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Purification, Characterization, and Cloning of a Cytosolic Aspartyl Aminopeptidase

1998; Elsevier BV; Volume: 273; Issue: 26 Linguagem: Inglês

10.1074/jbc.273.26.15961

1083-351X

Sherwin Wilk, Elizabeth Wilk, Ronald P. Magnusson,

Signaling Pathways in Disease

An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4.11.7) was purified to near homogeneity from rabbit brain cytosol. Its properties were similar to an enzyme described previously (Kelly, J. A., Neidle, E. L., and Neidle, A. (1983) J. Neurochem. 40, 1727–1734). Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates. Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5). The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis. The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags. Human and mouse clones described as “similar to a yeast vacuolar aminopeptidase” and containing full-length cDNAs were identified and sequenced. The human cDNA was expressed in Escherichia coli. The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases. Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates. Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism. An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4.11.7) was purified to near homogeneity from rabbit brain cytosol. Its properties were similar to an enzyme described previously (Kelly, J. A., Neidle, E. L., and Neidle, A. (1983) J. Neurochem. 40, 1727–1734). Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates. Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5). The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis. The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags. Human and mouse clones described as “similar to a yeast vacuolar aminopeptidase” and containing full-length cDNAs were identified and sequenced. The human cDNA was expressed in Escherichia coli. The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases. Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates. Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism. Aminopeptidases catalyze the sequential removal of amino acids from the unblocked N termini of peptides and proteins. These enzymes are widely distributed in eukaryotes and prokaryotes (1Taylor A. FASEB J. 1993; 7: 290-298Crossref PubMed Scopus (483) Google Scholar, 2Gonzales T. Robert-Baudroy J. FEMS Microbiol. Rev. 1996; 18: 319-344Crossref PubMed Google Scholar) as either integral membrane or cytosolic proteins. Aminopeptidases are generally classified in terms of their substrate specificities, i.e.preference for a neutral, acidic, or basic amino acid in the P1 position. In the case of X-Pro aminopeptidase (aminopeptidase P; E.C. 3.4.11.9), it is the amino acid in the P1′ position that governs specificity. Most aminopeptidases are metalloenzymes, although cysteine and serine aminopeptidases have been described (2Gonzales T. Robert-Baudroy J. FEMS Microbiol. Rev. 1996; 18: 319-344Crossref PubMed Google Scholar). In addition to their role in general protein and peptide metabolism, aminopeptidases have more specific functions. These include activation (3Cadel S. Pierotti A.R. Foulon T. Creminon C. Barre N. Segretain D. Cohen P. Mol. Cell Endocrinol. 1995; 110: 149-160Crossref PubMed Scopus (68) Google Scholar) and inactivation (4Hersh L.B. Aboukhair N. Watson S. Peptides. 1987; 8: 523-532Crossref PubMed Scopus (73) Google Scholar) of biologically active peptides, removal of the N-terminal methionine of newly synthesized proteins (5Kendall R.L. Bradshaw R.A. J. Biol. Chem. 1992; 267: 20667-20673Abstract Full Text PDF PubMed Google Scholar) and possibly in the trimming of antigens for presentation by the major histocompatibility complex-1 system (6Harris C.A. Hunte B. Krauss M.R. Taylor A. Epstein L.B. J. Biol. Chem. 1992; 267: 6865-6869Abstract Full Text PDF PubMed Google Scholar). The removal of N-terminal aspartyl and glutamyl residues from proteins and peptides in eukaryotes is catalyzed by glutamyl aminopeptidase (aminopeptidase A; EC 3.4.11.7), an enzyme first described by Glenner and Folk in 1961 (7Glenner G.G. Folk J.E. Nature. 1961; 192: 338-340Crossref PubMed Scopus (75) Google Scholar) (for a review, see Ref. 8Wilk S. Healy D.P. Adv. Neuroimmunol. 1993; 3: 195-207Abstract Full Text PDF Scopus (67) Google Scholar). This membrane-bound ectoenzyme is a member of the metalloproteinase family M1 and contains the HEXXH + E zinc binding ligands (9Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-228Crossref PubMed Scopus (695) Google Scholar). Glutamyl aminopeptidase, first cloned as the murine BP-1/6C3 antigen (10Wu Q. Lahti J.M. Air G.M. Burrows P.D. Cooper M.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 993-997Crossref PubMed Scopus (144) Google Scholar), is a protein of 945 amino acids with a molecular mass of 107.8 kDa. The purified porcine, human, rat, and mouse enzymes are isolated as homodimers (8Wilk S. Healy D.P. Adv. Neuroimmunol. 1993; 3: 195-207Abstract Full Text PDF Scopus (67) Google Scholar). A distinguishing feature of glutamyl aminopeptidase is its stimulation by Ca2+ (11Glenner G.G. McMillan P.J. Folk J.E. Nature. 1962; 194: 867-868Crossref PubMed Scopus (103) Google Scholar). The ability of glutamyl aminopeptidase to degrade angiotensins I and II is of considerable interest. The action of glutamyl aminopeptidase on angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) yields des-Asp angiotensin II, also known as angiotensin III. There is evidence that angiotensin III may mediate some of the effects of the renin-angiotensin system in the brain (12Wright J.W. Harding J.W. Brain Res. Rev. 1992; 17: 227-262Crossref PubMed Scopus (275) Google Scholar). A recent study utilizing 3-amino-4-thio-butyl sulfonate, a specific inhibitor of this enzyme, provided evidence for a predominant role of angiotensin III in the control of vasopressin release (13Zini S. Fournie-Zaluski M-C. Chauvel E. Roques B.P. Corvol P. Llorens-Cortes C. Proc. Natl. Acad. U. S. A. 1996; 93: 11968-11973Crossref PubMed Scopus (290) Google Scholar). The same inhibitor was used to establish a major role of glutamyl aminopeptidase in the metabolism of cholecystokinin-8 (14Migaud M. Durieux C. Viereck J. Soroca-Lucas E. Fournie-Zaluski M-C. Roques B. Peptides. 1996; 17: 601-607Crossref PubMed Scopus (54) Google Scholar). The literature also contains two reports of a cytosolic acidic amino acid preferring aminopeptidase, which is distinct from glutamyl aminopeptidase. Cheung and Cushman (15Cheung H.S. Cushman D.W. Biochim. Biophys. Acta. 1971; 242: 190-193Crossref PubMed Scopus (64) Google Scholar) described the partial purification of such an enzyme from the soluble fraction of a dog kidney extract. It is activated by preincubation with Mn2+and has a preference for Asp-2-naphthylamide (Asp-NA) 1The abbreviations used are: NA, 2-naphthylamide; THF, tetrahydrofuran; DMF, dimethylformamide; Boc,N-tert-butoxycarbonyl; t-Bu,tert-butyl; Fmoc, 9-fluorenylmethoxycarbonyl; SM, sulfamethoxazole; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; DTT, dithiothreitol; Bicine,N,N-bis(2-hydroxyethyl)glycine. over Glu-NA. Kellyet al. (16Kelly J.A. Neidle E.L. Neidle A. J. Neurochem. 1983; 40: 1727-1734Crossref PubMed Scopus (35) Google Scholar) have more extensively characterized a high molecular weight (450,000) acidic amino acid specific aminopeptidase in mouse brain cytosol. One of the features of the mouse brain enzyme is its inability to cleave simple NA substrates such as Asp-NA and Glu-NA. Due to its instability, this enzyme was not previously purified to homogeneity. In view of our interest in the role of angiotensin III in the brain renin-angiotensin system (17Song L. Wilk S. Healy D.P. Brain Res. 1997; 744: 1-6Crossref PubMed Scopus (38) Google Scholar), we sought to further study the latter enzyme. A new assay was developed to measure its activity. We report on the purification, specificity, properties, subunit structure, molecular cloning, and expression of the enzyme we call aspartyl aminopeptidase. All peptide intermediates were obtained from Bachem Inc,. (Philadelphia, PA). Frozen rabbit brains were obtained from Pel Freez (Rogers, AR). Freshly dissected rat tissues were obtained from rats sacrificed in the laboratory of Dr. Robert Blitzer of the Department of Pharmacology, Mount Sinai School of Medicine. The following compounds were obtained from Sigma: angiotensins II and III, 2-naphthylamine, aspartyl-2-naphthylamide, glutamyl-2-naphthylamide, phenylalanyl-naphthylamide, Lys-Ala-naphthylamide, leucine enkephalin, Pro-Leu-glycinamide, leucyl-p-nitroanilide, aspartyl-β-hydroxamate, sulfamethoxazole, puromycin, bacitracin, amastatin, and bestatin. The following compounds were obtained from Aldrich: 1-hydroxybenzotriazole, dicyclohexylcarbodiimide,N-methylmorpholine, isobutylchloroformate, Silica Gel Merck, grade 9385. Affi-Gel blue and Bio Gel HTP hydroxylapatite were products of Bio-Rad. Q-Sepharose, phenyl-Sepharose, and Superose 6 were obtained from Amersham Pharmacia Biotech. AcA-22 was obtained from Sepracor Inc. (Marlborough, MA). Oligonucleotide primers were obtained from IDT Inc. (Coralville, IA). Dipeptidyl-peptidase IV was purified to apparent homogeneity from rat kidney as described (18Li J. Wilk E. Wilk S. Arch. Biochem. Biophys. 1995; 323: 148-154Crossref PubMed Scopus (62) Google Scholar). A unit of dipeptidyl-peptidase IV activity is defined as the amount of enzyme releasing 1 μmol of NA/h from Ala-Pro-NA. Glutamyl aminopeptidase was purified to apparent homogeneity from rat kidney by immunoaffinity chromatography (8Wilk S. Healy D.P. Adv. Neuroimmunol. 1993; 3: 195-207Abstract Full Text PDF Scopus (67) Google Scholar). A mixture of 7 mmol (1 g) of NA and 7 mmol (1.5 g) of Boc-Pro in 10 ml of dry THF was cooled to −20 °C. Next 7 mmol each of isobutylchloroformate and N-methylmorpholine were added, and the reactants were stirred for 20 min at this temperature. The reaction mixture was allowed to come to room temperature and filtered. The precipitate was washed with THF, and the combined THF fractions were evaporated to dryness. The residue was dissolved in chloroform and sequentially washed with saturated NaHCO3, H2O, 10% citrate and H2O, dried over Na2SO4, and the chloroform was then removed by evaporation. The addition of ether to the residue resulted in crystallization of the product in 67% yield (1.6 g). The addition of 10 ml of 4 n HCl in dioxane to Boc-Pro-NA led to crystallization of Pro-NA·HCl from the reaction mixture. The product was obtained in quantitative yield following filtration and washing with ether. An equimolar amount of triethylamine was added to a suspension of 4.7 mmol of Pro-NA·HCl in 40 ml of DMF. The solution was then cooled to 0 °C, and equimolar amounts of dicyclohexylcarbodiimide and 1-hydroxybenzotriazole were added. After stirring for 15 h at 0 °C, the mixture was filtered, the filtrate was evaporated, and the residue was dissolved in chloroform. The chloroform was sequentially washed as described for Boc-Pro-NA. After drying over Na2SO4, the chloroform was removed by evaporation, and ether was added to the residue to facilitate crystallization of the product in quantitative yield. Treatment of Boc-Ala-Pro-NA with 10 ml of 4 n HCl in dioxane for 30 min at room temperature, followed by evaporation and the addition of ether to the residue, produced a quantitative yield of Ala-Pro-NA·HCl (compound 1). Triethylamine and Boc-Ala-N-hydroxysuccinimide ester (0.5 mmol each) were added to a stirred solution of 0.5 mmol of compound 1 in 10 ml of DMF. The mixture was allowed to stir overnight at room temperature. The solvent was then removed by evaporation, and the residue was dissolved in chloroform. The chloroform was sequentially washed with saturated NaHCO3, H2O, 10% citrate, and H2O. After drying over Na2SO4, the solvent was evaporated to yield 220 mg of Boc-Ala-Pro-NA. Treatment with trifluoroacetic acid for 30 min followed by evaporation and ether precipitation, yielded 180 mg of Ala-Ala-Pro-NA·trifluoroacetate (yield, 36%; M + 1 = 384). N-Benzyloxycarbonyl-Asp(O-t-Bu)-OH and triethylamine (0.5 mmol each) were added to a stirred solution of 0.5 mmol of compound 1 in 10 ml of DMF, followed by 0.5 mmol each of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide after the solution had cooled to 4 °C. The reaction was allowed to proceed at 4 °C for 18 h, after which time the mixture was filtered. The filtrate was evaporated to dryness, the residue was dissolved in chloroform, and the chloroform extract was washed sequentially with saturated NaHCO3, H2O, 10% citrate, and H2O. After drying over Na2SO4, the chloroform was removed by evaporation, and the residue was purified by silica gel chromatography. The productN-benzyloxycarbonyl-Asp(O-t-Bu)-Ala-Pro-NA was eluted with a solution of 95% chloroform, 5% ethanol. The purified product was hydrogenated in the presence of 10% Pd/C to yield 139 mg of Asp(O-t-Bu)-Ala-Pro-NA. Treatment of this product with trifluoroacetic acid for 30 min followed by evaporation and ether precipitation yielded Asp-Ala-Pro-NA (M +1 = 427). Equimolar amounts of Boc-Glu(O-t-Bu)-ONS (Bachem) and 1-hydroxybenzotriazole (0.5 mmol each) were added to 0.5 mmol of compound 1 dissolved in 20 ml of DMF. The mixture was stirred for 1 h at room temperature and then poured into a flask containing 200 ml of ethyl acetate and 30 ml of H2O. The organic layer was washed with 2 × 20 ml of saturated NaHCO3, 2 × 20 ml of H2O, and 20 ml of saturated NaCl. After drying over Na2SO4, the solvent was removed in vacuo. The residue was washed with 50 ml of ethyl ether and then treated with trifluoroacetic acid for 1 h. After removal of the trifluoroacetic acid under high vacuum, the product Glu-Ala-Pro-NA was obtained in 81% yield. M + 1 = 441. Equimolar amounts of triethylamine and Fmoc-Asp-(O-t-Bu)-OH (0.5 mmol) were added to a stirred solution of 0.5 mmol of compound 1 in 10 ml of DMF. The solution was cooled to 4 °C, and then 0.5 mmol each of dicyclohexylcarbodiimide and 1-hydoxybenzotriazole were added. The solution was stirred at this temperature for 18 h and then filtered. The solvent was evaporated, and the residue was dissolved in chloroform and then washed with 10% citrate and H2O. After drying over Na2SO4 and evaporation of solvent, a hygroscopic solid was obtained. Treatment with trifluoroacetic acid for 30 min followed by ether precipitation yielded 186 mg of Fmoc-Asp-Ala-Pro-NA as white crystalline material (yield 29%;M + 1 = 650). Equimolar amounts of triethylamine and Boc-Ala were added to 1.6 mmol of Pro-SM·trifluoroacetic acid (prepared by coupling Boc-Pro to SM and deprotecting with trifluoroacetic acid) and dissolved in 20 ml of THF. The solution was cooled to 4 °C, equimolar amounts of dicyclohexylcarbodiimide and 1-hydroxybenzotriazole were added, and the solution was stirred at this temperature for 18 h. The reaction mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in chloroform and sequentially washed with saturated NaHCO3, H2O, 10% citrate, and H2O. After drying over Na2SO4, evaporation yielded 220 mg (0.42 mmol) of Boc-Ala-Pro-SM. Treatment with 4 n HCl in dioxane, evaporation, and crystallization by ether addition yielded 151 mg of Ala-Pro-SM·HCl (0.33 mmol). Equimolar amounts of triethylamine and Fmoc-Asp(O-t-Bu)-OH were added to 0.33 mmol of Ala-Pro-SM dissolved in 10 ml of THF plus 10 ml of DMF. Coupling with dicyclohexylcarbodiimide and 1-hydroxybenzotriazole as described above yielded 201 mg of Fmoc-Asp(O-t-Bu)-Ala-Pro-SM (0.24 mmol). The Fmoc group was removed by treatment for 2 h with 5 ml each of DMF and diethylamine to yield 102 mg (0.16 mmol) of Asp(O-t-Bu)-Ala-Pro-SM. The final product was obtained in quantitative yield by treatment with trifluoroacetic acid. M + 1 = 537. A series of tetrapeptides were synthesized on an Applied Biosystems model 430A peptide synthesizer by Fmoc chemistry. Peptide purity was evaluated by HPLC (see below). The activity of aspartyl aminopeptidase was measured by a coupled enzymatic assay similar to that described for the measurement of pyroglutamyl peptidase II (19Friedman T.C. Wilk S. Biochem. Biophys. Res. Commun. 1985; 132: 787-794Crossref PubMed Scopus (21) Google Scholar). In this assay, the product of the action of aspartyl aminopeptidase is a substrate of dipeptidyl-peptidase IV (EC 3.4.14.5). The reaction sequence is as follows, Asp­Ala­Pro­C→Aspartyl aminopeptidaseAsp+Ala­Pro­C Ala­Pro­C→Dipeptidyl­peptidase IVAla­Pro+C REACTIONS1AND2where C represents a chromogen. Initially, we used 2-naphthylamine as chromogen, and more recently we have used the less toxic and more soluble sulfamethoxazole. The released chromogen is measured by a colorimetric procedure following its diazotization (20Goldbarg J.A. Rutenberg A.M. Cancer. 1958; 11: 283-291Crossref PubMed Scopus (557) Google Scholar). The reaction mixture contained 10 μl of a 10 mm solution of substrate in Me2SO, 0.5 units of dipeptidyl-peptidase IV, and 50 mm Tris-HCl, pH 7.5, in a total volume of 250 μl. Partially purified enzyme preparations were assayed in the presence of 0.4 mm puromycin to eliminate the possible contribution of puromycin-sensitive aminopeptidase (EC 3.4.11.14) to substrate hydrolysis. The substrate used to measure aspartyl aminopeptidase can also be cleaved in crude tissue samples by glutamyl aminopeptidase and possibly by the puromycin-sensitive aminopeptidase. Therefore, to measure aspartyl aminopeptidase in the supernatant fraction of homogenates of rat tissues, incubation buffers also contained Zn2+ and puromycin, each at a final concentration of 0.4 mm. We have determined that in crude tissue extracts, 0.4 mm Zn2+ totally inhibits glutamyl aminopeptidase but does not inhibit aspartyl aminopeptidase. Rat tissues were homogenized in 5 volumes of 25 mm Bicine buffer, pH 7.0, 5% glycerol, in a Potter-Elvejhem homogenizer fitted with a Teflon pestle. Homogenates were centrifuged for 30 min at 15,000 × g, and the supernatant was taken for analysis. Incubation mixtures (final volume of 125 μl) contained 5 μl of tissue supernatant, 0.5 units of dipeptidyl-peptidase IV, 5 μl of 10 mm Zn2+, 5 μl of 10 mmpuromycin, 5 μl of 10 mm Asp-Ala-Pro-SM in Me2SO, and Tris-HCl buffer (0.05 m, pH 7.5). Tubes were incubated at 37 °C for 5 min, and the reaction was stopped by the addition of 125 μl of 10% trichloroacetic acid. After centrifugation, 125 μl of supernatant was removed for analysis of chromogen. Protein was quantitated by the method of Lowry (21Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Protein elution from columns was monitored by absorbance at 280 nm. HPLC was performed on a Waters 600E liquid chromatograph equipped with a Vydac C4 protein column protected by a Vydac C4 5 μ guard column. For analysis of peptide purity, the column was equilibrated with 15% acetonitrile, 0.05% trifluoroacetic acid at a flow rate of 1 ml/min. Elution was carried out by linearly increasing the acetonitrile concentration to 70% over a period of 34 min. The products were monitored by measurement of absorbance at 210 nm. For analysis of angiotensin degradation, HPLC was run under isocratic conditions at a flow rate of 1 ml/min in a buffer consisting of 170 ml of acetonitrile and 5 ml of phosphoric acid diluted to 1 liter with H2O and adjusted to pH 3.0 with triethylamine. Peptides were detected by absorbance at 210 nm. SDS-PAGE was run on 10% gels according to the procedure of Laemmli (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). Electrospray mass spectrometry was run at The Mount Sinai School of Medicine. The molecular mass of the native enzyme was determined by FPLC on a calibrated Superose 6 column. The column was equilibrated with 50 mm Bicine, pH 7.0, 0.1 m NaCl, 10% glycerol and calibrated with a high molecular weight calibration kit consisting of thyroglobulin, catalase, ferritin, and aldolase (Amersham Pharmacia Biotech). The subunit molecular mass was determined by SDS-PAGE. A calibration kit supplied by Amersham Pharmacia Biotech contained marker proteins ranging in molecular mass from 94.4 to 14.4 kDa. K m and V max were obtained by a least squares analysis of the Lineweaver-Burk plot. Determination of K i was by the method of Dixon (23Dixon M. Biochem. J. 1953; 55: 170-171Crossref PubMed Scopus (3290) Google Scholar). Each determination was conducted at two substrate concentrations and at six concentrations of competing peptide. Frozen rabbit brains (50 g) were partially defrosted and homogenized in a Waring blender with 4 volumes of 25 mmBicine, pH 7.0, 5% glycerol (buffer A). The homogenate was centrifuged for 1 h at 100,000 × g, and the supernatant was retained. The pellet was washed with an equal volume of buffer A and centrifuged for 1 h at 100,000 × g, and the supernatant fractions were combined. The supernatant was passed over a 2.5 × 20-cm column of Affi-Gel blue, 100–200 mesh, equilibrated with buffer A. The enzymatically active fractions of the effluent were combined. The enzymatically active fractions were applied to a 5 × 15-cm Q-Sepharose column, equilibrated with buffer A, and the column was then washed with 1500 ml of buffer A. The enzyme was eluted with a 2-liter 0–0.4 m NaCl gradient in buffer A. The active fractions were combined and concentrated in an Amicon ultrafiltration cell fitted with a PM-10 membrane. The concentrated enzyme was applied to a 2.5 × 90-cm AcA-22 column equilibrated with 50 mm Bicine, pH 7.0, 10% glycerol, 0.1 m NaCl. The active fractions were combined, and NaCl was added to a final concentration of 0.3 m. The active fractions were chromatographed on a 1 × 7-cm phenyl-Sepharose column equilibrated with 50 mm Bicine, pH 7.0, 10% glycerol, 0.3 m NaCl (buffer B). The column was washed with 30 ml of buffer B. The enzyme was eluted with 30 ml of 50 mm Bicine, pH 7.0, 60% glycerol. The active fractions were dialyzed against 10 mm sodium phosphate buffer, pH 7.0, 10% glycerol (buffer C). The dialyzed enzyme was applied to a 2.5 × 1-cm column of hydroxylapatite equilibrated with buffer C, and the column was washed with this buffer. The effluent was collected, and fractions containing active enzyme were concentrated and then stored at −80 °C. Microsequencing was conducted at the W. M. Keck Foundation Biotechnology Resource Laboratory of Yale University. Gel slices containing protein were digested with trypsin, and the eluate was subjected to microbore HPLC. Matrix-assisted laser desorption/ionization mass spectrometry was used to evaluate peak purity and identify candidate peaks suitable for sequencing. Two sequences were obtained, and a third sequence of a tryptic peptide was obtained from the Baker Medical Research Institute (Prahran, Australia). Nucleotide sequencing of ATCC clone 355377 was performed by the Biotechnology Center, Utah State University (Logan, UT) using sequence-specific oligonucleotide primers synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The cDNA coding for aspartyl aminopeptidase was excised from ATCC clone 355377 (lafmid BA plasmid) by first digesting withNotI and then partially digesting with BstXI to release a 1.55-kilobase pair piece containing the entire coding region minus 174 bp from the 5′-end. An adaptor was synthesized containing a 5′-overhang complementary to an NcoI digestion site on the 5′-end and a 3′-overhang complementary to theBstXI-generated overhang on the 3′-end. This adaptor contained the ATG start codon followed by six histidine codons, a glycine codon, and then the codon for asparagine normally present after the putative start codon in the aspartyl aminopeptidase sequence. The sequences of the oligonucleotides annealed to make the adaptor were 5′-CATGCACCATCACCACCATCACGGGCAG-3′ and 5′-GGCACTACCACCACTACCAC-3′. TheEscherichia coli expression plasmid pSE-420 (a gift from Dr. J. Brosius) was digested with NcoI and NotI. The digested pSE-420, the BstXI-NotI insert, and the adaptor were ligated to yield the plasmid pSE-6HDAP. The plasmid was transfected into XL-2 blue cells (Stratagene). The presence of the adaptor and insert was confirmed by restriction digestion. The cells containing the plasmid were grown in the presence of 2 mmisopropyl-1-thio-β-d-galactopyranoside, and aspartyl aminopeptidase activity was determined. Total RNA was prepared essentially by the method of Chomczynski and Sacchi (24Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar) from freshly frozen rat tissues. Formaldehyde gel electrophoresis and transfer of 20 μg of RNA/lane were performed using the methods described in Sambrook et al. (25Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The RNA was transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech) and hybridized with 40 μCi of a mouse aspartyl aminopeptidase cDNA probe from ATCC clone 355377, which had been labeled to a specific activity of approximately 0.1 μCi/ng with [α-32P]dCTP using the NEBlot kit from New England Biolabs. Prehybridization and hybridization at 42 °C were carried out in 20 ml of 6× SSPE, 5× Denhardt's solution, 50% formamide, and 100 μg/ml sheared salmon sperm DNA. Washes were in 1× SSC, 0.1% SDS at 42 and 55 °C followed by a high stringency wash in 0.1× SSC, 0.01% SDS at 55 °C An RNA ladder from Life Technologies, Inc. was also electrophoresed for calculation of molecular size. The cytosol of a rabbit brain homogenate contained an enzyme that cleaved the substrate Asp-Ala-Pro-NA and that, in the presence of excess dipeptidyl-peptidase IV, liberated free naphthylamine. Enzymatic activity was optimal in the neutral pH range (Fig. 1). This enzyme having the properties of an aspartyl aminopeptidase (see below) was purified 722-fold by conventional chromatographic techniques from a rabbit brain supernatant, with an overall yield of 3.8% (TableI). Starting with 50 g of rabbit brain, 112 μg of enzyme protein was obtained. Examination of the enzyme by SDS-PAGE revealed a highly purified preparation containing a major protein band and trace amounts of higher molecular weight bands (Fig. 2).Table IPurificationStepVolumeActivityaEnzyme through step 4 was assayed in the presence of 0.4 mm puromycin.ProteinTotal activitySpecific activityRecoverymlunits/mlmg/mlunitsunits/mg%1. Supernatant3182.46.77630.361002. Affi-Gel blue3362.01.76721.2883. Q-Sepharose3601.50.295405.2714. AcA-22603.50.1721020.6285. Phenyl-Sepharose249.40.122694306. Hydroxylapatite2.810.40.04292603.8Purification was from 50 g of rabbit brain. One unit catalyzes the degradation of 1 μmol of Asp-Ala-Pro-sulfamethoxazole/h.a Enzyme through step 4 was assayed in the presence of 0.4 mm puromycin. Open table in a new tab Figure 2SDS-PAGE (10% gel) of aspartyl aminopeptidase after the last step of purification. Proteins were visualized with silver staining. Lane 1, molecular mass markers (kDa); lane 2, aspartyl aminopeptidase.View Large Image Figure ViewerDownload (PPT) Purification was from 50 g of rabbit brain. One unit catalyzes the degradation of 1 μmol of Asp-Ala-Pro-sulfamethoxazole/h. The molecular mass of the native enzyme was determined by gel filtration chromatography on a calibrated FPLC Superose 6 column. The enzyme eluted identically with ferritin, establishing a molecular mass of 440 kDa. This value is virtually identical to the estimate of 450 kDa reported by Kelly et al. (16Kelly J.A. Neidle E.L. Neidle A. J. Neurochem. 1983; 40: 1727-1734Crossref PubMed Scopus (35) Google Scholar). SDS-PAGE gave a monomer molecular mass of 55 kDa. The specificity of the enzyme was explored with a series of peptides. Asp-Ala-Pro-SM and Asp-Ala-Pro-NA used for assay and purification were cleaved at the Asp-Ala bond, since release of the chromogen was dependent upon the presence of dipeptidyl-peptidase IV. Thek cat/K m ratio for Asp-Ala-Pro-NA exceeded that of the corresponding glutamyl peptide. The difference was primarily reflected in K m values with the glutamyl peptide binding very poorly to the enzyme. The enzyme required the presence of a free α-amino group. When the N terminus of the substrate Asp-Ala-Pro-NA was blocked by an Fmoc group, there was no cleavage. The enzyme also required the presence of an acidic amino acid at the N terminus for optimal activity. There was no measurable cleavage (<1% of Asp-Ala-Pro-NA) for Ala-Ala-Pro-NA, Lys-Ala-NA, Pro-Leu-Gly-NH2, leucine enkephalin (Tyr-Gly-Gly-Phe-Leu), Ile-