Characterization of Peptidylglycine α-Amidating Activities in Rat Pituitary, Brain and Small Intestine Using Glycine-Extended C-Terminal Analogues of Vasoactive Intestinal Polypeptide as Substrate

MASATO NOGUCHI; KENICHI TAKAHASHI; HIROSHI OKAMOTO

doi:10.1620/tjem.156.191

MASATO NOGUCHI, KENICHI TAKAHASHI, HIROSHI OKAMOTO

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Keywords: vasoactive intestinal polypeptide, rat α-amidating activity

JOURNAL FREE ACCESS

1988 Volume 156 Issue 2 Pages 191-207

DOI https://doi.org/10.1620/tjem.156.191

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Published: 1988 Received: December 01, 1987 Available on J-STAGE: August 31, 2006 Accepted: September 08, 1988 Advance online publication: - Revised: -
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Date of correction: August 31, 2006 Reason for correction: - Correction: ABSTRACT Details: Wrong : NOGUCHI, M., TAKAHASHI, K. and OKAMOTO, H. Characterization of Peptidylglycine α-Amidating Activities in Rat Pituitary, Brain and Small Intestine Using Glycine-Extended C-Terminal Analogues of Vasoactive Intestinal Polypeptide as Substrate. Tohoku J. exp. Med., 1988, 156 (2), 191-207 - Peptidylglycine α-amidating activities from rat pituitary, brain and small intestine were compared, utilizing C-terminal analogues of vasoactive intestinal polypeptide (VIP), D-Tyr-Leu-Asn-Gly and D-Tyr-Asn-Gly, and C-terminal analogue of α-MSH, D-Tyr-Val-Gly. The three tissues had enzymic activities capable of converting the glycine-extended peptides to the corresponding α-amidated ones. In other words, all of three peptides could serve as substrates for the enzymes from both neural and gastrointestinal tissues. The activities were stimulated in the presence of copper and ascorbate; the optimal concentration of each cofactor was roughly equal for the three enzymes; similar pH profiles (a neutral pH optimum at 6.5-7 and another one at 8-8.5) were also observed. Desamide VIP-Gly was proved to be a potent inhibitor of the α-amidating activities from the tissues, but VIP was not, indicating that the α-amidating enzymes from these tissues in common have a recognition site for the C-terminal glycine of the glycine-extended precursor regardless of the length and nature of the sequence. No fundamental differences were observed between the catalytic properties of the α-amidating activities from these three tissues, raising the possibility that similar enzymes, which may or may not be a single species, are functioning in tissues that produce α-amidated polypeptides in vivo.
Right : Peptidylglycine α-amidating activities from rat pituitary, brain and small intestine were compared, utilizing C-terminal analogues of vasoactive intestinal polypeptide (VIP), D-Tyr-Leu-Asn-Gly and D-Tyr-Asn-Gly, and C-terminal analogue of α-MSH, D-Tyr-Val-Gly. The three tissues had enzymic activities capable of converting the glycine-extended peptides to the corresponding α-amidated ones. In other words, all of three peptides could serve as substrates for the enzymes from both neural and gastrointestinal tissues. The activities were stimulated in the presence of copper and ascorbate; the optimal concentration of each cofactor was roughly equal for the three enzymes; similar pH profiles (a neutral pH optimum at 6.5-7 and another one at 8-8.5) were also observed. Desamide VIP-Gly was proved to be a potent inhibitor of the α-amidating activities from the tissues, but VIP was not, indicating that the α-amidating enzymes from these tissues in common have a recognition site for the C-terminal glycine of the glycine-extended precursor regardless of the length and nature of the sequence. No fundamental differences were observed between the catalytic properties of the α-amidating activities from these three tissues, raising the possibility that similar enzymes, which may or may not be a single species, are functioning in tissues that produce α-amidated polypeptides in vivo.

Date of correction: August 31, 2006 Reason for correction: - Correction: CITATION Details: Wrong : 1) Bateman, R.C., Jr., Youngblood, W.W., Busby, W.H. & Kizer, J.S. (1985) Nonenzymatic peptide α-amidation. Implications for a novel enzyme mechanism. J. biol. Chem., 260, 9088-9091.
2) Bensadoun, A. & Weinstein, D. (1976) Assay of proteins in the presence of interfering materials. Anal. Biochem., 70, 241-250.
3) Bradbury, A.F., Finnie, M.D.A. & Smyth, D.G. (1982) Mechanism of C-terminal amide formation by pituitary enzymes. Nature (Lond.), 298, 686-688.
4) Diliberto, E.J., Jr. & Allen, P.L. (1981) Mechanism of dopamine-β-hydroxylation. Semidehydroascorbate as the enzymic oxidation product of ascorbate. J. biol. Chem., 256, 3385-3393.
5) Eipper, B.A., Glembotski, C.C. & Mains, R.E. (1983a) Selective loss of α-melanotropin-amidating activity in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7292-7298.
6) Eipper, B.A., Mains, R.E. & Glembotski, C.C. (1983b) Identification in pituitary tissue of a peptide α-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proc. nat. Acad. Sci. USA, 80, 5144-5148.
7) Eipper, B.A., Myers, A.C. & Mains, R.E. (1985) Peptidyl-glycine α-amidation activity in tissues and serum of the adult rat. Endocrinology, 116, 2497-2504.
8) Emeson, R.B. (1984) Hypothalamic peptidyl-glycine α-amidating monooxygenase: Preliminary characterization. J. Neurosci., 4, 2604-2613.
9) Friedman, S. & Kaufman, S. (1965) 3, 4-Dihydroxyphenylethylamine β-hydroxylase. J. biol. Chem., 240, 4763-4773.
10) Glembotski, C.C., Eipper, B.A. & Mains, R.E. (1983) Adrenocortiicotropin (1-14) OH-related molecules in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7299-7304.
11) Glembotski, C.C., Eipper, B.A. & Mains, R.E. (1984) Characterization of a peptide α-amidation activity from rat anterior pituitary. J. biol. Chem., 259, 6385-6392.
12) Halliwell, B. & Gutteridge, J.M.C. (1984) Oxygen toxicity, oxygen radicals, transtion metals and disease. Biochem. J., 219, 1-14.
13) Itoh, N., Obata, K., Yanaihara, N. & Okamoto, H. (1983) Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature (Lond.), 304, 547-549.
14) Kizer, J.S., Busby, W.H., Jr., Cottle, C. & Youngblood, W.W. (1984) Glycine-directed peptide amidation: Presence in rat brain of two enzymes that convert p-Glu-His-Pro-GIy-OH into p-Glu-His-Pro-NH₂ (thyrotropin-releasing hormone). Proc. nat. Acad. Sci. USA, 81, 3228-3232.
15) Kizer, J.S., Bateman, R.C., Jr., Miller, C.R., Humm, J., Busby, W.H., Jr. & Youngblood, W.W. (1986) Purification and characterization of a peptidyl glycine monooxygenase from porcine pituitary. Endocrinology, 118, 2262-2267.
16) Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem., 193, 265-275.
17) May, V. & Eipper, B.A. (1985) Regulation of peptide amidation in cultured pituitary cells. J. biol. Chem., 260, 16224-16231.
18) Mains, R.E., Eipper, B.A., Glembotski, C.C. & Dores, R.M. (1983) Strategies for the biosynthesis of bioactive peptides. Trends Neurosci., 6, 229-235.
19) Miyake, S., Shimo, T., Kitamura, Y., Nojyo, T., Nakamura, S., Imashuku, S. & Abe, T. (1973) Characteristics of continuous and functional cell line NB-I, derived from a human neuroblastoma. Autonomic Nervous System, 10, 115-120. (Japanese)
20) Mizuno, K., Sakata, J., Kojima, M., Kangawa, K. & Matsuo, H. (1986) Peptide C-terminal α-amidating enzyme purified to homogeneity from Xenopus laevis skin. Biochem. biophys. Res. Commun., 137, 984-991.
21) Murthy, A.S.N., Mains, R.E. & Eipper, B.A. (1986) Purification and characterization of peptidylglycine α-amidating monooxygenase from bovine neurointermediate pituitary. J. biol. Chem., 261, 1815-1822.
22) Mutt, V. & Said, S.I. (1974) Structure of the porcine vasoactive intestinal octacosape-ptide. The amino-acid sequence. Use of kallikrein in its determination. Europ. J. Biochem., 42, 581-589.
23) Nishizawa, M., Hayakawa, Y., Yanaihara, N. & Okamoto, H. (1985) Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett., 183, 55-59.
24) Ouafik, L'H. Dutour, A., Salers, P., Giraud, P., Boudouresque, F., Castanas, E. & Oliver, C. (1986) Evidence for high peptide α-amidation activity in the neonatal rat pancreas. Biochem. biophys. Res. Commun., 138, 179-184.
25) Ouafik, L'H., Giraud, P., Salers, P., Dutour, A., Castanas, E., Boudouresque, F. & Oliver, C. (1987) Evidence for high peptide α-amidating activity in the pancreas from neonatal rats. Proc. nat. Acad. Sci. USA, 84, 261-264.
26) Paquette, T.L., Gingerich, R. & Scharp, D. (1981) Altered amidation of pancreatic polypeptide in clutured dog islet tissue. Biochemistry, 20, 7403-7408.
27) Said, S.I. (1982) Vasoactive Intestinal Peptide in Hormone Research Series, edited by S.I. Said, Raven Press, New York.
28) Steiner, D.F., Quinn, P.S., Chan, S.J., Marsh, J. & Tager, H.S. (1980) Processing mechanisms in the biosynthesis of proteins. Ann. N. Y. Acad. Sci., 343, 1-16.
29) von Zastrow, M., Tritton, T.R. & Castle, J.D. (1986) Exocrine secretion granules contain peptide amidation activity. Proc. nat. Acad. Sci. USA, 83, 3297-3301.
30) Yamagami, T., Ohsawa, K., Nishizawa, M., Inoue, C., Gotoh, E., Yanaihara, N., Yamamoto, H. & Okamoto, H. (1988) Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter. Ann. N. Y. Acad. Sci., 527, 87-102.

Right : 1) Bateman, R. C., Jr., Youngblood, W. W., Busby, W. H. & Kizer, J. S. (1985) Nonenzymatic peptide α-amidation. Implications for a novel enzyme mechanism. J. biol. Chem., 260, 9088-9091.
2) Bensadoun, A. & Weinstein, D. (1976) Assay of proteins in the presence of interfering materials. Anal. Biochem., 70, 241-250.
3) Bradbury, A. F., Finnie, M. D. A. & Smyth, D. G. (1982) Mechanism of C-terminal amide formation by pituitary enzymes. Nature (Lond.), 298, 686-688.
4) Diliberto, E. J., Jr. & Allen, P. L. (1981) Mechanism of dopamine-β-hydroxylation. Semidehydroascorbate as the enzymic oxidation product of ascorbate. J. biol. Chem., 256, 3385-3393.
5) Eipper, B. A., Glembotski, C. C. & Mains, R. E. (1983a) Selective loss of α-melanotropin-amidating activity in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7292-7298.
6) Eipper, B. A., Mains, R. E. & Glembotski, C. C. (1983b) Identification in pituitary tissue of a peptide α-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proc. nat. Acad. Sci. USA, 80, 5144-5148.
7) Eipper, B. A., Myers, A. C. & Mains, R. E. (1985) Peptidyl-glycine α-amidation activity in tissues and serum of the adult rat. Endocrinology, 116, 2497-2504.
8) Emeson, R. B. (1984) Hypothalamic peptidyl-glycine α-amidating monooxygenase: Preliminary characterization. J. Neurosci., 4, 2604-2613.
9) Friedman, S. & Kaufman, S. (1965) 3, 4-Dihydroxyphenylethylamine β-hydroxylase. J. biol. Chem., 240, 4763-4773.
10) Glembotski, C. C., Eipper, B. A. & Mains, R. E. (1983) Adrenocorticotropin (1-14) OH-related molecules in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7299-7304.
11) Glembotski, C. C., Eipper, B. A. & Mains, R. E. (1984) Characterization of a peptide α-amidation activity from rat anterior pituitary. J. biol. Chem., 259, 6385-6392.
12) Halliwell, B. & Gutteridge, J. M. C. (1984) Oxygen toxicity, oxygen radicals, transtion metals and disease. Biochem. J., 219, 1-14.
13) Itoh, N., Obata, K., Yanaihara, N. & Okamoto, H. (1983) Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature (Lond.), 304, 547-549.
14) Kizer, J. S., Busby, W. H., Jr., Cottle, C. & Youngblood, W. W. (1984) Glycinedirected peptide amidation: Presence in rat brain of two enzymes that convert p-Glu-His-Pro-Gly-OH into p-Glu-His-Pro-NH₂ (thyrotropin-releasing hormone). Proc. nat. Acad. Sci. USA, 81, 3228-3232.
15) Kizer, J. S., Bateman, R. C., Jr., Miller, C. R., Humm, J., Busby, W. H., Jr. & Youngblood, W. W. (1986) Purification and characterization of a peptidyl glycine monooxygenase from porcine pituitary. Endocrinology, 118, 2262-2267.
16) Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem., 193, 265-275.
17) May, V. & Eipper, B. A. (1985) Regulation of peptide amidation in cultured pituitary cells. J. biol. Chem., 260, 16224-16231.
18) Mains, R. E., Eipper, B. A., Glembotski, C. C. & Dores, R. M. (1983) Strategies for the biosynthesis of bioactive peptides. Trends Neurosci., 6, 229-235.
19) Miyake, S., Shimo, T., Kitamura, Y., Nojyo, T., Nakamura, S., Imashuku, S. & Abe, T. (1973) Characteristics of continuous and functional cell line NB-I, derived from a human neuroblastoma. Autonomic Nervous System, 10, 115-120. (Japanese)
20) Mizuno, K., Sakata, J., Kojima, M., Kangawa, K. & Matsuo, H. (1986) Peptide C-terminal α-amidating enzyme purified to homogeneity from Xenopus laevis skin. Biochem. biophys. Res. Commun., 137, 984-991.
21) Murthy, A. S. N., Mains, R. E. & Eipper, B. A. (1986) Purification and characterization of peptidylglycine α-amidating monooxygenase from bovine neurointermediate pituitary. J. biol. Chem., 261, 1815-1822.
22) Mutt, V. & Said, S. I. (1974) Structure of the porcine vasoactive intestinal octacosape- ptide. The amino-acid sequence. Use of kallikrein in its determination. Europ. J. Biochem., 42, 581-589.
23) Nishizawa, M., Hayakawa, Y., Yanaihara, N. & Okamoto, H. (1985) Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett., 183, 55-59.
24) Ouafik, L'H. Dutour, A., Salers, P., Giraud, P., Boudouresque, F., Castanas, E. & Oliver, C. (1986) Evidence for high peptide α-amidation activity in the neonatal rat pancreas. Biochem. biophys. Res. Commun., 138, 179-184.
25) Ouafik, L'H., Giraud, P., Salers, P., Dutour, A., Castanas, E., Boudouresque, F. & Oliver, C. (1987) Evidence for high peptide α-amidating activity in the pancreas from neonatal rats. Proc. nat. Acad. Sci. USA, 84, 261-264.
26) Paquette, T. L., Gingerich, R. & Scharp, D. (1981) Altered amidation of pancreatic polypeptide in clutured dog islet tissue. Biochemistry, 20, 7403-7408.
27) Said, S. I. (1982) Vasoactive Intestinal Peptide in Hormone Research Series, edited by S. I. Said, Raven Press, New York.
28) Steiner, D. F., Quinn, P. S., Chan, S. J., Marsh, J. & Tager, H. S. (1980) Processing mechanisms in the biosynthesis of proteins. Ann. N. Y. Acad. Sci., 343, 1-16.
29) von Zastrow, M., Tritton, T. R. & Castle, J. D. (1986) Exocrine secretion granules contain peptide amidation activity. Proc. nat. Acad. Sci. USA, 83, 3297-3301.
30) Yamagami, T., Ohsawa, K., Nishizawa, M., Inoue, C., Gotoh, E., Yanaihara, N., Yamamoto, H. & Okamoto, H. (1988) Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter. Ann. N. Y. Acad. Sci., 527, 87-102.

Date of correction: August 31, 2006 Reason for correction: - Correction: PDF FILE Details: -

Date of correction: August 31, 2006 Reason for correction: - Correction: CITATION Details: Wrong : 1) Bateman, R. C., Jr., Youngblood, W. W., Busby, W. H. & Kizer, J. S. (1985) Nonenzymatic peptide α-amidation. Implications for a novel enzyme mechanism. J. biol. Chem., 260, 9088-9091.
2) Bensadoun, A. & Weinstein, D. (1976) Assay of proteins in the presence of interfering materials. Anal. Biochem., 70, 241-250.
3) Bradbury, A. F., Finnie, M. D. A. & Smyth, D. G. (1982) Mechanism of C-terminal amide formation by pituitary enzymes. Nature (Lond.), 298, 686-688.
4) Diliberto, E. J., Jr. & Allen, P. L. (1981) Mechanism of dopamine-β-hydroxylation. Semidehydroascorbate as the enzymic oxidation product of ascorbate. J. biol. Chem., 256, 3385-3393.
5) Eipper, B. A., Glembotski, C. C. & Mains, R. E. (1983a) Selective loss of α-melanotropin-amidating activity in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7292-7298.
6) Eipper, B. A., Mains, R. E. & Glembotski, C. C. (1983b) Identification in pituitary tissue of a peptide α-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proc. nat. Acad. Sci. USA, 80, 5144-5148.
7) Eipper, B. A., Myers, A. C. & Mains, R. E. (1985) Peptidyl-glycine α-amidation activity in tissues and serum of the adult rat. Endocrinology, 116, 2497-2504.
8) Emeson, R. B. (1984) Hypothalamic peptidyl-glycine α-amidating monooxygenase: Preliminary characterization. J. Neurosci., 4, 2604-2613.
9) Friedman, S. & Kaufman, S. (1965) 3, 4-Dihydroxyphenylethylamine β-hydroxylase. J. biol. Chem., 240, 4763-4773.
10) Glembotski, C. C., Eipper, B. A. & Mains, R. E. (1983) Adrenocorticotropin (1-14) OH-related molecules in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7299-7304.
11) Glembotski, C. C., Eipper, B. A. & Mains, R. E. (1984) Characterization of a peptide α-amidation activity from rat anterior pituitary. J. biol. Chem., 259, 6385-6392.
12) Halliwell, B. & Gutteridge, J. M. C. (1984) Oxygen toxicity, oxygen radicals, transtion metals and disease. Biochem. J., 219, 1-14.
13) Itoh, N., Obata, K., Yanaihara, N. & Okamoto, H. (1983) Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature (Lond.), 304, 547-549.
14) Kizer, J. S., Busby, W. H., Jr., Cottle, C. & Youngblood, W. W. (1984) Glycinedirected peptide amidation: Presence in rat brain of two enzymes that convert p-Glu-His-Pro-Gly-OH into p-Glu-His-Pro-NH₂ (thyrotropin-releasing hormone). Proc. nat. Acad. Sci. USA, 81, 3228-3232.
15) Kizer, J. S., Bateman, R. C., Jr., Miller, C. R., Humm, J., Busby, W. H., Jr. & Youngblood, W. W. (1986) Purification and characterization of a peptidyl glycine monooxygenase from porcine pituitary. Endocrinology, 118, 2262-2267.
16) Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem., 193, 265-275.
17) May, V. & Eipper, B. A. (1985) Regulation of peptide amidation in cultured pituitary cells. J. biol. Chem., 260, 16224-16231.
18) Mains, R. E., Eipper, B. A., Glembotski, C. C. & Dores, R. M. (1983) Strategies for the biosynthesis of bioactive peptides. Trends Neurosci., 6, 229-235.
19) Miyake, S., Shimo, T., Kitamura, Y., Nojyo, T., Nakamura, S., Imashuku, S. & Abe, T. (1973) Characteristics of continuous and functional cell line NB-I, derived from a human neuroblastoma. Autonomic Nervous System, 10, 115-120. (Japanese)
20) Mizuno, K., Sakata, J., Kojima, M., Kangawa, K. & Matsuo, H. (1986) Peptide C-terminal α-amidating enzyme purified to homogeneity from Xenopus laevis skin. Biochem. biophys. Res. Commun., 137, 984-991.
21) Murthy, A. S. N., Mains, R. E. & Eipper, B. A. (1986) Purification and characterization of peptidylglycine α-amidating monooxygenase from bovine neurointermediate pituitary. J. biol. Chem., 261, 1815-1822.
22) Mutt, V. & Said, S. I. (1974) Structure of the porcine vasoactive intestinal octacosape- ptide. The amino-acid sequence. Use of kallikrein in its determination. Europ. J. Biochem., 42, 581-589.
23) Nishizawa, M., Hayakawa, Y., Yanaihara, N. & Okamoto, H. (1985) Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett., 183, 55-59.
24) Ouafik, L'H. Dutour, A., Salers, P., Giraud, P., Boudouresque, F., Castanas, E. & Oliver, C. (1986) Evidence for high peptide α-amidation activity in the neonatal rat pancreas. Biochem. biophys. Res. Commun., 138, 179-184.
25) Ouafik, L'H., Giraud, P., Salers, P., Dutour, A., Castanas, E., Boudouresque, F. & Oliver, C. (1987) Evidence for high peptide α-amidating activity in the pancreas from neonatal rats. Proc. nat. Acad. Sci. USA, 84, 261-264.
26) Paquette, T. L., Gingerich, R. & Scharp, D. (1981) Altered amidation of pancreatic polypeptide in clutured dog islet tissue. Biochemistry, 20, 7403-7408.
27) Said, S. I. (1982) Vasoactive Intestinal Peptide in Hormone Research Series, edited by S. I. Said, Raven Press, New York.
28) Steiner, D. F., Quinn, P. S., Chan, S. J., Marsh, J. & Tager, H. S. (1980) Processing mechanisms in the biosynthesis of proteins. Ann. N. Y. Acad. Sci., 343, 1-16.
29) von Zastrow, M., Tritton, T. R. & Castle, J. D. (1986) Exocrine secretion granules contain peptide amidation activity. Proc. nat. Acad. Sci. USA, 83, 3297-3301.
30) Yamagami, T., Ohsawa, K., Nishizawa, M., Inoue, C., Gotoh, E., Yanaihara, N., Yamamoto, H. & Okamoto, H. (1988) Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter. Ann. N. Y. Acad. Sci., 527, 87-102.

Right : 1) Bateman, R. C., Jr., Youngblood, W. W., Busby, W. H. & Kizer, J. S. (1985) Nonenzymatic peptide α-amidation. Implications for a novel enzyme mechanism. J. biol. Chem., 260, 9088-9091.
2) Bensadoun, A. & Weinstein, D. (1976) Assay of proteins in the presence of interfering materials. Anal. Biochem., 70, 241-250.
3) Bradbury, A. F., Finnie, M. D. A. & Smyth, D. G. (1982) Mechanism of C-terminal amide formation by pituitary enzymes. Nature (Lond.), 298, 686-688.
4) Diliberto, E. J., Jr. & Allen, P. L. (1981) Mechanism of dopamine-β-hydroxylation. Semidehydroascorbate as the enzymic oxidation product of ascorbate. J. biol. Chem., 256, 3385-3393.
5) Eipper, B. A., Glembotski, C. C. & Mains, R. E. (1983a) Selective loss of α-melanotropin-amidating activity in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7292-7298.
6) Eipper, B. A., Mains, R. E. & Glembotski, C. C. (1983b) Identification in pituitary tissue of a peptide α-amidation activity that acts on glycine-extended peptides and requires molecular oxygen, copper, and ascorbic acid. Proc. nat. Acad. Sci. USA, 80, 5144-5148.
7) Eipper, B. A., Myers, A. C. & Mains, R. E. (1985) Peptidyl-glycine α-amidation activity in tissues and serum of the adult rat. Endocrinology, 116, 2497-2504.
8) Emeson, R. B. (1984) Hypothalamic peptidyl-glycine α-amidating monooxygenase: Preliminary characterization. J. Neurosci., 4, 2604-2613.
9) Friedman, S. & Kaufman, S. (1965) 3, 4-Dihydroxyphenylethylamine β-hydroxylase. J. biol. Chem., 240, 4763-4773.
10) Glembotski, C. C., Eipper, B. A. & Mains, R. E. (1983) Adrenocorticotropin (1-14) OH-related molecules in primary cultures of rat intermediate pituitary cells. J. biol. Chem., 258, 7299-7304.
11) Glembotski, C. C., Eipper, B. A. & Mains, R. E. (1984) Characterization of a peptide α-amidation activity from rat anterior pituitary. J. biol. Chem., 259, 6385-6392.
12) Halliwell, B. & Gutteridge, J. M. C. (1984) Oxygen toxicity, oxygen radicals, transtion metals and disease. Biochem. J., 219, 1-14.
13) Itoh, N., Obata, K., Yanaihara, N. & Okamoto, H. (1983) Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27. Nature (Lond.), 304, 547-549.
14) Kizer, J. S., Busby, W. H., Jr., Cottle, C. & Youngblood, W. W. (1984) Glycinedirected peptide amidation: Presence in rat brain of two enzymes that convert p-Glu-His-Pro-Gly-OH into p-Glu-His-Pro-NH₂ (thyrotropin-releasing hormone). Proc. nat. Acad. Sci. USA, 81, 3228-3232.
15) Kizer, J. S., Bateman, R. C., Jr., Miller, C. R., Humm, J., Busby, W. H., Jr. & Youngblood, W. W. (1986) Purification and characterization of a peptidyl glycine monooxygenase from porcine pituitary. Endocrinology, 118, 2262-2267.
16) Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem., 193, 265-275.
17) May, V. & Eipper, B. A. (1985) Regulation of peptide amidation in cultured pituitary cells. J. biol. Chem., 260, 16224-16231.
18) Mains, R. E., Eipper, B. A., Glembotski, C. C. & Dores, R. M. (1983) Strategies for the biosynthesis of bioactive peptides. Trends Neurosci., 6, 229-235.
19) Miyake, S., Shimo, T., Kitamura, Y., Nojyo, T., Nakamura, S., Imashuku, S. & Abe, T. (1973) Characteristics of continuous and functional cell line NB-I, derived from a human neuroblastoma. Autonomic Nervous System, 10, 115-120. (Japanese)
20) Mizuno, K., Sakata, J., Kojima, M., Kangawa, K. & Matsuo, H. (1986) Peptide C-terminal α-amidating enzyme purified to homogeneity from Xenopus laevis skin. Biochem. biophys. Res. Commun., 137, 984-991.
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Date of correction: August 31, 2006 Reason for correction: - Correction: PDF FILE Details: -

Abstract

Peptidylglycine α-amidating activities from rat pituitary, brain and small intestine were compared, utilizing C-terminal analogues of vasoactive intestinal polypeptide (VIP), D-Tyr-Leu-Asn-Gly and D-Tyr-Asn-Gly, and C-terminal analogue of α-MSH, D-Tyr-Val-Gly. The three tissues had enzymic activities capable of converting the glycine-extended peptides to the corresponding α-amidated ones. In other words, all of three peptides could serve as substrates for the enzymes from both neural and gastrointestinal tissues. The activities were stimulated in the presence of copper and ascorbate; the optimal concentration of each cofactor was roughly equal for the three enzymes; similar pH profiles (a neutral pH optimum at 6.5-7 and another one at 8-8.5) were also observed. Desamide VIP-Gly was proved to be a potent inhibitor of the α-amidating activities from the tissues, but VIP was not, indicating that the α-amidating enzymes from these tissues in common have a recognition site for the C-terminal glycine of the glycine-extended precursor regardless of the length and nature of the sequence. No fundamental differences were observed between the catalytic properties of the α-amidating activities from these three tissues, raising the possibility that similar enzymes, which may or may not be a single species, are functioning in tissues that produce α-amidated polypeptides in vivo.

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