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• W2141150756 abstract "In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (kcat/Km) between 9.4 × 104m−1 s−1(Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp; where Abz is οrtho-aminobenzoic acid and EDDnp isN-(2,4-dinitrophenyl)ethylenediamine)) and 0.24 × 104m−1 s−1(Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 × 103m−1s−1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 × 104m−1s−1 (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited kcat of less than 0.05 s−1. Substitution of ornithine for Lys at the P4 position did not significantly affect the kcat but increased the Km 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1. These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters. In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. In this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (kcat/Km) between 9.4 × 104m−1 s−1(Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp; where Abz is οrtho-aminobenzoic acid and EDDnp isN-(2,4-dinitrophenyl)ethylenediamine)) and 0.24 × 104m−1 s−1(Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 × 103m−1s−1 (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 × 104m−1s−1 (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited kcat of less than 0.05 s−1. Substitution of ornithine for Lys at the P4 position did not significantly affect the kcat but increased the Km 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1. These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters. Prohormones and proneuropeptides are synthesized as inactive large precursors that are proteolytically cleaved during intracellular transport to generate active peptide forms for extracellular release (1Mains R.E. Dickerson I.M. May V. Stoffers D.A. Perkins S.N. Ouafik L. Husten E.J. Eipper B. Front. Neuroendocrinol. 1990; 11: 52-89Google Scholar, 2Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (311) Google Scholar). Pairs of basic amino acid residues such as Lys-Arg and Arg-Arg- and to a certain extent Lys-Lys and Arg-Lys- have been recognized as consensus sites of proteolytic cleavage (1Mains R.E. Dickerson I.M. May V. Stoffers D.A. Perkins S.N. Ouafik L. Husten E.J. Eipper B. Front. Neuroendocrinol. 1990; 11: 52-89Google Scholar, 2Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (311) Google Scholar). Primary and secondary structures in proproteins are thought to be important for enzyme recognition and selectivity of cleavage (1Mains R.E. Dickerson I.M. May V. Stoffers D.A. Perkins S.N. Ouafik L. Husten E.J. Eipper B. Front. Neuroendocrinol. 1990; 11: 52-89Google Scholar, 2Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (311) Google Scholar, 3Brakch N. Rholam M. Boussetta H. Cohen P. Biochemistry. 1993; 32: 4925-4930Crossref PubMed Scopus (53) Google Scholar). The involvement of prohormone convertases 1 and 2 (PC1 1The abbreviations used are: PC1prohormone convertase 1PC2prohormone convertase 2IQinternally quenchedAbzοrtho-aminobenzoic acidMCAmethylcoumarin amideEDDnpN-(2,4-dinitrophenyl)ethylenediamine)HPGPChigh pressure gel permeation chromatographyLeu-enkLeu-enkephalinMCAmethylcoumarin amide<Glupyroglutamic acidBoct-butoxycarbonylMet-enkmet-enkephalinBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPEproenkephalinRIAradioimmunoassayMALDI-TOFmass spectroscopy laser desorption ionization-time of flightHPLChigh pressure liquid chromatography.1The abbreviations used are: PC1prohormone convertase 1PC2prohormone convertase 2IQinternally quenchedAbzοrtho-aminobenzoic acidMCAmethylcoumarin amideEDDnpN-(2,4-dinitrophenyl)ethylenediamine)HPGPChigh pressure gel permeation chromatographyLeu-enkLeu-enkephalinMCAmethylcoumarin amide<Glupyroglutamic acidBoct-butoxycarbonylMet-enkmet-enkephalinBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolPEproenkephalinRIAradioimmunoassayMALDI-TOFmass spectroscopy laser desorption ionization-time of flightHPLChigh pressure liquid chromatography. and PC2), serine proteinases expressed in neuroendocrine tissues, in precursor cleavage has been widely accepted (1Mains R.E. Dickerson I.M. May V. Stoffers D.A. Perkins S.N. Ouafik L. Husten E.J. Eipper B. Front. Neuroendocrinol. 1990; 11: 52-89Google Scholar, 2Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (311) Google Scholar) although other enzymes have recently also been reported to be involved in opioid peptide precursor cleavage (4Hook V.Y.H. Schiller M.R. Azaryan A.V. Arch. Biochem. Biophys. 1996; 328: 107-114Crossref PubMed Scopus (31) Google Scholar). prohormone convertase 1 prohormone convertase 2 internally quenched οrtho-aminobenzoic acid methylcoumarin amide N-(2,4-dinitrophenyl)ethylenediamine) high pressure gel permeation chromatography Leu-enkephalin methylcoumarin amide pyroglutamic acid t-butoxycarbonyl met-enkephalin 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol proenkephalin radioimmunoassay mass spectroscopy laser desorption ionization-time of flight high pressure liquid chromatography. prohormone convertase 1 prohormone convertase 2 internally quenched οrtho-aminobenzoic acid methylcoumarin amide N-(2,4-dinitrophenyl)ethylenediamine) high pressure gel permeation chromatography Leu-enkephalin methylcoumarin amide pyroglutamic acid t-butoxycarbonyl met-enkephalin 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol proenkephalin radioimmunoassay mass spectroscopy laser desorption ionization-time of flight high pressure liquid chromatography. Previous reports from our laboratory using cell-based systems have provided support for the idea that PC1 is the chief enzyme concerned with the generation of intermediate-sized peptides from proenkephalin (PE) and that PC2 is mostly responsible for the production of small, bioactive opioid peptides (5Breslin M.B. Lindberg I. Benjannet S. Mathis J.P. Lazure C. Seidah N.G. J. Biol. Chem. 1993; 268: 27084-27093Abstract Full Text PDF PubMed Google Scholar, 6Johanning K. Mathis J.P. Lindberg I. J. Neurochem. 1996; 66: 898-907Crossref PubMed Scopus (38) Google Scholar). The profile of peptides generated in PE-expressing AtT-20 cells (7Mathis J. Lindberg I. Endocrinology. 1992; 131: 2287-2296Crossref PubMed Scopus (40) Google Scholar), which contain high quantities of endogenous PC1 (8Seidah N.G. Gaspar L. Mion P. Marcinkiewicz M. Mbikay M. Chretien M. DNA Cell Biol. 1990; 9: 415-424Crossref PubMed Scopus (364) Google Scholar), consists mainly of 3–18-kDa enkephalin-containing peptides, although some mature enkephalins such as Met-enk-Arg-Phe and Met-enk are also present (7Mathis J. Lindberg I. Endocrinology. 1992; 131: 2287-2296Crossref PubMed Scopus (40) Google Scholar). In contrast, the major PE products in PC2-containing AtT-20/PE cells, as well as in PC2-expressing Rin-PE cells, are the fully processed opioid peptides Met-enk-Arg-Phe, Met-enk-Arg-Gly-Leu, Met-enk, and Leu-enk (6Johanning K. Mathis J.P. Lindberg I. J. Neurochem. 1996; 66: 898-907Crossref PubMed Scopus (38) Google Scholar, 7Mathis J. Lindberg I. Endocrinology. 1992; 131: 2287-2296Crossref PubMed Scopus (40) Google Scholar). In agreement with the notion that PC2 expression is correlated with more complete processing of PE, antisense experiments have shown that PC2 is largely responsible for the processing of PE into smaller opioid peptides in Rin cells (6Johanning K. Mathis J.P. Lindberg I. J. Neurochem. 1996; 66: 898-907Crossref PubMed Scopus (38) Google Scholar). These data imply that PC2 can cleave at a wider range of sites within PE than PC1; however, the structural factors that differentiate PC1 from PC2 cleavage sites remain unclear. Since the PCs have only recently become available in recombinant form (9Zhou Y. Lindberg I. J. Biol. Chem. 1993; 268: 5615-5623Abstract Full Text PDF PubMed Google Scholar, 10Jean F. Basak A. Rondeau N. Benjannet S. Hendy G.N. Seidah N.G. Chretien M. Lazure C. Biochem. J. 1993; 292: 891-900Crossref PubMed Scopus (87) Google Scholar, 11Rufaut N.W. Brennan S.O. Hakes D.J. Dixon J.E. Birch N.P. J. Biol. Chem. 1993; 268: 20291-20298Abstract Full Text PDF PubMed Google Scholar, 12Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (85) Google Scholar), there is limited information on their reaction and kinetics with natural substrates. Examples of studies on the cleavage of naturally occurring peptides by recombinant PC1 include the cleavage of proopiomelanocortin (13Friedman T.C. Loh Y.P. Birch N.P. Endocrinology. 1994; 135: 854-862Crossref PubMed Scopus (40) Google Scholar), anthrax toxin-protective antigen (14Friedman T.C. Gordon V.M. Leppla S.H. Klimpel K.R. Birch N.P. Loh Y.P. Arch. Biochem. Biophys. 1995; 316: 5-13Crossref PubMed Scopus (14) Google Scholar) pro-thyrotropin-releasing hormone by PC1 (15Nillni E.A. Friedman T.C. Todd R.B. Birch N.P. Loh Y.P. Jackson I.M. J. Neurochem. 1995; 65: 2462-2472Crossref PubMed Scopus (54) Google Scholar), prodynorphin (16Dupuy A. Lindberg I. Zhou Y. Akil H. Lazure C. Chretien M. Seidah N.G. Day R. FEBS Lett. 1994; 337: 60-65Crossref PubMed Scopus (72) Google Scholar), and proalbumin (17Ledgerwood E.C. Brennan S.O. Birch N.P. George P.M. Biochem. Mol. Biol. Int. 1996; 39: 1167-1176PubMed Google Scholar). Studies of recombinant PC2 cleavage on natural substrates include proglucagon (18Rothenberg M.E. Eilertson C.D. Klein K. Zhou Y. Lindberg I. McDonald J.K. Mackin R.B. Noe R.B. J. Biol. Chem. 1995; 270: 10136-10146Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 19Rouille Y. Bianchi M. Irminger J.C. Halban P.A. FEBS Lett. 1997; 413: 119-123Crossref PubMed Scopus (55) Google Scholar), cholecystokinin-33 (20Wang W. Beinfeld M.C. Biochem. Biophys. Res. Commun. 1997; 231: 149-152Crossref PubMed Scopus (19) Google Scholar), and prodynorphin (21Day R. Lazure C. Basak A. Boudreault A. Limperis P. Dong W. Lindberg I. J. Biol. Chem. 1998; 273: 829-836Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Comparative work on both enzymes includes reports on the cleavage of proneuropeptide Y (22Brakch N. Rist B. Beck-Sickinger A.G. Goenaga J. Wittek R. Burger E. Brunner H.R. Grouzmann E. Biochemistry. 1997; 36: 16309-16320Crossref PubMed Scopus (41) Google Scholar) and proinsulin (23Furuta M. Carroll R. Martin S. Swift H.H. Ravazzola M. Orci L. Steiner D.F. J. Biol. Chem. 1998; 273: 1-7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 24Bailyes E.M. Shennan K.I.J. Usac E.F. Arden S.D. Guest P.C. Docherty K. Hutton J.C. Biochem. J. 1995; 309: 587-594Crossref PubMed Scopus (17) Google Scholar). Taken together, this work supported the idea that both prohormone convertases prefer paired basic cleavage sites containing a P4 basic residue and can cleave at single basic residues given the presence of additional amino-terminal basic residues. However, in general, work with natural substrates has not revealed additional preferences for residues surrounding the cleavage site. Synthetic fluorogenic and internally quenched peptides and methylcoumarin amide (MCA)-containing substrates have been used to determine kinetic parameters of subtilisin-like enzymes and to explore the structural features surrounding the dibasic cleavage sites that contribute to enzyme specificity (25Jean F. Basak A. Dimaio J. Seidah N.G. Lazure C. Biochem. J. 1995; 307: 689-695Crossref PubMed Scopus (45) Google Scholar, 26Basak A. Schmidt C. Ismail A.F. Seidah N.G. Chretien M. Lazure C. Int. J. Pept. Protein Res. 1995; 46: 228-237Crossref PubMed Scopus (28) Google Scholar). In this study we have investigated the kinetic properties of PC2 against proenkephalin-related internally quenched substrates and a series of fluorogenic peptides. In addition, we examined the hydrolysis of recombinant PE in vitro by PC1 and PC2. Finally, we have used PC2 knock-out mice (27Furuta M. Yano H. Zhou A. Rouille Y. Holst J.J. Carroll R. Ravazzola M. Orci L. Furuta H. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6646-6651Crossref PubMed Scopus (346) Google Scholar) to confirm the involvement of this enzyme in the natural processing of PE. To determine the specificity of PC2 for proenkephalin-derived peptides, internally quenched Abz-peptidyl-EDDnp substrates (where Abz isortho-aminobenzoic acid and EDDnp is N-(2, 4-dinitrophenyl)ethylenediamine) were synthesized and purified as described previously (28Hirata Y. Cezari M.H.S. Nakaie C.R. Boschcov P. Ito A.S. Juliano M.A. Juliano L. Lett. Peptide Sci. 1994; 1: 299-308Crossref Scopus (199) Google Scholar, 29Del Nery E. Juliano M.A. Meldal M. Svendsen I. Scharfstein J. Walmsley A. Juliano L. Biochem. J. 1997; 323: 427-433Crossref PubMed Scopus (85) Google Scholar). The molecular weight and purity of synthesized peptides were checked by mass spectroscopy laser desorption ionization-time of flight (MALDI-TOF) using TofSpec-E from Micromass. In these intramolecularly quenched peptides the Abz group is attached to the amino terminus and the EDDnp moiety to a carboxyl-terminal glutamine, necessary for the solid-phase peptide synthesis (29Del Nery E. Juliano M.A. Meldal M. Svendsen I. Scharfstein J. Walmsley A. Juliano L. Biochem. J. 1997; 323: 427-433Crossref PubMed Scopus (85) Google Scholar). A total of 11 substrates, 11–12 amino acids in length, was synthesized corresponding to the sequences surrounding PE cleavage sites (Fig. 1). In addition, another set of 10 substrates was synthesized that corresponded to analogs of a 12-residue peptide B-like sequence (Abz-VPEMEKRYGGFMQ-EDDnp). In the latter peptides, Arg and Ala were substituted at various positions from P3 to P7 to determine the effect of sequence preference. To investigate the potential requirement for length, an extended peptide B-related substrate was synthesized (Abz-LPSDEEGESYSKEVPEMEKRYGGFMQ-EDDnp). Recombinant mPC2 was overexpressed in Chinese hamster ovary cells using the dihydrofolate reductase-coupled amplification method previously described (30Lindberg I. Shaw E. Finley J. Leone D. Deininger P. Endocrinology. 1991; 128: 1849-1856Crossref PubMed Scopus (25) Google Scholar); cells were subsequently stably supertransfected with cDNAs encoding 21-kDa rat 7B2 (12Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (85) Google Scholar). Cells were grown in a Cellmax artificial capillary cell culture system (Cellco, Germantown, MD). PC2 was purified from 20 ml of conditioned media (in which the primary proteins present were the 71- and 75-kDa proenzyme forms) diluted 1:3 in buffer A on a 5 × 50-mm Protein-Pak anion-exchange column (Waters Chromatography, Milford, MA) using a step gradient first from 0 to 35% B in 175 min at a flow rate of 0.25 ml/min, followed by a further gradient to 100% B in 50 min at 0.50 ml/min. Buffer A was 20 mm Bis-Tris, 0.1% Brij, pH 6.5, and buffer B was 1 m sodium acetate, 20 mm Bis-Tris, 0.1% Brij, pH 6.5. Two-ml fractions were collected and assessed for purity on 8.8% gels using Coomassie staining. Recombinant rat PE was overexpressed in Chinese hamster ovary cells using the dihydrofolate-reductase amplification method (30Lindberg I. Shaw E. Finley J. Leone D. Deininger P. Endocrinology. 1991; 128: 1849-1856Crossref PubMed Scopus (25) Google Scholar). Recombinant PE was purified from the conditioned medium essentially as described previously (30Lindberg I. Shaw E. Finley J. Leone D. Deininger P. Endocrinology. 1991; 128: 1849-1856Crossref PubMed Scopus (25) Google Scholar) using a 4.6 × 25-mm Vydac semi-preparative C4 column (Vydac, Hesperia, CA) by elution with 80% acetonitrile in 0.1% trifluoroacetic acid. Recombinant PC1 was produced by the same method, but cells were grown in a Cellmax artificial capillary cell culture system as for PC2 (Cellco, Germantown, MD). PC1 was purified from 20 ml of conditioned medium diluted 1:3 in buffer A on a 5 × 50-mm Protein-Pak anion-exchange column (Waters Chromatography, Milford, MA) using a gradient from 0 to 100% B in 120 min. The buffers used were identical to those employed for PC2. The flow rate was 0.50 ml/min, and 1-ml fractions were collected. PC1 fractions were assessed for purity as above. The actual concentration of each substrate was determined using a spectrophotometer by measuring the EDDnp absorbance at 365 nm (EDDnp extinction coefficient, 17, 300m−1). A stock solution of 1 mmpeptide was prepared in dimethyl sulfoxide. Internally quenched substrates (final dilutions of 2 μm and 200 nm) were subjected to digestion by recombinant mouse PC2 at 37 °C in a buffer containing 100 mm sodium acetate, pH 5.0, 5 mm calcium chloride, and 0.1% Brij in a total volume of either 1 ml or 250 μl. Cuvettes and buffer were kept at 37 °C prior to the addition of substrate. Recombinant pro-PC2 was diluted 1:3 with the above buffer and incubated for 20 min at 37 °C to obtain the 66-kDa autoactivated form of this enzyme (12Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (85) Google Scholar). The specific activity of the preparation under saturating substrate concentrations was 29 μmol/h/mg. Substrate was added to reaction buffer in the cuvette, and the cuvette was placed in the thermostated fluorometer (37 °C) for equilibration. Abz fluorescence of each substrate was measured with a Perkin-Elmer fluorometer (λexcitation = 320 nm; λemission = 420 nm) at time 0 and was recorded at various points after the addition of PC2 (36 nm final concentration). These reaction conditions represent pseudo first-order conditions. Fluorescence data were fitted to a first-order curve by nonlinear regression (one phase exponential decay) using GraphPad version 2.0 (ISI Software, CA) as shown in Equation 1.Y=Spane−kt+plateauEquation 1 where Y is the amplitude of the fluorescence change; k is the apparent first- order rate constant; and plateau is the fluorescence at the end point of the reaction. The resultant apparent first-order rate constants were divided by the moles of enzyme (as calculated from the protein concentration and assuming a molecular mass of 66 kDa for activated PC2). These experiments were always performed in duplicate and, for the three best substrates of each group, at two different substrate concentrations. In addition, six IQ substrates (Fig. 1, 1, 2, 5, 7, 9, and 10) were subjected to PC1 digestion under the same pseudo first-order rate conditions (2 μm substrate). PC1 (115 nmfinal) was preincubated in 100 mm sodium acetate, pH 5.5, 5 mm calcium chloride, 0.1% Brij for 1 h in order to attain linear cleavage rates (9Zhou Y. Lindberg I. J. Biol. Chem. 1993; 268: 5615-5623Abstract Full Text PDF PubMed Google Scholar). The enzyme was then added to the substrate as described above for PC2 digestions in a total volume of 1 ml. To verify cleavage at the dibasic pair, each Abz-peptidyl-EDDnp substrate (100 μm) was incubated overnight with PC2 (5 nm) in the same reaction mixture as described above for subsequent isolation of the product by HPLC and was identified by mass spectroscopy (MALDI-TOF). Custom synthesis of Cbz-Arg-Ser-Lys-Arg-MCA (where Cbz is benzyloxycarbonyl) was performed by Enzyme Systems Products (Dublin, CA) and verified by amino acid composition. The 7B2 hCT1-31 peptide was synthesized by LSUMC Core Laboratories. Boc-Gly-Arg-Arg-MCA, Boc-Gly-Lys-Arg-MCA, Boc-Val-Pro-Arg-MCA, Boc-Val-Leu-Lys-MCA, and <Glu-Arg-Thr-Lys-Arg-MCA were purchased from Peptides International, Inc. (Louisville, KY). All other fluorogenic substrates were synthesized at the Clinical Research Institute of Montreal, Canada, as described elsewhere (31Jean F. Boudreault A. Basak A. Seidah N.G. Lazure C. J. Biol. Chem. 1995; 270: 19225-19231Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). A series of MCA-based fluorogenic substrates was tested for hydrolysis rates by PC2 (3.7 to 260 ng, depending on the rate of hydrolysis) in 100 mm sodium acetate buffer, pH 5.0, containing 5 mm calcium chloride, 0.1% Brij, and 100 nm 21-kDa 7B2 (which stabilizes the enzyme; Ref. 12Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (85) Google Scholar) at 37 °C. Various concentrations of the different peptides were used, as indicated in the figure legends, and the rates of hydrolysis were determined by fluorometry. The background fluorescence in each substrate at the start of incubation was subtracted from the final values. Km and Vmax values were obtained by applying Michaelis-Menten kinetics to the data using the Enzfitter program (Elsevier-Biosoft). PC assays were performed in duplicate at 37 °C in a buffer consisting of 100 mmsodium acetate, 5 mm calcium chloride, and 0.1% Brij (final concentrations); the pH of the sodium acetate buffer in PC1-containing reactions was 5.5, and the pH of PC2-containing reactions was 5.0. Activity was estimated using the fluorogenic substrate <Glu-Arg-Thr-Lys-Arg-MCA. Forkcat/Km determinations, pro-PC2 was preincubated in assay buffer for 20 min at 37 °C for conversion to the 66-kDa active form (12Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (85) Google Scholar). The reactions were initiated by the addition of substrate. All reactions were carried out in a 50-μl total reaction volume in a polypropylene microtiter plate as described previously (12Lamango N.S. Zhu X. Lindberg I. Arch. Biochem. Biophys. 1996; 330: 238-250Crossref PubMed Scopus (85) Google Scholar). Free amino methyl coumarin was measured with a Cambridge Technology microtiter plate fluorometer (Watertown, MA) at λexcitation = 380 nm and λemission = 460 nm. For the time course digestion experiment PC1 and PC2 (0.24 μg; 55 and 72 nmfinal concentrations, respectively) were preincubated at the appropriate pH values (1 h for PC1 at pH 5.5 and 20 min for PC2 at pH 5.0) as described above in a 50-μl reaction volume; recombinant rat PE (1.25 μg; 0.91 μm final concentration) was then added. The reaction mixtures were incubated for 0, 10, 30, 60, 90 and 120 min, at which time 1/10 volume of a 10× solution of Laemmli sample buffer (0.5 m Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 10% glycerol, 2% SDS, and 0.06 mg/ml bromphenol blue) was added. Samples were then boiled, and a tenth of the reaction mixture was subjected to electrophoresis on a 10–20% SDS-polyacrylamide gradient gel (Bio-Rad). Proteins were then transferred to nitrocellulose and subjected to Western blotting as described previously (6Johanning K. Mathis J.P. Lindberg I. J. Neurochem. 1996; 66: 898-907Crossref PubMed Scopus (38) Google Scholar). The antiserum used was raised against peptide F (32Christie D.L. Birch N.P. Aitken J.F. Harding D.R. Hancock W.S. Biochem. Biophys. Res. Commun. 1984; 120: 650-656Crossref PubMed Scopus (7) Google Scholar) and recognizes proenkephalin as well as PE cleavage products containing peptide F. Chromaffin granule total protein (about 20 μg) was subjected to electrophoresis to compare the pattern of natural cleavage products to those resulting from the digestion with PC1 and PC2. All experiments were repeated at least twice. PE (450 nm final concentration) was incubated with preincubated PC2 (40 nm) in 100 mm acetate buffer, pH 5.0, at 37 °C for 30 min. The reaction mixture was then acidified with trifluoroacetic acid (0.1% final concentration) and frozen until HPGPC, performed as described previously (33Johanning K. Mathis J.P. Lindberg I. J. Biol. Chem. 1996; 271: 27871-27878Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). HPGPC fractions were collected into polypropylene tubes to which 5 μg of bovine serum albumin had been added as carrier. Duplicate aliquots of each fraction were subjected to enkephalin radioimmunoassay as described previously (34Lindberg I. Yang H.Y. Brain Res. 1984; 299: 73-78Crossref PubMed Scopus (41) Google Scholar), either untreated or following treatment with trypsin and carboxypeptidase B (35Lindberg I. Thomas G. Endocrinology. 1990; 126: 480-487Crossref PubMed Scopus (13) Google Scholar) to reveal cryptic enkephalin sequences and to remove carboxyl-terminal basic amino acids that interfere with immunoreactivity. Enkephalin antisera used were raised against Met-enk-Arg-Phe (JAS; Ref. 36Mocchetti I. Giorgi O. Schwartz J.P. Costa E. Eur. J. Pharmacol. 1984; 106: 427-430Crossref PubMed Scopus (27) Google Scholar), Met-enk (RB4; Ref. 37Giraud P. Eiden L.E. Audigier Y. Gillioz Conte-Devols B. Bourdouresque F. Eskay R. Oliver C. Neuropeptides. 1981; 1: 237-252Crossref Scopus (36) Google Scholar), and Leu-enk (38Lindberg I. Shaw E. J. Neurochem. 1992; 58: 448-453Crossref PubMed Scopus (10) Google Scholar). PE digestions with PC2 and RIAs were carried out at least twice with similar results. Frozen brains from PC2-knock-out and wild-type control mice were thawed and immediately homogenized in 5 volumes of ice-cold 1n acetic acid, 20 mm HCl, and 0.1% β-mercaptoethanol. After centrifugation at 10,000 × g for 30 min, half of the supernatant was lyophilized and resuspended in 250 μl of 32% acetonitrile containing 0.1% trifluoroacetic acid for gel permeation analysis. High pressure gel permeation was carried out as described previously (6Johanning K. Mathis J.P. Lindberg I. J. Neurochem. 1996; 66: 898-907Crossref PubMed Scopus (38) Google Scholar) except that the flow rate was 0.40 ml/min, and 0.40 ml was collected per fraction. Aliquots of fractions were assayed for the various enkephalins by RIA, in certain cases following tryptic digestion and carboxypeptidase B trimming as described previously (35Lindberg I. Thomas G. Endocrinology. 1990; 126: 480-487Crossref PubMed Scopus (13) Google Scholar), and the results were reported per fraction. The experiment was repeated once with a separate set of brains. Fig. 1 is a diagrammatic representation of rat PE that depicts the different cleavage sites and peptides known to result from proteolytic cleavage of this precursor molecule (39Udenfriend S. Kilpatrick D.L. Arch. Biochem. Biophys. 1983; 221: 309-323Crossref PubMed Scopus (205) Google Scholar). The numbers above the PE structure indicate the 11–12-residue internally quenched peptides used for studies of PC2 specificity. As shown in the diagram, peptide B is an example of an intermediate that results from cleavage at a paired basic site and that serves as a substrate for generation of the opioid peptide Met-enk-Arg-Phe. Peptide E, an intermediate-sized peptide, can give rise to both Met-enk and Leu-enk. Whereas internally quenched peptide substrate series represent an effective means to determine enzyme preference at various subsites (25Jean F. Basak A. Dimaio J. Seidah N.G. Lazure C. Biochem. 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• W2141150756 title "Specificity of Prohormone Convertase 2 on Proenkephalin and Proenkephalin-related Substrates" @default.
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