Neuropeptides (1991) 19, 163-168 0 Longman Group UK Ltd 1991
Human Brain Leucyl Aminopeptidase: Isolation, Characterization and Specificity Against Some Neuropeptides A. M. GIBSON, J. A. BIGGINS,
B. LAUFFART”,
D. MANTLE*
and J. R. McDERMOTT
MRC Neurochemical Pathology Unit, *Regional Neurological Centre, Newcastle Newcastle upon Tyne NE4 6BE, UK (Reprint requests to JRM)
General Hospital,
Abstract-In order to obtain a greater understanding of the role of aminopeptidases in the degradation of peptides and proteins in the nervous system, we have isolated and characterized leucyl aminopeptidase (EC 3.4.11 .l) from human cerebral cortex and studied its action on some physiologically important neuropeptides. The enzyme has a low specificity constant for the hydrolysis of Leu-7-amido-4-methylcoumarin (69s-‘M-‘1 but the peptides Tyr-Gly-Gly and Tyr-Gly-Gty-Phe-Leu (Let?-enkephalin) were much better substrates (specificity constants 8300 and 18050~~‘M-’ respectively). Optimum activity for the degradation of Leu-enkephalin was obtained at pH10.5 in the presence of 5mM-Mn+‘. A sharp drop in specificity constant occurred with increasing chain length in the series Leu-enkephalin, dynorphin l-8, l-10 and 1-13, suggesting that the enzyme functions only as an oligopeptidase. Other neuropeptides were poor substrates (cholecystokinin octapeptide, angiotensin-I) or not hydrolysed at all (somatostatin, Arg8-vasopressin).
Introduction Cellular aminopeptidases (EC 3.4.11) are thought to mediate the degradation of peptides in the later stages of cellular protein turnover, returning essential amino acids to the amino acid pool. They may also have more specialized functions, for example in the inactivation of neuropeptides in the brain (1). Four cytosolic aminopeptidases have been identified by ion exchange chromatography
Date received 24 January 1991 Date accepted 17 February 1991
of human brain extracts: alanyl-, arginyl-, leucyland pyroglutamyl- aminopeptidases (2-5). The relative amounts of these enzymes in brain is very similar to that seen in muscle extracts (6) suggesting a fundamental role for these peptidases in cellular protein catabolism. The specificity of brain alanyl aminopeptidase (Ala-AP; EC 3.4.11.14) against neuropeptides has been examined in some detail (2,7) and the results suggest an upper limit of around 13 amino acids for peptide substrates. Hydrophobic amino acids at the N-terminus or the adjacent position favour hydrolysis. Brain arginyl aminopeptidase (EC 3.4.11.6) has a much more 163
164 limited aminopeptidase specificity, hydrolysing only Arg- or Lys- residues at the N-terminus of diand tripeptides (3). Leucyl aminopeptidase (EC 3.4.11.1) has been isolated from several tissues and species (8) including human liver (9), but the purified brain enzyme has not been studied. It is of particular interest because aminopeptidases have been implicated in the degradation of several neuropeptides including the enkephalins. We report here on the purification and characterization of human brain leucyl aminopeptidase (LAP) and on its action against some neuropeptides.
Materials and Methods Peptides were obtained from Peninsula Labs Europe, St Helens, UK; other chemicals were from Sigma, London, UK or BDH, Poole, UK and were of analytical grade where available. Enzyme isolation
The method has been described previously (4). Briefly, human cerebral cortex (35 g of mixed grey and white matter obtained at autopsy within 15h post-mortem) was homogenized in 4 vols of 50mM-glycylglycine buffer (pH 7.5), centrifuged (2OOOOg,30min) and the supernatant subjected to anion exchange chromatography on a column (50 x 3cm) of DEAE-Sephadex A50, eluting with 0.1 M-NaCl in extraction buffer. Column fractions were assayed for LAP activity as described below. Fractions containing activity were pooled, concentrated by ultrafiltration (Amicon YMlOO membrane) and subjected to gel filtration on a column (90 x 1.5cm) of Sephacryl S300 column. The single peak of activity was concentrated by ultrafiltration, and further purified by preparative polyacrylamide gel electrophoresis (5% slab gel) as described previously (10). Assay of leucyl aminopeptidase activity
Enzyme solution (0.1 ml) was incubated for 1 h at 37” in SOmM-glycine/NaOH (pH9.5 at 37”) containing SmM-MgClz and 1.25 mM-leucyl-7-amido4-methylcoumarin (Leu-AMC) . Total assay volume was 0.3ml. Ethanol (0.6ml) was added to stop the reaction and the fluorescence of the
NEUROPEFTIDES
liberated AMC measured (LX 380nm; h,, 440nm). Assay conditions were modified for samples with high enzyme activity so that the extent of substrate hydrolysis did not exceed 15%.
Action of leucyl aminopeptidase on peptides
Peptide (3-3OOpM) was incubated with enzyme solution (10 ~1) in SOmM-glycine/NaOH buffer, pH 9.5, containing 5 mM-Mn*+ (total ~0150 ~1) for l-5h at 37°C. The reaction was stopped with 0.1% -trifluoroacetic acid (TFA) (150 ~1). The extent of peptide degradation was analysed by reversed-phase HPLC on a Nova-pak Cl8 cartridge fitted to a Z module radial compression unit (Waters Associates) eluting with a linear 20min acetonitrile gradient (2-49%) containing llmM-TFA at lml/min. Peptides were detected by UV absorbance at 214nm. Kinetic data for the hydrolysis of enkephalin and dynorphin peptides were analysed by Lineweaver-Burk plots as described previously (7). Amino acid analysis was carried out on hydrolysates (6M HCl; llO”, 18h) of peptide fragments using precolumn derivatization with phenylisothiocyanate followed by reversed-phase HPLC (Waters Pica-Tag method).
Protein
Protein was determined by the Coomassie Blue G binding method of Sedmak and Grossberg (1977) using bovine serum albumin as standard.
Results Isolation of leucyl aminopeptidase brain
from
human
On anion exchange chromatography, leucyl aminopeptidase activity eluted from the column in 0.1 M NaCl and was completely separated from alanyl aminopeptidase (6). The enzyme eluted from the Sephacryl S300 column with a Kav of 0.33 corresponding to a molecular weight of 270000 Da. A single protein staining band coinciding with the enzyme activity was obtained on preparative polyacrylamide gel electrophoresis under non-denaturing conditions.
165
HUMAN BRAIN LEUCYL AMINOPEPTIDASE
Table 1 Action of human brain LAP on neuropeptides Peptide Leu-enkephalin Dynorphin (1-8) Dynorphin (l-10) Dynorphin (1-13) Dynorphin (1-17) i3-endorphin Proctohn LH-RH (7-10) Desacetyl-a-MSH CCK-8 Angiotensin-I P-casomorphin (1-3) Somatostatin Args-vasopressin Bradykinin TRH aMSH
Residues
N-terminal sequence
5 8 10 13 17 31 5 4 13 8 10 3 14 8 8 3 13
Tyr-GIyTyr-GlyTyr-GlyTyr-GlyTyr-GlyTyr-GlyArg-TyrLeu-ArgSer-TyrAsp-Tyr Asp-ArgTyr-Pro Ala-Gly Cys-TyrArg-PropGlu-His Ac-Ser-Tyr
Rate of degradation (nmopmin) 32.4 2.1 0.34 0.20 0.26
Human Brain Leucyl Aminopeptidase: Isolation, Characterization and Specificity Against Some Neuropeptides A. M. GIBSON, J. A. BIGGINS,
B. LAUFFART”,
D. MANTLE*
and J. R. McDERMOTT
MRC Neurochemical Pathology Unit, *Regional Neurological Centre, Newcastle Newcastle upon Tyne NE4 6BE, UK (Reprint requests to JRM)
General Hospital,
Abstract-In order to obtain a greater understanding of the role of aminopeptidases in the degradation of peptides and proteins in the nervous system, we have isolated and characterized leucyl aminopeptidase (EC 3.4.11 .l) from human cerebral cortex and studied its action on some physiologically important neuropeptides. The enzyme has a low specificity constant for the hydrolysis of Leu-7-amido-4-methylcoumarin (69s-‘M-‘1 but the peptides Tyr-Gly-Gly and Tyr-Gly-Gty-Phe-Leu (Let?-enkephalin) were much better substrates (specificity constants 8300 and 18050~~‘M-’ respectively). Optimum activity for the degradation of Leu-enkephalin was obtained at pH10.5 in the presence of 5mM-Mn+‘. A sharp drop in specificity constant occurred with increasing chain length in the series Leu-enkephalin, dynorphin l-8, l-10 and 1-13, suggesting that the enzyme functions only as an oligopeptidase. Other neuropeptides were poor substrates (cholecystokinin octapeptide, angiotensin-I) or not hydrolysed at all (somatostatin, Arg8-vasopressin).
Introduction Cellular aminopeptidases (EC 3.4.11) are thought to mediate the degradation of peptides in the later stages of cellular protein turnover, returning essential amino acids to the amino acid pool. They may also have more specialized functions, for example in the inactivation of neuropeptides in the brain (1). Four cytosolic aminopeptidases have been identified by ion exchange chromatography
Date received 24 January 1991 Date accepted 17 February 1991
of human brain extracts: alanyl-, arginyl-, leucyland pyroglutamyl- aminopeptidases (2-5). The relative amounts of these enzymes in brain is very similar to that seen in muscle extracts (6) suggesting a fundamental role for these peptidases in cellular protein catabolism. The specificity of brain alanyl aminopeptidase (Ala-AP; EC 3.4.11.14) against neuropeptides has been examined in some detail (2,7) and the results suggest an upper limit of around 13 amino acids for peptide substrates. Hydrophobic amino acids at the N-terminus or the adjacent position favour hydrolysis. Brain arginyl aminopeptidase (EC 3.4.11.6) has a much more 163
164 limited aminopeptidase specificity, hydrolysing only Arg- or Lys- residues at the N-terminus of diand tripeptides (3). Leucyl aminopeptidase (EC 3.4.11.1) has been isolated from several tissues and species (8) including human liver (9), but the purified brain enzyme has not been studied. It is of particular interest because aminopeptidases have been implicated in the degradation of several neuropeptides including the enkephalins. We report here on the purification and characterization of human brain leucyl aminopeptidase (LAP) and on its action against some neuropeptides.
Materials and Methods Peptides were obtained from Peninsula Labs Europe, St Helens, UK; other chemicals were from Sigma, London, UK or BDH, Poole, UK and were of analytical grade where available. Enzyme isolation
The method has been described previously (4). Briefly, human cerebral cortex (35 g of mixed grey and white matter obtained at autopsy within 15h post-mortem) was homogenized in 4 vols of 50mM-glycylglycine buffer (pH 7.5), centrifuged (2OOOOg,30min) and the supernatant subjected to anion exchange chromatography on a column (50 x 3cm) of DEAE-Sephadex A50, eluting with 0.1 M-NaCl in extraction buffer. Column fractions were assayed for LAP activity as described below. Fractions containing activity were pooled, concentrated by ultrafiltration (Amicon YMlOO membrane) and subjected to gel filtration on a column (90 x 1.5cm) of Sephacryl S300 column. The single peak of activity was concentrated by ultrafiltration, and further purified by preparative polyacrylamide gel electrophoresis (5% slab gel) as described previously (10). Assay of leucyl aminopeptidase activity
Enzyme solution (0.1 ml) was incubated for 1 h at 37” in SOmM-glycine/NaOH (pH9.5 at 37”) containing SmM-MgClz and 1.25 mM-leucyl-7-amido4-methylcoumarin (Leu-AMC) . Total assay volume was 0.3ml. Ethanol (0.6ml) was added to stop the reaction and the fluorescence of the
NEUROPEFTIDES
liberated AMC measured (LX 380nm; h,, 440nm). Assay conditions were modified for samples with high enzyme activity so that the extent of substrate hydrolysis did not exceed 15%.
Action of leucyl aminopeptidase on peptides
Peptide (3-3OOpM) was incubated with enzyme solution (10 ~1) in SOmM-glycine/NaOH buffer, pH 9.5, containing 5 mM-Mn*+ (total ~0150 ~1) for l-5h at 37°C. The reaction was stopped with 0.1% -trifluoroacetic acid (TFA) (150 ~1). The extent of peptide degradation was analysed by reversed-phase HPLC on a Nova-pak Cl8 cartridge fitted to a Z module radial compression unit (Waters Associates) eluting with a linear 20min acetonitrile gradient (2-49%) containing llmM-TFA at lml/min. Peptides were detected by UV absorbance at 214nm. Kinetic data for the hydrolysis of enkephalin and dynorphin peptides were analysed by Lineweaver-Burk plots as described previously (7). Amino acid analysis was carried out on hydrolysates (6M HCl; llO”, 18h) of peptide fragments using precolumn derivatization with phenylisothiocyanate followed by reversed-phase HPLC (Waters Pica-Tag method).
Protein
Protein was determined by the Coomassie Blue G binding method of Sedmak and Grossberg (1977) using bovine serum albumin as standard.
Results Isolation of leucyl aminopeptidase brain
from
human
On anion exchange chromatography, leucyl aminopeptidase activity eluted from the column in 0.1 M NaCl and was completely separated from alanyl aminopeptidase (6). The enzyme eluted from the Sephacryl S300 column with a Kav of 0.33 corresponding to a molecular weight of 270000 Da. A single protein staining band coinciding with the enzyme activity was obtained on preparative polyacrylamide gel electrophoresis under non-denaturing conditions.
165
HUMAN BRAIN LEUCYL AMINOPEPTIDASE
Table 1 Action of human brain LAP on neuropeptides Peptide Leu-enkephalin Dynorphin (1-8) Dynorphin (l-10) Dynorphin (1-13) Dynorphin (1-17) i3-endorphin Proctohn LH-RH (7-10) Desacetyl-a-MSH CCK-8 Angiotensin-I P-casomorphin (1-3) Somatostatin Args-vasopressin Bradykinin TRH aMSH
Residues
N-terminal sequence
5 8 10 13 17 31 5 4 13 8 10 3 14 8 8 3 13
Tyr-GIyTyr-GlyTyr-GlyTyr-GlyTyr-GlyTyr-GlyArg-TyrLeu-ArgSer-TyrAsp-Tyr Asp-ArgTyr-Pro Ala-Gly Cys-TyrArg-PropGlu-His Ac-Ser-Tyr
Rate of degradation (nmopmin) 32.4 2.1 0.34 0.20 0.26
