Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1991-05-24
1999-02-16
Naff, David M.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435195, 435227, 435228, 435471, C12N 948, C12N 914, C12N 978, C12N 980
Patent
active
058719954
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of Application No. PCT/JP90/01036, filed Aug. 14, 1990.
TECHNICAL FIELD
This invention relates to a novel enzyme participating in a C-terminal amidation of a peptide C-terminal glycine adduct, a method of preparing same, and the use thereof. The term "participating in a C-terminal amidation" as used herein means possessing an action promoting any step for converting a peptide C-terminal glycine adduct into its peptide C-terminal amidated compound.
BACKGROUND ART
In the past, the enzyme participating in the enzymatic reaction in vivo, i.e., a C-terminal amidation of a C-terminal glycine adduct of peptides (compound in which glycine is peptide-bonded to C-terminal residue) is called peptidylglycine-.alpha.-amidating monooxygenase (C-terminal amidating enzyme) (EC.1.14.17.3) (Bradbury et. al., Nature, 298, 686, 1982: Glembotski et. al., J. Biol. Chem., 259. 6385, 1984), and is considered to catalyze the following reaction: ##STR1##
To clarify the amidation mechanism in vivo and utilize the enzyme for the method of converting the peptides which exhibit a physilogical activity for the first time by an amidation of the C-terminal with the peptide produced by the recombinant DNA technique, for example, calcitonin and gastrin, in vitro, attempts have been made to purify this enzyme. For example, there have been reported those derived from bovine pituitary gland middle lobe (Murthy et. al., J. Biol. Chem., 261, 1815, 1986), porcine pituitary gland (Kizer et. al., Endocrinology, 118, 2262, 1986; Bradbury et. al., Eur. J. Biochem., 169, 579, 1987), porcine heart atrium (Kojima et. al., J. Biochem., 105, 440, 1989), Xenopus body skin (Mizuno et. al., Biochem., Biophys. Res. Commun., 137, 984, 1986), rat thyroid gland tumor (Mehta et. al., Arch. Biochem., Biophys., 261, 44, 1988).
On the other hand, since it is difficult to procure a large amount of these purified enzymes, attempts have been made to isolate the cDNA's necessary for expression of these enzymes by use of the recombinant DNA technique generally practiced in recent years, and the production of the enzymes by utilizing same. For example, Eipper B. A, et. al. in Mol. Endocrinol. 1, 777-790, 1987, Ohsuye K. et. al. in Biochem. Biophys. Res. Commun., 150, 1275-1281, 1988, Stoffers, D. A. et. al. in Proc. Natl. Acad. Sci., USA, 86, 735-739, 1989, and Glauder, J. et. al. in Biochem. Biophys. Res. Commun., 169, 551-558, 1990, have reported peptide C-terminal amidating enzyme cDNA's derived from bovine pituitary gland, frog skin, rat atrium and human thyroid gland cell, respectively. Further, although not necessarily having a satisfactory productivity, there are also known examples of peptide C-terminal amidating enzymes produced by using of the recombinant DNA technique utilizing the cDNA derived from frog and bovine (e.g., see Japanese Unexamined Patent Publication (Kokai) No. 1-104168, Published International Application: WO89/02460, and Perkins et. al., Mol. Endocrinol., 4, 132-139, 1990).
On the other hand, these proteins have been reported to have molecular weights of 38, 42 or 54 kDa in bovine, 39 kDa in frog, and 41, 50, or 75 kDa in rat, which are very different from each other, depending on the collecting methods, etc. For example, the literature of Bradbury et. al., described above, Ramer et. al., 110, 8526-8532 (1988) and Young et. al., J. Am. Chem. Soc. 111, 1933-1934 (1989) suggest the existence of reaction intermediatesm, but there are no current examples which clarify the isolation of an intermediate, and the relationship between the intermediate and the amidating enzyme.
As described above, the peptide C-terminal amidating enzyme exhibits a very interesting action in vivo, and a composition having a constant purity derived from a specific living body organ is known. Nevertheless, these compositions cannot be used for the production of a peptide C-terminal amidated compound in vivo, as the purity and stability as well as to production costs thereof are not satisfactory. To solve these problems, on the premise that it
REFERENCES:
patent: 4693985 (1987-09-01), Degen et al.
patent: 4708934 (1987-11-01), Gilligan et al.
A. Bradbury et al., "Mechanism of C-terminal amide Formation By Pituitary Enzymes," Nature, vol. 298, Aug. 12, 1982, pp. 686-688.
K. Ohsuye et al., "Cloning of cDNA Encoding A New Peptide C-Terminal .alpha.-Amidating Enzyme . . . ," BBRC, vol. 150, Feb. 15, 1988, pp. 1275-1281.
K. Mizuno et al., "Peptide C-Terminal .alpha.-Amidating Enzyme Purified to Homogeneity . . . ," BBRC, vol. 137, Jun. 30, 1986, pp. 984-991.
A. Katopodis et al., "A Novel Enzyme from Bovine Neurointermediate Pituitary Catalyzes Dealkylation of .alpha.-Hydroxyglycine Derivatives, Thereby Functioning Sequentially with Peptidylglycine .alpha.-Amidating Monooxygenase in Peptide Amidation", Biochemistry, vol. 29, No. 26, Jul. 3, 1990, pp. 6115-6120.
K. Takahashi et al., "Peptidylglycine .alpha.-Amidating Reaction: Evidence for A Two-Step Mechanism Involving a Stable Intermediate at Neutral pH", Biochemical and Biophysical Research Communications, vol. 169, No. 2, Jun. 15, 1990, pp. 524-530.
J. Glauder et al., "Human Peptidylglycime .alpha.-Amidating Monooxygenase: cDNA, Cloning and Functional Expression of a Truncated Form in Cos Cells", Biochemical and Biophysical Research Communications, vol. 169, No. 2, Jun. 15, 1990, pp. 551-558.
A. Murthy et al., "Purification and Characterization of Peptidylglycine .alpha.-Amidating Monooxygenase from Bovine Neurointermediate Pituitary", The Journal of Biological Chemistry, vol. 261, No. 1., Feb. 5, 1986, pp. 1815-1822.
Stoffers et al., Proc. Natl. Acad. Sci., USA, Jan. 1989, vol. 86, pp. 735-739.
Betty A. Eipper et al., "Structure of the Precursor to an Enzyme Mediating COOH-Terminal Amidation in Peptide Biosynthesis", Molecular Endocrinology, vol. 1 No. 11, pp. 777-790 (1987).
Doris A. Stoffers et al., "Alternative mRNA splicing generates multiple forms of peptidyl-glycine .alpha.-amidating monooxygenase in rat atrium", Proc. Natl' Acad. Sci. USA, vol. 86, pp. 735-739 (Jan. 1989).
Masahiro Tajima et al., "The Reaction Product of Peptidylglycine .alpha.-Amidating Enzyme Is a Hydroxyl Derivative at .alpha.-Carbon of the Carboxyl-terminal Glycine", The Journal of Biological Chemistry, vol. 265, No. 17, pp. 9602-9605, Issue of Jun. 15, 1990.
D. Stoffers et al., "Alternative mRNA Splcing Generates Multiple Forms of Peptidyl-glycine .alpha.-amidating Monooxygenase in Rate Atrium", Proc. Natl. Acad. Sci. USA, vol. 86, Jan. 1989, pp. 735-739.
Young et al., "Enzymatic Peptidyl .alpha.-Amidation Proceeds through Formation of an .alpha.-Hydroxyglycine Intermediate", J. Am. Chem. Soc., vol. 111, No. 5, Mar. 1989, pp. 1933-1934.
Biochemical and Biophysical Research Communication, vol. 151, No. 1 (1988), A. G. Katopodis, et. al., pp. 499-505.
Biochemistry, vol. 29, No. 26, (1990), A. G. Katopodis, et al., pp. 6115-6120.
Fuse Yuka
Ifuku Ohji
Iida Toshii
Kaminuma Toshihiko
Kato Ichiro
Naff David M.
Shiseido Company Ltd.
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