Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1990-01-29
1994-10-11
Marx, Irene
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435183, 435 681, C12P 2100, C12N 900, C12N 902
Patent
active
053546750
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a C-terminal amidating enzyme composition derived from serum or plasma, a process for preparing the same and a method of production a peptide amidated at the C-terminal or a derivative thereof utilizing the same.
BACKGROUND ART
Peptides, which exhibit biological activity for the first time when C-terminal is amidated, for example, calcitonin, gastrin, secretin, vasoactive intestinal polypeptide, growth hormone-releasing factor, corticotropin-releasing factor, etc. have been known to be formed from glycine adducts through enzymatic reactions in vivo. Many of these biologically active peptides are useful as pharmaceuticals, and presently calcitonin, secretin, among others, are commercially available as pharmaceuticals.
These peptides have been obtained primarily by separation and purification from living bodies, but the steps are cumbersome and living bodies as sources are obtained only with difficulty. Therefore, the above presently commercially available peptides are very expensive.
Accordingly, in recent years, attempts have been made to produce these biologically active peptides by using a recombinant DNA technique. But according to the recombinant DNA technique using Escherichia coli, yeast, Bacillus subtilis, and the like as the host, an C-terminal amidation of the peptide produced cannot be effected, which has been an obstacle to producing the above peptide. Accordingly, there is a demand for a technique whereby C-terminal amidation is effected easily and inexpensively in vitro.
The enzyme participating in such amidation is called peptidylglycine-.alpha.-amidating monoxygenase (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:
To clarify the amidation mechanism in vivo and amidate the peptide produced by the recombinant technique at the C-terminal in vitro, attempts have been made to purify this enzyme. As examples which have a purified specific activity to 100-fold based source or higher, 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. Blochem., 169, 579, 1987), porcine heart atrium (Kojima et al, J. Blochem., 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). But, except for the method of Kizer et al, the operation is cumbersome, involving 5 to 6 purification steps. Also, according to the method of Kizer et al, there is the step of Sephadex G-100 (produced by Pharmacia) gel filtration, and this takes a long time for elution, and at the same time it is difficult to treat a large amount of the product.
In addition, concerning the existence of the C-terminal amidating enzyme in blood, there are reports of rat (Eipper et al, Endocrinology, 116, 2497, 1985) and human (Wand et al, Metabolism, 34, 1044, 1985), but both have low specific activities and no attempt at purification thereof have been made.
As described above, no purification method capable of simple and bulk treatment of the C-terminal amidating enzyme has been established. Also, no purification has been done from serum and plasma, and no method of producing a peptide amidated at the C-terminal or derivative thereof at low cost and in large which utilizing a C-terminal amidating enzyme in vitro, is known.
DISCLOSURE OF INVENTION
As described above, the 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, the use of these enzyme compositions for practical reactions in vitro, partly because of the difficulty of the availability thereof, is not always satisfactory.
On the other hand, the present inventors studied intensively to find an enzy
REFERENCES:
Murthy et al, (1987) Mol. Endrocriol., 1(4), 290-299.
Eipper et al, (1987) Mol. Endocrinol., 1(11), 777-790.
Pesce et al, (1967) J. Biol. Chem., 242(9), 2151-2167.
Sakata et al. (1986) Biochem Biophys. Res. Comm., 140(1), 230-236.
Stoffers et al. (1989) Proc. Nat. Acad. Sci. USA, 86, 735-739.
J. Biol. Chem., vol. 261, No. 4, (1986), A. S. N. Murthy, et al. [Purification and Characterization of Peptidylglycine .alpha.-Amidating Monooxygenase from Bovine Neurointermediate Pituitary] pp. 1815-1822.
Endocrinology, vol. 118, No. 6, (1986), J. S. Kizer, et al [Purification and Characterization of a Peptidyl Glycine Monooxygenase from Porcine Pituitary] pp. 2262-2267.
Endocrinology, vol. 116, No. 6, (1985), B. A. Eipper, et al [Peptidyl-Glycine .alpha.-Amidation Activity in Tissues and Serum of the Adult Rat] pp. 2497-2504.
Metabolism, vol. 34, No. 11, (1985), G. S. Wand, et al [Characterization of a Peptide Alpha-Amidation Activity In Human Plasma and Tissues] pp. 1044-1052.
Gastroenterology, vol. 92, No. 5, (1987), T. Azuma, et al [COOH-Terminal Glycine Extended Progastrin(Gastrin-G) and Peptidyl-Glycine .alpha.-Amidating Monooxigenase(PAM)Activity in Rat Serum] p. 1302.
Fuse Yuka
Iida Toshii
Okamoto Hiroshi
Tajima Masahiro
Yanagi Mitsuo
Marx Irene
Shiseido Company Ltd.
Weber Jon P.
LandOfFree
C-terminal amidating enzyme composition, process for preparing, does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with C-terminal amidating enzyme composition, process for preparing, , we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and C-terminal amidating enzyme composition, process for preparing, will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1658054