The Development of Two Fluorimetric Assays for the Determination of Pyroglutamyl Aminopeptidase Type-II Activity

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

250, 1–9 (1997)

AB972195

The Development of Two Fluorimetric Assays for the Determination of Pyroglutamyl Aminopeptidase Type-II Activity Sea´n P. Gallagher, Rhona M. O’Leary, and Brendan O’Connor1 School of Biological Sciences, Dublin City University, Ireland

Received February 11, 1997

Two fluorimetric assays for the determination of pyroglutamyl aminopeptidase type-II activity have been developed. The assays are based on hydrolysis of the quenched-fluorimetric substrate õGlu-His-Pro-7amino-4-methylcoumarin. Following the removal of the N-terminal õGlu by pyroglutamyl aminopeptidase type-II, liberation of 7-amino-4-methylcoumarin from the metabolite His-Pro-7-amino-4-methylcoumarin is catalyzed by one of two methods: (i) the addition of partially purified bovine serum dipeptidyl aminopeptidase type-IV or (ii) by incubating the reaction mixture for up to 2 h at 807C, thus promoting the nonenzymatic cyclization of His-Pro-7-amino-4-methylcoumarin to cyclo His-Pro and free 7-amino-4-methylcoumarin. Pyroglutamyl aminopeptidase type-II from bovine brain is used to establish appropriate assay conditions. These fluorimetric assays offer expeditious alternatives to the existing radiolabeled thyrotropin-releasing hormone assays for the determination of PAPII activity. q 1997 Academic Press

Pyroglutamyl aminopeptidases (PAPs)2 are omega peptidases which specifically remove the L-pyroglutamyl residue from the amino-terminus of polypeptides by hydrolysis. To date, two classes of pyroglutamyl aminopeptidase have been characterized. The first in1 To whom correspondence should be addressed at School of Biological Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. Fax: /353-1-7045412. 2 Abbreviations used: PAP, pyroglutamyl aminopeptidase; TRH, thyrotropin-releasing hormone; bNA, b-napthylamide; pNA, p-nitroanilide; MCA, 7-amino-4-methylcoumarin; DAP, dipeptidyl aminopeptidase; Z, N-benzyloxycaronyl; DMSO, dimethyl sulfoxide; SP-, sulfopropyl-; Q-, quaternary anion-; BCA, bicinchoninic acid; FmocPro-Pro-CN, 9-fluorenylmethyloxycarbonyl-prolyl-pyrrolidine-2-nitrile; BSA, bovine serum albumin; SEM, standard error of the mean.

cludes bacterial PAP and animal type-I PAP (PAPI), a cytosolic enzyme with biochemical characteristics similar to the bacterial enzyme. The second class includes animal type-II PAP (PAPII), a membrane-bound enzyme, and serum PAP, also known as thyroliberinase (1, 2). PAPI (EC 3.4.19.3) has been found to be present in all mammalian tissues tested, with the exception of blood (3). This cysteine proteinase is a monomeric enzyme with a molecular mass of 22,000 to 25,000 Da (4– 10). A distinctive feature of PAPI is its broad substrate specificity. The enzyme has been shown to cleave Nterminal õGlu from a range of biologically active peptides including thyrotropin-releasing hormone (TRH), acid TRH, luliberin, neurotensin, and bombesin. õGluPro bonds are not hydrolyzed by the enzyme (7, 8). Initial PAP assays were based on the cleavage of pyroglutamyl dipeptides such as õGlu-Ala and measurement of amino acid release by the ninhydrin method (4). The development of the colourimetric and fluorimetric substrates õGlu-b-napthylamide (õGlu-bNA) (3), õGlu-p-nitroanilide (õGlu-pNA), and õGlu-7amino-4-methylcoumarin (õGlu-MCA) (11) resulted in PAPI assays with greatly increased sensitivity. PAPII (EC 3.4.19.6) is an ectoenzyme (i.e., an integral membrane protein with an extracellularly localized active site) (12–15). Although PAPII has been shown to be present in many mammalian tissues, it is predominantly an enzyme of the central nervous system with highest levels of activity in the brain (15– 18). Within the brain the distribution of the enzyme is heterogeneous (16). Studies on primary cell cultures of rodent brain have shown that while a relatively high level of PAPII activity is found on neuronal cells, glial cells are almost devoid of activity. In the pituitary, PAPII is localized preferentially on lactotrophs (12, 14). It is likely that serum PAP is a secreted form of liver 1

0003-2697/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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PAPII (15, 19, 20). Both enzymes are metallopeptidases (1, 15, 22–29) of relatively large molecular size, with estimates of 230,000 to 240,000 Da for PAPII (23, 26– 28) and 260,000 Da for the serum enzyme (1, 21). Probably the most interesting feature of PAPII and serum PAP is their extraordinarily high degree of substrate specificity. Both enzymes have been shown to remove the N-terminal õGlu from only TRH (õGluHis-Pro-NH2) or closely related peptides (2, 23, 24, 26, 28, 30–32). There is considerable evidence that the inactivation of neuronally released TRH is catalyzed by PAPII (12, 33) and it has been suggested that this enzyme may be the first neuropeptide-specific peptidase to be characterized (34). Due to the unique specificity of these enzymes, the PAPI substrates õGlu-bNA, õGlupNA, and õGlu-MCA are extremely poor substrates and can only be used to determine the activity of purified samples containing relatively high levels of enzyme activity (2, 24–27, 29). The determination of PAPII and serum PAP activity has been achieved by many researchers using radiolabeled TRH (for example, [3H-Pro]TRH, [14C-õGlu]TRH, or [3H-His]TRH) as a substrate. A number of variations of the assay have been described in which, following hydrolysis of the substrate, the degradation products are separated by paper chromatography, thinlayer chromatography, column chromatography, immunoaffinity, or HPLC (12, 18, 21, 23–25, 35, 36). Following metabolite separation, the enzyme activity of the sample is quantified by measuring the radioactivity to determine either the amount of TRH remaining or the amount of the metabolites õGlu, His-Pro-NH2 or cyclo His-Pro produced. The development of a coupled enzyme assay based on the hydrolysis of the TRH substrate analogue õGluHis-Pro-bNA in the presence of excess dipeptidyl aminopeptidase type-IV (DAPIV; EC 3.4.14.5) has facilitated the colorimetric detection of PAPII activity (18). The assay is based on the following reaction sequence: PAPII

õGlu-His-Pro-bNA r õGlu / His-Pro-bNA DAPIV

His-Pro-bNA

r

His-Pro / bNA

The inclusion of N-benzyloxycarbonyl-Pro-prolinal (Z-Pro-Prolinal), a specific inhibitor of prolyl endopeptidase (EC 3.4.21.26), in the assay mixture blocks the cleavage of the Pro-bNA bond, and the inclusion of õGlu-diazomethylketone prevents hydrolysis by PAPI. A modification of this procedure has been described which utilizes the quenched fluorimetric substrate õGlu-His-Pro-MCA (28). Although DAPIV is not commercially available and must therefore be purified in the laboratory, these coupled enzyme assays are less

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labor-intensive and expensive than the radiolabeled assays. They have the added advantage of avoiding the use of radioactivity. In this paper we describe two new fluorimetric assays, based on the substrate õGlu-His-Pro-MCA, for the determination of PAPII activity. In the first of these, a modification of the coupled enzyme assay, the reactions of PAPII and DAPIV are uncoupled in order to ensure the complete liberation of MCA from the metabolite His-Pro-MCA, produced by the action of PAPII. The second assay is based on the nonenzymatic cyclization of His-Pro-MCA to cyclo His-Pro, thus releasing MCA. MATERIALS AND METHODS

Materials 2-Pyrrolidone was obtained from Aldrich Chemical Company (Poole, Dorset, England). õGlu-His-ProMCA, õGlu-MCA, Gly-Pro-MCA, and Z-Gly-Pro-MCA were obtained from Bachem Feinchemikalein AG (Bubendorf, Switzerland). Acetic acid, DMSO, glycerol, and polyethylene glycol 6000 were obtained from BDH Chemicals Ltd. (Poole, Dorset, England). Ammonium sulfate and NaOH were obtained from Merck Chemical Company (Frankfurt, Germany). Chelating Sepharose Fast Flow, phenyl Sepharose CL-4B, Sephacryl S200 HR, SP-Sepharose Fast Flow, and Q-Sepharose High Performance were obtained from Pharmacia Fine Chemical Company (Uppsala, Sweeden). BCA reagent was obtained from Pierce Chemical Company (Illinois). Bovine blood and brains were obtained from Kepak Ltd., meat processors (Dublin, Ireland). Fmoc-Pro-ProCN was generously donated by Dr. S. Wilk (Mount Siani School of Medicine, New York, NY). All other chemicals were obtained from Sigma Chemical Company (Poole, Dorset, England). Methods Buffers. The principal buffers used had the following composition: buffer A, 20 mM potassium phosphate, pH 7.5; buffer B, 100 mM potassium phosphate; buffer C, 5 mM potassium phosphate, pH 7.5. Enzyme assays. PAPI activity was determined by hydrolysis of 0.1 mM õGlu-MCA in buffer A containing 2% (v/v) DMSO, 2 mM dithiothreitol, and 2 mM EDTA, according to the method of Cummins and O’Connor (8), a modification of the original procedure of Fujiwara and Tsuru (11). Prolyl endopeptidase activity was determined by hydrolysis of 0.1 mM Z-Gly-Pro-MCA in buffer A containing 4% (v/v) DMSO, 2 mM dithiothreitol, and 2 mM EDTA, according to a modification of the original procedure of Yoshimoto et al. (37). DAPIV activity was determined by hydrolysis of 0.1 mM Gly-

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Pro-MCA in buffer A, according to a modification of the original procedure of Kato et al. (38). Prolyl aminopeptidase activity (EC 3.4.11.5) was determined by hydrolysis of 0.1 mM Pro-MCA in buffer A, according to a modification of the original procedure of Yoshimoto et al. (39). Alanyl aminopeptidase activity (EC 3.4.11.2) was determined by hydrolysis of 0.1 mM Ala-MCA in buffer A, according to a modification of the original procedure of Mantle et al. (40). One hundred microlites of enzyme containing sample was incubated for 1 h at 377C with 400 ml of the appropriate substrate. The reaction was terminated by the addition of 1 ml of 1.5 M acetic acid. All assays were performed in triplicate and suitable negative controls were prepared by incubating substrate and sample separately for the duration of the assay, followed by the addition of acetic acid to the sample immediately before the addition of substrate. Liberated MCA was measured using a Perkin–Elmer LS-50 fluorescence spectrophotometer with excitation and emission wavelengths of 370 and 440 nm, respectively. Samples containing particulate material were centrifuged at 13,000 rpm for 10 min, using a Heraeus Sepatech Biofuge A, prior to fluorescence determination. Enzyme activity was quantified by reference to an appropriate MCA standard curve prepared under corresponding conditions. A unit of enzyme activity was defined as that which liberates one picomole of MCA per minute at 377C. Protein determination. Using BSA as standard, protein concentration was determined using the BCA protein assay (41). The absorbance at 560 nm was measured using a TiterTek Multiscan PLUS spectrophotometric plate reader. Partial purification of DAPIV from bovine serum. This procedure was carried out at 47C unless otherwise stated. Bovine whole blood was collected from a freshly killed animal. The whole blood was transported to a 47C cold room and the clot was allowed to shrink for 24 h. The remaining unclotted whole blood was then decanted and centrifuged at 6000 rpm (4100g) for 1 h using a Beckman J2-MC refrigerated centrifuge fitted with a JA-21 rotor at 47C. The serum thus produced was divided into 20-ml aliquots and stored at 0207C. Twenty milliliters of serum was dialyzed for 12 h into 4 L of 100 mM monobasic potassium phosphate buffer (KH2PO4) and was subsequently centrifuged for 30 min at 15,000 rpm (27,200g) using a Beckman J2MC refrigerated centrifuge fitted with a JA-20 rotor, to remove postdialysis precipitate. The resulting supernatant was applied to a 20-ml SP-Sepharose Fast Flow cation-exchange column (2.5 1 4.1 cm), equilibrated with 100 ml of buffer B at pH 5.5. After washing the column with 40 ml of equilibration buffer, bound pro-

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tein was eluted with 50 ml of 500 mM NaCl in buffer B at pH 7.5. Four-milliliter fractions were collected throughout the procedure, which was carried out at a flowrate of 2 ml/min (24.4 cm/h). The DAPIV containing fractions were pooled and 6.607 g of dry ammonium sulphate was added under continuous stirring. The pH of the pool was adjusted to 7.0 using 1 M NaOH, and the volume was adjusted to 50 ml with buffer B at pH 7.0, thus resulting in a final concentration 1 M ammonium sulfate in buffer B at pH 7.0. Following the application of this sample to a 20-ml Phenyl Sepharose CL-4B hydrophobic interaction column (2.5 1 4.1 cm), equilibrated with 100 ml of 1 M ammonium sulfate in buffer B at pH 7.0, the column was washed with 60 ml of equilibration buffer. Bound protein was eluted with 60 ml of buffer C. Five-milliliter fractions were collected throughout the procedure, which was carried out at a flowrate of 2 ml/min (24.4 cm/h). Fractions containing the highest DAPIV activity were pooled. Post-phenyl Sepharose CL-4B DAPIV (partially purified DAPIV) was dialyzed extensively against buffer B at pH 7.5 to remove ammonium sulfate. The postdialysis sample was concentrated by reverse osmosis with polyethylene glycol until the activity reached approximately 2500 units/ml. Following further dialysis of the concentrated sample against buffer B at pH 7.5, to remove polyethylene glycol, glycerol was added to a final concentration of 10% (v/v) and 1-ml aliquots were stored at 0207C. PAPII preparation. Twenty-five grams of bovine brain was homogenized in 100 ml of buffer A with the aid of a Sorvall Omni Mixer. Tissue was disrupted by three 5-s pulses at speed setting four, with a 10-s pause between pulses. PAPII was solubilized from washed bovine brain membranes using trypsin and was subsequently purified using Q-Sepharose anion exchange, immobilized zinc affinity, calcium phosphate cellulose, and Sephacryl S200 gel filtration chromatography, resulting in a 3000-fold purification with a 24% recovery of enzyme activity (unpublished work). Development of fluorimetric PAPII assays. Two fluorimetric assays were developed for the determination of PAPII activity. Both assays are based on the hydrolysis of 0.1 mM õGlu-His-Pro-MCA in buffer A, in the presence of 2-pyrrolidone, a reversible PAPI inhibitor (42), and/or Fmoc-Pro-Pro-CN, a specific prolyl endopeptidase inhibitor (43). DAPIV-catalyzed assay. One hundred microliters of bovine brain homogenate, 20 ml of 26 mM Fmoc-ProPro-CN (final concentration, 1 mM), and 20 ml of 1.32 M 2-pyrrolidone (final concentration, 50 mM) was incubated for 1 h at 377C with 400 ml of 0.1 mM õGlu-HisPro-MCA. The reaction was terminated by incubating

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the mixture at 807C for 2 min. When reaction mixture had cooled to 377C, 150 ml of a range of dilutions from 0 to 2526 units/ml of partially purified DAPIV (final concentration, 0–549 units/ml) was added. Following further incubation for 1 h at 377C the reaction was terminated by the addition of 1.5 M acetic acid to a final volume of 1.5 ml. Liberated MCA was determined as previously described. Eighty microliters of purified PAPII containing 1% (w/v) BSA and 20 ml of 26 mM Fmoc-Pro-Pro-CN (final concentration, 1.04 mM) was incubated for 1 h at 377C with 400 ml of 0.1 mM õGlu-His-Pro-MCA. The reaction was terminated by incubating the mixture at 807C for 2 min. When the reaction mixture had cooled to 377C, 300 ml of a range of dilutions from 0–2526 units/ ml of partially purified DAPIV (final concentration, 0– 947 units/ml) was added. Following further incubation for 1 h at 377C, the reaction was terminated by the addition of 1.5 M acetic acid to a final volume of 1.5 ml. Liberated MCA was determined as previously described. All assays were performed in triplicate. A suitable negative control for each sample was prepared by incubating substrate separate from the other components of the incubation mixture for 1 h at 377C and subsequently for 2 min at 807C. Following the addition of substrate, the appropriate concentration of DAPIV was added and following 1 h further incubation at 377C, the appropriate volume of acetic acid was added. Nonenzymatic cyclization assay. Following the reaction of bovine brain homogenate or purified PAPII with õGlu-His-Pro-MCA as previously described, the reaction was terminated by the addition of 1.5 M acetic acid to a final volume of 1.5 ml. The reaction mixture was subsequently incubated at 807C for a range of times from 0 to 180 min, following which the samples were cooled by incubation in an ice-water bath. Liberated MCA was determined as previously described. All assays were performed in triplicate. A suitable negative control for each sample was prepared by incubating substrate separate from the other components for 1 h at 377C, followed by the addition of acetic acid immediately prior to the addition of substrate and subsequent incubation for the appropriate length of time at 807C. Linearity of PAPII activity with respect to time. One hundred microliters of bovine brain homogenate or 100 ml of purified PAPII containing 1% (w/v) BSA, 20 ml of 26 mM Fmoc-Pro-Pro-CN, and 20 ml of 1.32 M 2-pyrrolidone was incubated for a range of times from 0 to 120 min at 377C with 400 ml of 0.1 mM õGlu-His-Pro-MCA in buffer A. The reaction was terminated by the addition of 960 ml of 1.5 M acetic acid. The reaction mixture was incubated at 807C for 30 min, following which the

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samples were cooled by incubation in an ice-water bath. Liberated MCA was determined as previously described. Linearity of the nonenzymatic cyclization PAPII assay with respect to enzyme concentration. A range of dilutions of purified PAPII was prepared using buffer A as diluant. Similarly, a range of dilutions of purified PAPII containing 1% (w/v) BSA was prepared using 1% (w/v) BSA in buffer A as diluant. One hundred microliters of each dilution, 20 ml of 26 mM Fmoc-Pro-ProCN, and 20 ml of 1.32 M 2-pyrrolidone was incubated for 1 h at 377C with 400 ml of 0.1 mM õGlu-His-ProMCA in buffer A. The reaction was terminated by the addition of 960 ml of 1.5 M acetic acid. The reaction mixture was incubated at 807C for 30 min or 2 h, following which the samples were cooled by incubation in an ice-water bath. Liberated MCA was determined as previously described. RESULTS AND DISCUSSION

Assays based on the hydrolysis of radiolabeled TRH have been used by many researchers for the determination of PAPII activity. Although sensitive and accurate for the quantitation of enzyme activity, these assays are labor-intensive and expensive. In 1986, Friedman and Wilk (18) developed a colorimetric, coupled enzyme assay for the determination of PAPII activity, based on hydrolysis of the substrate õGlu-His-Pro-bNA in the presence of excess DAPIV. A modification of this assay was described by O’Leary and O’Connor (28), using the quenched fluorimetric substrate õGlu-His-Pro-MCA. In order for assays based on the cleavage of TRH or the TRH substrate analogue, õGlu-His-Pro-MCA, to be specific for PAPII, it is important to ensure that other enzymes capable of its hydrolysis (prolyl edopeptidase and PAPI) are inhibited under assay conditions. Total inhibition of prolyl edopeptidase was achieved using Fmoc-Pro-Pro-CN, while PAPI was inhibited by 2-pyrrolidone. The inhibition of PAPI is particularly important when assaying crude homogenates which contain high levels of the cytosolic enzyme. Washed membrane preparations and further purified samples are less likely to contain PAPI activity; however, bacterial contamination of samples, thereby resulting in the presence of the bacterial PAP, has been observed (results not shown). Prolyl edopeptidase, although described as a predominantly cytosolic enzyme (44), has also been shown to be present in serum and in the particulate fraction of tissue homogenates, including synaptosomal membranes (45–47). Inhibition of this enzyme under assay conditions is therefore important when determining PAPII activity in serum and washed membrane fractions as well as in tissue homogenates. The DAPIV-catalyzed PAPII assay described herein

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Partial Purification of DAPIV from Bovine Serum Sample

Total activity (Units)

Total protein (mg)

Specific activity (Units/mg)

Purification factor (fold)

Yield (%)

Serum Post-phenyl Sepharose (concentrated)

116,389 73,253

1,616 205.7

72.02 356.12

1 4.9

100 62.9

is a modification of that previously described by O’Leary and O’Connor (28). In order to ensure that the assay is quantitative for the determination of PAPII activity, i.e., that MCA is liberated from all of the HisPro-MCA produced by the action of PAPII on the substrate õGlu-His-Pro-MCA, the two enzyme reactions were uncoupled. Following the termination of the PAPII reaction by heating the reaction mixture to 807C, DAPIV was added to catalyze His-Pro-MCA hydrolysis. This reaction was subsequently terminated by the addition of acetic acid. This procedure differs from that described by Friedman and Wilk (18) in which the PAPII and DAPIV reactions proceed simultaneously, thereby assuming that as His-Pro-bNA is being produced by the action of PAPII on the substrate, DAPIV catalyzes the liberation of bNA. Due to the fact that DAPIV is not commercially available, it was necessary to purify this enzyme in the laboratory for use in the DAPIV-catalyzed PAPII assay. Bovine serum was chosen as the source of the enzyme since it is readily available in large quantities and is rich in DAPIV activity. From 4.5 L of whole blood collected, 1 L of unclotted blood was obtained after 24 h at 47C. Following centrifugation, 800 ml of serum was obtained. Dialysis of 20 ml of bovine serum and subsequent centrifugation to remove the postdialysis precipitate, resulted in 21.5 ml of clear supernatant at pH 5.3. When this supernatant was applied to SP-Sepharose under the conditions described, the majority of DAPIV activity did not bind to the column, but merely ran through with the bulk of the protein. The bulk of the Z-Gly-Pro-MCA degrading activity, however, bound to the column and was eluted with 500 mM NaCl. Fractions containing the highest DAPIV activity were pooled (35 ml) and adjusted to a final concentration of 1 M ammonium sulfate in buffer B at pH 7.0. The sample (50 ml adjusted volume) was then subjected to hydrophobic interaction chromatography on phenyl Sepharose. This step successfully separated the remaining Z-Gly-Pro-MCA degrading activity from the greater part of the DAPIV activity which, once again, ran through the column. Fractions containing the highest DAPIV activity were pooled (45 ml), dialyzed, and concentrated to a final volume of 26 ml by reverse osmosis with polyethylene glycol. Following further dialysis

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into buffer B at pH 7.5, glycerol was added to a final concentration of 10% (v/v) (final volume, 29 ml). Despite the coelution of the major DAPIV peak with the major protein peak from both columns, thus reducing the purification achieved, it was decided that removal of contaminating prolyl endopeptidase activity was the most important consideration. An overall purification factor of only 4.9-fold was achieved using this procedure; however, a relatively high yield of 62.9% was obtained. The purification procedure is summarized in Table 1. Due to the fact that this partially purified DAPIV preparation was shown to be free from aminopeptidase activity (Pro-MCA and Ala-MCA degrading activities) and enzymes capable of õGlu-HisPro-MCA hydrolysis, PAP, and prolyl endopeptidase, it was decided that this material was suitable for use in the PAPII assay. Figure 1. illustrates the effect of increasing amounts of DAPIV on MCA release, following the reaction of brain homogenate or purified PAPII with õGlu-HisPro-MCA. The addition of 260 units of DAPIV activity (180 units/ml under assay conditions) is sufficient to catalyze the liberation of MCA from all of the His-ProMCA produced by the reaction of brain homogenate with the substrate. However, the DAPIV reaction increases the concentration of free MCA by only 4%. The liberation of such a high concentration of MCA without the addition of DAPIV can be explained by the fact that brain homogenate contains endogenous DAPIV and DAPII (EC 3.4.14.2) activities (44). Following the reaction of purified PAPII with õGlu-His-Pro-MCA, 1000 units of DAPIV activity (800 units/ml under assay conditions) must be added to catalyze the complete hydrolysis of His-Pro-MCA. Since the purified PAPII preparation contains no DAP activity, it is likely that the low concentration of MCA observed when no exogenous DAPIV is added occurs as a result of non-enzymatic cyclization of His-Pro-MCA to cyclo His-Pro and free MCA during the 2-h incubation. Although the DAPIV-catalyzed assay described has distinct advantages over radiolabeled assays, there are nonetheless some disadvantages. As mentioned previously, DAPIV is not commercially available and it must therefore be purified in the laboratory. Moreover, the use of a coupled enzyme assay may cause complica-

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His-Pro has been shown to occur in aqueous solution at pH 2–10 (48, 49). Following the termination of the PAPII reaction by the addition of acetic acid, the cyclization of His-Pro-MCA was promoted by incubating the reaction mixture at 807C. The effect of incubation time at 807C on MCA release, following the reaction of brain homogenate or purified PAPII with õGlu-His-ProMCA, is illustrated in Fig. 2. Following the reaction of brain homogenate with the substrate, only a short

FIG. 1. The effect of DAPIV on MCA release. Following the reaction of (A) bovine brain homogenate (l) or (B) purified PAPII (s) with õGlu-His-Pro-MCA, varying amounts of partially purified DAPIV were added as described under Materials and Methods. After further incubation for 1 h at 377C, liberated MCA was determined as described under Materials and Methods. Error bars represent the SEM of triplicate readings.

tions in PAPII characterization studies. For instance, when investigating the effect of pH, functional reagents, or metals on enzyme activity or in the determination of Ki values of peptides, it may be difficult to determine whether an observed result is due to an effect on PAPII, DAPIV, or both. For these reasons it was decided to develop a fluorimetric assay based on the nonenzymatic cyclisation of His-Pro-MCA to cyclo His-Pro and free MCA. The nonenzymatic cyclization of His-Pro-NH2 to cyclo

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FIG. 2. The effect of time at 807C on MCA release. Following the reaction of (A) bovine brain homogenate (l) or (B) purified PAPII (s) with õGlu-His-Pro-MCA, samples were incubated for a range of times at 807C as described under Materials and Methods. After cooling in an ice-water bath, liberated MCA was determined as described under Materials and Methods. Error bars represent the SEM of triplicate readings.

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FIG. 3. Linearity of the nonenzymatic cyclization PAPII assay with respect to enzyme concentration. A range of dilutions of purified PAPII containing (A) no BSA or (B) 1% (w/v) BSA were incubated for 1 h at 377C with õGlu-His-Pro-MCA. Following termination of the reaction by the addition of acetic acid, samples were incubated for 30 min (l) or 2 h (s) at 807C. After cooling in an ice-water bath, liberated MCA was determined as described under Materials and Methods. Error bars represent the SEM of triplicate readings.

incubation of 15–20 min at 807C is required to promote the complete cyclisation of His-Pro-MCA. An increase in the MCA concentration of only 10% was observed as a result of this incubation, indicating that the majority of His-Pro-MCA was already hydrolyzed by endogenous DAP activity in this sample. An incubation of 2 h at 807C was required for the complete cyclization of HisPro-MCA produced by the reaction of purified PAPII with õGlu-His-Pro-MCA. In the absence of endogenous

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DAP activity in this purified sample, the extremely low concentration of MCA observed when the reaction mixture was not incubated at 807C can be accounted for by a slow rate of His-Pro-MCA cyclization during the initial reaction at 377C. The possibility of shortening the incubation time necessary for the complete cyclization of His-Pro-MCA was considered. In view of the fact that the cyclisation of His-Pro-NH2 has been shown to occur more rapidly at pH 6–7 (48), the possibility of terminating the PAPII reaction by means other than the addition of acetic acid, which reduces the pH of the reaction mixture to approximately 2.2, was considered. The PAPII reaction could be terminated by transferring the reaction mixture directly to an 807C water bath where the cyclization reaction could proceed at pH 7.5. However, this option was not pursued further since it was decided that the addition of acetic acid negated the possibility of intersample pH variations affecting the rate of cyclization. Slight pH variations within the pH range 6–8 have profound effects on the rate of cyclisation of HisPro-NH2 , while slight pH variations within the pH range 2–3 have little or no effect on the rate of cyclization (48). Since all of the enzyme assays described herein are based on the release of MCA from specific substrates, a brief discussion on fluorescence spectrometry is warranted. Liberated MCA was quantified by reference to standard curves, prepared under assay conditions. The inclusion of biological samples such as 100 ml of bovine serum or bovine brain homogenate in the standards, results in a decrease of 26% and 6% respectively in the slope of the standard curve (data not shown). This decrease is due primarily to two factors; the inner filter effect and quenching. (50). Failure to prepare MCA standard curves under conditions identical to those in the assay would have resulted in erroneous determination of enzyme activity and, therefore, misinterpretation of results. Due to the nature of the assays described for the fluorimetric determination of PAPII activity, both of which require a second step after the initial reaction of the enzyme with the substrate, continuous monitoring of activity is not possible. These methods therefore represent discontinuous assays in which the amount of product formed by the enzyme is measured at one discrete point on the reaction curve. With such assay systems it is important to ensure that the measurement is taken from the linear part of the progress curve, thereby representing the initial velocity of the reaction (51). Using either brain homogenate or purified PAPII as the enzyme source and employing a 30-min incubation at 807C to promote cyclisation of His-Pro-MCA, a linear relationship between product formed (MCA) and incu-

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bation time at 377C was observed over a 2-h period (data not shown). It is therefore clear that the assays described give a true measure of the initial activity of PAPII since an incubation time of only 1 h at 377C is employed. In order to ensure that the nonenzymatic cyclization assay is quantitative for the measurement of PAPII activity, standard curves of enzyme concentration versus MCA release were constructed. Figure 3(A) illustrates a nonlinear relationship between MCA release and the concentration of purified PAPII. An upward curvature such as this is usually indicative of the presence of an enzyme activator, dilution of the enzyme sample reducing the concentration of the activator below that at which optimal enzyme activity is expressed (52). In this case, protein can be considered to be the enzyme activator or, perhaps more correctly, the absence of protein–protein interactions in the dilute sample causes enzyme inactivation. The presence of 1% (w/v) BSA in the enzyme results in a linear relationship between product formed and enzyme concentration, when cyclization of His-Pro-MCA is promoted by either 30 min or 2 h incubation at 807C, as illustrated in Fig. 3(B). The results presented demonstrate that the nonenzymatic cyclization assay is suitable for the quantitative determination of PAPII activity in both crude and purified samples. Following the initial reaction of PAPII with the substrate õGlu-His-Pro-MCA, the cyclization of His-Pro-MCA may be promoted by a 2-h incubation period at 807C when an absolute value of enzyme activity is required. Where relative values of enzyme activity are sufficient, the shorter assay, in which the reaction mixture is incubated for 30 min at 807C, may be employed. Both the DAPIV catalyzed assay and the spontaneous cyclisation assays described herein offer expeditious alternatives to radiolabeled TRH-based assays for the determination of PAPII activity. It is hoped that the development of these fluorimetric assays will aid further characterization of PAPII, ultimately leading to the elucidation of its physiological role. ACKNOWLEDGMENTS We gratefully acknowledge the financial assistance of Forbairt, Ireland, for awarding maintenance grants to Sea´n Gallagher and Rhona O’Leary and the Health Research Board, Ireland for awarding a maintenance grant and Rhona O’Leary. Our thanks also to Dr. Sherwin Wilk for generously providing Fmoc-Pro-Pro-CN.

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