Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1995-06-21
1998-03-17
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435195, 435 41, 435 89, C12N 914, C12N 916, C12P 1930, C12P 100
Patent
active
057285632
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a novel cyclic nucleotide phosphodiesterase and a method for production thereof.
BACKGROUND ART
Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) have been found to be intracellular messenger so that their importance in regulating various cell functions has been revealed. Cyclic nucleotide phosphodiesterases are known as enzymes for degrading cAMP or cGMP and play an important role in vivo involving cyclic nucleotide metabolism. Accordingly, inhibition of cyclic nucleotide phosphodiesterases by chemicals seriously affects the cyclic nucleotide metabolism, resulting eventually in change in cell functions.
Five isozyme families from I to V are currently reported of the cyclic nucleotide phosphodiesterase, each family composed of several subtypes (Tanaka et al., Folia pharmacol. japon., 100, 249-258, 1992).
In recent years, cyclic nucleotide phosphodiesterase inhibitors have been developed as new drugs. In order to screen such inhibitors and clarify their mechanism, analysis on a selective inhibition activity against the cyclic nucleotide phosphodiesterase is important as a basis for the development.
An object of the present invention is to provide a novel cyclic nucleotide phosphodiesterase for the purpose of developing a new drug.
SUMMARY OF THE INVENTION
In view of the foregoing object, extensive studies have been made; as a result, the present inventor has discovered a novel cyclic nucleotide phosphodiesterase in animal organs or tissues, which is dissimilar to known isozyme families. For the first time, the present inventor has isolated and purified the enzyme and clarified its properties.
The present invention is directed to a cyclic nucleotide phosphodiesterase characterized by at least the following physicochemical properties (1) through (7) (hereinafter simply referred to as the enzyme of the present invention). The present invention is also directed to a method for producing the enzyme which comprises excising animal organs or tissues and eluting the resulting homogenate with a linear gradient elution technique using a Mono-Q HR ion exchange column.
The enzyme acts on cAMP and cGMP to form 5'-AMP and 5'-GMP, respectively.
The Km values for cAMP and cGMP show 0.11 .mu.M and 1.78 .mu.M, respectively.
The optimum pH for cAMP hydrolysis is 10.
The initial velocities of the enzymatic activity at 37.degree. C. are greater than those at 30.degree. C. The enzymatic activity is lost by heating at 60.degree. C. for 10 minutes.
IC.sub.50 is >300 .mu.M with nicardipine, >300 .mu.M with Cilostazol and 5-(4-acetamidophenyl)pyrazin-1H-one (SK&F94120), and >300 .mu.M with rolipram and 4-(3-buthoxy-4-methoxybenzyl)-2-imidazolidinone (Ro20-1724). The enzyme is calcium-calmodulin-insensitive and cGMP-insensitive.
Magnesium is the most efficient metal cation for the enzyme of the present invention.
298000.+-.8000 (Superose 12HR 10/30, gel filtration)
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows relationship between pH and the enzymatic activity of the present enzyme.
FIG. 2 shows effects of temperature on the enzymatic activity of the present enzyme.
FIG. 3 shows effects of temperature on the enzymatic activity of the present enzyme.
FIG. 4 shows effects of metal ions on the enzymatic activity of the present enzyme.
FIG. 5 shows relationship between the fraction and the enzymatic activity, when applied to a Q-Sepharose Fast Flow anionic exchange column.
FIG. 6 shows relationship between the fraction and the enzymatic activity, when applied to a calmodulin-Sepharose affinity column.
FIG. 7 shows relationship between the fraction and the enzymatic activity, when applied to a Mono Q HR10/10 anionic exchange column.
BEST MODE FOR CARRYING OUT THE INVENTION
According to the present invention, the enzyme of the present invention is widely found particularly in mammalian brains and also distributed in animal organs or tissues such as cardiac muscle, lung, kidney, smooth muscle or platelet, which can produce cy
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The Journal of Biological Chemistry, Col 263, No. 30, Issue of Oct. 25, 1988, pp. 15681-15687.
Mukai et al. Br. J. Pharmacol. vol. 111(2) 1994 pp. 389-390 -Abstract Enclosed.
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Lankford , Jr. Leon B.
Taisho Pharmaceutical Co. Ltd.
Tate Christohpher R.
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