PRCP) in myocardial ischemia/reperfusion (I/R) injury is unclear. Herein, we aimed to evaluate the protective effect of the PRCP–angiotensin-(1–7) [Ang-(1–7)]/bradykinin-(1–9) [BK-(1–9)] axis on myocardial I/R injury and identify the mechanisms involved. Plasma PRCP level and activity, as well as Ang-(1–7) and BK-(1–9) levels, were compared in healthy subjects, patients with unstable angina, and those with ST-segment–elevated acute myocardial infarction (AMI). Thereafter, the effects of PRCP overexpression and knockdown on left ventricular function, mitophagy, and levels of Ang-(1–7) and BK-(1–9) were examined in rats during myocardial I/R. Finally, the effects of Ang-(1–7) and BK-(1–9) on I/R-induced mitophagy and the signaling pathways involved were investigated in vitro in rat cardiomyocytes. AMI patients showed increased plasma level and activity of PRCP and levels of Ang-(1–7) and BK-(1–9) as compared with healthy subjects and those with unstable angina. PRCP protected against myocardial I/R injury in rats by paradoxical regulation of cardiomyocyte mitophagy during the ischemia and reperfusion phases, which was mediated by downstream Ang-(1–7) and BK-(1–9). We further depicted a possible role of activation of AMPK in mitophagy induction during ischemia and activation of Akt in mitophagy inhibition during reperfusion in the beneficial effects of Ang-(1–7) and BK-(1–9). Thus, the PRCP–Ang-(1–7)/BK-(1–9) axis may protect against myocardial I/R injury by paradoxical regulation of cardiomyocyte mitophagy during ischemia and reperfusion phases.

Dysregulation of the renin-angiotensin system during lung ischemia-reperfusion injury

ABSTRACTAim/Purpose of the Study: Activation of the renin-angiotensin system leading to increased angiotensin-(1–7) (Ang-(1–7)) and decreased angiotensin 2 (Ang 2) levels may be a new therapeutic approach to reduce acute lung injury. Prolylcarboxypeptidase (PRCP) and prolyloligopeptidase (PREP) are capable of hydrolyzing Ang 2 into Ang-(1–7). However, their relation with circulating Ang 2 levels after lung ischemia–reperfusion injury (LIRI) has never been explored. This study determines whether the activity and expression of PRCP and PREP in plasma and lung tissue is related to circulating Ang 2 levels in a murine model of LIRI. Materials and Methods: LIRI in Swiss mice (6 animals per group) was induced by temporary left lung hilar clamping (1 h) followed by 0, 1 or 24 h of reperfusion. Animals in the sham group received thoracotomy only. PRCP activity was measured via RP-HPLC, PREP activity using a fluorogenic substrate and plasma Ang 2 levels via ELISA. Western blotting was used to determine the PRCP and PREP protein expression profiles in left lung tissue. Results: Plasma Ang 2 levels significantly rise after lung ischemia and remain increased after 1 h and 24 h of reperfusion compared to the sham group. While a significant decrease in plasma PREP activity was found after 24 h of reperfusion, a transient increase in plasma PRCP activity was observed after ischemia. However, no correlation with plasma Ang 2 levels could be demonstrated. The activity profiles of PRCP and PREP and the protein expression of PRCP in the lung tissues remained unchanged after LIRI. Conclusions: LIRI causes a dysregulation of circulating Ang 2 levels and plasma PREP activity, although no direct link between both phenomena could be shown. The activity profile of pulmonary PRCP and PREP was not significantly changed after LIRI, which implies a minor role for local PRCP and PREP in the ischemic lung itself.

Prolyl carboxypeptidase activity decline correlates with severity and short-term outcome in acute ischemic stroke.

Prolyl carboxypeptidase (PRCP) is an enzyme associated with cerebrovascular risk factors such as hypertension, diabetes mellitus, obesity and hyperlipidemia. We aim to evaluate the relation between serum PRCP activity and severity, evolution and outcome of acute ischemic stroke. We used a specific RP-HPLC activity assay to measure PRCP activity in serum of 50 stroke patients at admission, and at 24 h, 72 h and 7 days after stroke onset to assess correlations with stroke severity based on the National Institutes of Health Stroke scale score (NIHSS), infarct volume on brain MRI scan, stroke outcome based on the modified Rankin scale (mRS) and mortality at 3 months after stroke. The average PRCP activity in serum decreased significantly the first 24 h after stroke onset and returned to baseline values at day 7. High NIHSS scores and infarct volumes at admission were related with a more pronounced decrease of PRCP in the first 24 h after stroke (ΔPRCP24, r = 0.31, P < 0.05; r = 0.30, P < 0.05). In addition, patients who displayed a more pronounced decrease in PRCP levels during the first 24 h after stroke were more likely to be institutionalized upon discharge (n = 21) (ΔPRCP24 ± SD, 0.05 ± 0.10 U/L vs. 0.17 ± 0.14 U/L, P = 0.001). The decrease in PRCP levels in the first 24 h after stroke onset is associated with stroke severity and an unfavourable short-term stroke outcome.

Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements

Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-l-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis–Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65±0.02 and 0.72±0.03U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP’s role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome.

Abstract 630: Blood Pressure and Kidney Function in PRCP deficient Mice

Changes in the enzymes involved in Ang II degradation may influence disease conditions such as hypertension and kidney disease. The enzyme prolylcarboxypeptidase (PRCP) catalyzes the conversion of Ang II to Ang(1-7) and its deficiency results in a phenotype characterized by a prothrombotic state, hypertension, and impaired wound healing. Here, we investigated kidney function and blood pressure in young mice, 10-16 weeks of age, with a subtotal PRCP deficiency (PRCP genetrap, PRCPgt). Enzyme activity based on a synthetic fluorogenic peptide was used to measure PRCP activity. The activity of recombinant PRCP was strongly pH-dependent with an optimum in the acidic range (pH 5). In keeping with this, recombinant PRCP degraded Ang II at pH 5, but not at pH 7.4 in-vitro. PRCP activity was detected in serum and in kidney of WT mice whereas PRCPgt/gt mice displayed highly diminished serum PRCP activity (about 5% of the WT). Kidney function was assessed by urinary protein/creatinine ratio and GFR. At 10 weeks of age, neither female nor male PRCPgt/gt animals showed elevations in urinary protein/creatinine-ratio (female PRCPgt/gt 19.9±1.4 vs. WT 23.5±1.9 and male PRCPgt/gt 32.4±3.5 vs. WT 34.8±1.8). At 16 weeks of age, the GFR (measured by clearance of FITC-Inulin) was not different (female PRCPgt/gt 4.10±0.32 vs. WT 4.33±0.29 and male PRCPgt/gt 4.41±0.50 vs. WT 4.28±0.31 ul/min/g). Systolic blood pressure was significantly elevated in PRCPgt/gt animals as compared to the respective WT controls (female PRCPgt/gt 147.8±3.4 vs. WT 117.3±7.1 mmHg, male 138.1±8.4 vs. 120.5±3.6 mmHg, p&lt;0.05). PRCP deficient mice have normal GFR but elevated basal blood pressure. Whether PRCP deficiency causes hypertension as a result of impaired Ang II degradation or other mechanisms needs to be determined.

Discovery of benzodihydroisofurans as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase inhibitors

A series of benzodihydroisofurans were discovered as novel, potent, bioavailable and brain-penetrant prolylcarboxypeptidase (PrCP) inhibitors. The structure–activity relationship (SAR) is focused on improving PrCP activity and metabolic stability, and reducing plasma protein binding. In the established diet-induced obese (eDIO) mouse model, compound ent-3a displayed target engagement both in plasma and in brain. However, this compound failed to induce significant body weight loss in eDIO mice in a five-day study.

Prolylcarboxypeptidase Regulates Proliferation, Autophagy, and Resistance to 4-Hydroxytamoxifen-induced Cytotoxicity in Estrogen Receptor-positive Breast Cancer Cells

Endocrine therapy with tamoxifen (TAM) significantly improves outcomes for patients with estrogen receptor-positive breast cancer. However, intrinsic (de novo) or acquired resistance to TAM occurs in a significant proportion of treated patients. To identify genes involved in resistance to TAM, we introduced full-length cDNA expression library into estrogen receptor-positive MCF7 cells and exposed them to a cytotoxic dose of 4-hydroxytamoxifen (4OHTAM). Four different library inserts were isolated from surviving clones. Re-introduction of the genes individually into naive MCF7 cells made them resistant to 4OHTAM. Cells overexpressing these genes had an increase in acidic autophagic vacuoles induced by 4OHTAM, suggesting their role in autophagy. One of them, prolylcarboxypeptidase (PRCP), was investigated further. Overexpression of PRCP increased cell proliferation, boosted several established markers of autophagy, including expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA in an ER-α dependent manner, and increased resistance to 4OHTAM. Conversely, knockdown of endogenous PRCP in MCF7 cells increased cell sensitivity to 4OHTAM and at the same time decreased cell proliferation and expression of LC3-2, sequestration of monodansylcadaverine, and proteolysis of BSA. Inhibition of enzymatic activity of PRCP enhanced 4OHTAM-induced cytotoxicity in MCF7 cells. Cells with acquired resistance to 4OHTAM exhibited increased PRCP activity, although inhibition of PRCP prevented development of 4OHTAM resistance in parental MCF7 cells and restored response to 4OHTAM in MCF7 cells with acquired resistance to 4OHTAM. Thus, we have for the first time identified PRCP as a resistance factor for 4OHTAM resistance in estrogen receptor-positive breast cancer cells.

Prolylcarboxypeptidase regulates food intake by inactivating α-MSH in rodents

The anorexigenic neuromodulator alpha-melanocyte-stimulating hormone (alpha-MSH; referred to here as alpha-MSH1-13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of alpha-MSH1-13 maturation and inactivation is incompletely understood. Here we have provided insight into alpha-MSH1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of alpha-MSH1-13, producing alpha-MSH1-12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where alpha-MSH1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active alpha-MSH1-13 levels.

High-performance liquid chromatographic determination of prolylcarboxypeptidase activity in monkey kidney.

A simple HPLC procedure for the determination of prolylcarboxypeptidase activity in monkey kidney was established with Cbz-Pro-Ala used as substrate. Decrease of the substrate and increase of the product were stoichiometrically related to each other. Heat treatment at 60 degrees C freed the enzyme preparation of contaminating activities. Data on substrate specificity and influence of inhibitors suggested this method was sensitive for the determination of prolylcarboxypeptidase without the use of a radioactive substrate.

Angiotensin II 1 receptor-mediated contraction of pulmonary artery and its modulation by prolylcarboxypeptidase.

To determine the subtype of angiotensin II (ANG II) receptor involved in the contraction of pulmonary artery and to elucidate its possible modulation by endogenous peptidases, we studied canine isolated pulmonary arterial rings under isometric conditions in vitro. Addition of ANG II caused a concentration-dependent contraction, an effect that was not altered by the ANG II 2 receptor antagonist EXP655 but was depressed by the ANG II 1 receptor antagonist DuP 753 so that the ANG II response curves were displaced to higher concentration by 1.5-2.0 log U (P < 0.001). Pretreatment of tissues with the prolylcarboxypeptidase (PCP) inhibitor p-methylphenyl sulfonyl-fluoride potentiated the ANG II-induced contraction, with the concentration required to produce a half-maximal effect of ANG II being decreased from 4.1 +/- 0.9 x 10(-9) to 3.8 +/- 0.5 x 10(-10) M (P < 0.001), whereas other peptidase inhibitors such as p-chloromercuriphenyl sulfonic acid, amastatin, and phosphoramidon had no effect. The p-methylphenyl sulfonylfluoride-induced potentiation was abolished by the removal of endothelium, but it was still observed in the presence of NG-nitro-L-arginine methyl ester in the endothelium-intact tissues. The PCP activity in the tissues was reduced by the removal of endothelium from 645 +/- 88 to 91 +/- 29 nmol.mg protein-1.h-1 (P < 0.001), and cultured endothelium had the activity of 404 +/- 39 nmol.mg protein-1.h-1. These results suggest that ANG II contracts pulmonary artery via ANG II 1 receptor and that PCP localized to the endothelium may have a modulatory role in the ANG II-induced pulmonary vasoconstriction.

High levels of exopeptidase activity are present in rat and canine bronchoalveolar lavage fluid

Although reduced loss from porteolytic activity is a potential advantage of the repiratory route for peptide and protein drug delivery, except for elastase and enkephalinase, few data are availabe regarding relevamt enzyme levels. Protease activity in brochoalvelar lavage (BAL) fuid from rat and dog has been examined to determined whether degradation is potential limiting factor of peptide s deliverd via the lung in these animal models. Normal levels of endopeptidases cleaving substrates preferred by elastase, trypsin, and chymotrypsin are low in BAL fluid of both species, consistent with preliminary published data indicating that the stability of proteins may be much greater in the respiratory than the gastrointestinal tract. Aminopeptidase, dipeptidyl peptidase IV, and prolyl carboxypeptidase activity are found in BAL fluid: aminopeptidase levels are suffciently high to be a potential limiting factor for peptide absorption after pulmonary administration in either species. In situ assays confirm that aminopeptidase activity detected in rat lavage fluids is only a fraction of that present at the surface of cells lining the respiratory tract. Whil low levels of active endoproteases probably do not pose a major enzymatic barrier, peptides susceptible to exopeptidase may require protection against degradation e.g., by structural modification or protease inhibition, for optimal delivery via the respiratory tact.

Rapid radioassay for prolylcarboxypeptidase (angiotensinase C)

A simple, sensitive assay for prolylcarboxypeptidase (PCP) is described. It utilizes a radiolabeled substrate, benzyloxycarbonyl- l-prolyl- l-[ 3H]alanine, and the details of its synthesis are also reported here. The hydrolysis of the dipeptide substrate is linear with respect to time or protein concentration until 10% of the substrate has been cleaved. Kinetic analysis yielded a K m of 4.7 m m. The assay can be used to measure PCP activity in small amounts of biological fluid, homogenized tissue or cultured cells. Measurements of PCP activity in various cultured human cells showed endothelial cells from umbilical veins to have the highest activity (1625 ± 151 nmol/mg/h) followed by endothelial cells from umbilical artery (1017 ± 46 nmol/mg/h), human foreskin fibroblasts (719 ± 39 nmol/mg/h), and pulmonary artery endothelial cells (352 nmol/mg/h).

Prolylcarboxypeptidase (angiotensinase C) in human lung and cultured cells.

The activity of prolylcarboxypeptidase (PCP), or angiotensinase C, was measured in lung tissues, leukocytes, and cultured human cells using Cbz-Pro-[14C]Ala as a substrate. A lysosomal fraction of homogenized rat or human lung contained most of the PCP activity in that tissue. Polymorphonuclear neutrophils, macrophages, and lymphocytes isolated from human blood had PCP activity. Fibroblasts cultured from human tissues had the highest activity (0.56-1.15 mumol/h per 10(6) cells), more than endothelial cells cultured from human pulmonary arteries. PCP of cultured human fibroblasts was similar to the human renal enzyme because it was resistant to moderate heating and was not inhibited by p-chloromercuriphenyl sulfonic acid. These properties and the substrate specificity distinguish PCP from cathepsin A, which is also in fibroblasts. Antibody to human renal PCP reacted with fibroblast PCP in immunofluorescence, indicating common antigenic determinants. Hydrocortisone changed PCP activity in fibroblasts in parallel with changes in beta-glucuronidase activity and cell-protein concentration; the activity was depressed at low concentration of the hormone. PCP activity was also found in synovial fluid from arthritic joints and in fibroblasts from the synovium. That PCP is found in both inflammatory exudates and in cells that appear at sites of inflammation indicates that, in addition to inactivating angiotensins, this enzyme may have a role in inflammation.

38] Human prolylcarboxypeptidase

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.