Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1993-10-18
1996-09-03
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 691, 435195, 435227, 4353201, 4352402, 4352523, 5303871, 536 221, 536 231, 536 232, 536 237, C12Q 168, C12P 2106, C12N 120, C07H 1900
Patent
active
055522734
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to polypeptides containing sequences characteristic of pyrrolidone carboxylyl peptidases, to polynucleotides containing a sequence coding for such polypeptides, and to their use, in particular for diagnostic purposes.
Pyrrolidone carboxylyl peptidases are exopeptidasee which specifically remove 2-pyrrolidone-5-carboxylic acid (PCA) residues from the NH.sub.2 -terminal end of polypeptide chains and proteins (Doolittle and Armentrout, Biochemistry, 1968, 7, 516-521). These enzymes are also referred to as pyrrolidonyl peptidases (Szewczuk and Mulczyk, Eur. J. Biochem, 1969, 8, 63-67) or more commonly PYRases (Mitchell et al., Diagn. Microbiol. Infect. Dis., 1987, 6, 283-286). These enzymes are specific for the L-PCA-L-amino acid optical isomers (Uliana and Doolittle, Arch. Biochem. Biophys., 1969, 131, 561-656), and the rate of hydrolysis depends on the amino acid adjacent to the PCA residue (Fujiwara et al., Biochim. Biophys. Acta, 1979, 570, 140-148). These enzymes also belong to the arylamidase group, since they are capable of hydrolysing the peptide bond of the synthetic chromogenic substrate L-pyroglutamyl-.beta.-naphthylamide (Patterson et al., J. Biol. Chem., 1963, 238, 3611-3620). These enzymes were first described in bacteria (Mulczyk and Szewczuk, J. Gen. Microb., 1970, 61, 9-13), but they are also found to be present in animal and plant tissues and in man (Szewczuk and Kwiatkowska, Eur. J. Biochem., 1970, 15, 92-96). PYRases prove, in addition, to be a specific means for removing PCA residues blocking the NH.sub.2 -terminal end of peptides, and to be very useful for determining the amino acid sequence of proteins, since peptides not possessing a free .alpha.-NH.sub.2 group cannot be sequenced according to the usual Edman degradation. The biochemical and physicochemical properties of PYRases are quite well known, since they have been studied for more than about twenty years. Enzymes having PYRase activity have been isolated and partially purified from several microorganisms, in particular from bacteria such as Pseudomonas fluorescens (Armentrout and Doolittle, Arch. Biochem. Biophys., 1969, 132, 80-90; Doolittle, Meth. Enzymol. 1970, 19, 555-569), Bacillus subtilis (Szewczuk and Mulczyk, Eur. J. Biochem., 1969, 8, 63-67), Bacillus amyloliquefaciens (Tsuru etal., J. Biochem, 1978, 84, 467-476), Klebsiella cloacas (Kwiatkowska et al., J. Biol. Chem., 1974, 249, 7729-7736) and Enterococcus faecium (Sullivan etal., Aust. J. Biol. Sci., 1977, 30, 543-552). Conventional purification methods have not, to date, enabled the pyrrolidone carboxylyl peptidases to be purified to homogeneity from these various organisms. Peptide sequencing of them could consequently not be performed. It is found, moreover, that the molecular masses of PYRases vary greatly according to the microorganisms from which they originate. As an example, there may be mentioned Enterococcus faecium PYRase, which possesses a molecular mass of approximately 42 kDa, whereas Bacillus amyloliquefaciens PYRase possesses a molecular mass of approximately 24 kDA.
These enzymes, apart from their known peptidase activity, constitute a major criterion for the differentiation of enterobacteria (Mutczyk and Szewczuk, J. Gen. Microbiol, 1970, 61, 9-13), from staphylococci (Mulzcyk and Szewczuk, J. Gen. Microbiol., 1970, 70, 383-384), as well as an important factor in presumption of Group A streptococci and enterococci (Ellner et al., J. Clin. Microbiol., 1985, 22, 880-881).
On studying the genetics of these enzymes, the Applicant discovered, surprisingly, that the nucleotide sequences coding for the PYRases are very different for each of the species of microorganism studied, and that, in addition, the genes coding for PYRases originating from different microbial organisms, though having very divergent nucleotide sequences, code, in fact, for proteins having very convergent peptide sequences.
A considerable homology is, in effect, observed between the peptide sequences of PYRases extracted, for example, from Streptococcus pyo
REFERENCES:
A. Szewczuk et al., "Pyrrolidonyl Peptidase in Bacteria", European Journal Of Biochemistry, vol. 8, 1969, pp. 63-67.
P. D. Ellner et al., "Preliminary Evaluation of a Rapid Colorimetric Method for the Presumptive Identification of Group A Streptococci and Enterococci," Journal Of Clinical Microbiology, vol. 22, No. 5, Nov. 1985, pp. 70-74.
A. Awade et al., "One Step Purification and Characterization of the Pyrrolidone Carboxyl Peptidase of Streptococcus pyogenes Over-expressed in E. coli", Febs Letters, vol. 308, No. 1, Aug. 1992, pp. 70-74.
A. Awade et al. "Characterization of the PCP Gene Encoding the Pyrrolidone Carboxyl Peptidase of Bacillus subtilis", Febs Letters, vol. 305, No. 1, Jun. 1992, pp. 67-73.
P. Cleuziat et al., "Molecular Characterization of PCP, the Structural Gene Encoding the Pyrrolidone Carboxylyl Peptidase from Streptococcus pyogenes", Molecular Microbiology, vol. 6, No. 15, 1992, pp. 2051-2063.
R. W. Armentrout et al., "Pyrrolidonecarboxylyl Peptidase: Stabilization and Purification", Archives Of Biochemistry And Biophysics, vol. 132, No. 1, Jun. 1969, pp. 80-90.
R. F. Doolittle, "Pyrrolidonecarboxylyl Peptidase", Methods In Enzymology, vol. 19, 1970, pp. 555-569.
Kauthold et al "Few minute tests for the identificati . . . " Zentralbl Bakteriol. 272 (2) 1989 pp. 191-195.
Glover "Principles of cloning DNA" Gene Cloning pp. 1-20 1984.
Lerner "Tapping the immunological repertoire . . . " Nature 299 (14) pp. 592-596 1982.
Awade Abalo
Cleuziat Philippe L.
Gayral Jean-Pierre
Robert-Baudouy Jeannine
Bio Merieux
Kim Hyosuk
Wax Robert A.
LandOfFree
Polypeptides containing sequences characteristic of pyrrolidone does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Polypeptides containing sequences characteristic of pyrrolidone , we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polypeptides containing sequences characteristic of pyrrolidone will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1949061