Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-10-01
2001-06-26
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S024000, C435S004000, C435S183000, C435S219000, C435S814000
Reexamination Certificate
active
06251623
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a quick assay method for the activity of a plant-derived, asparagine residue-specific endoprotease and more particularly to a method for quickly assaying the activity of a plant-derived, asparagine residue-specific endoprotease utilizing a fluorescence quenching substrate having a specified structure.
BACKGROUND OF THE INVENTION
Asparagine residue-specific endoproteases refer to enzymes which split an amino acid sequence of peptide or protein at the C-terminal side of asparagine residue. Among them, the asparagine residue-specific endoprotease derived from plants is called legumaturain.
Legumaturain is an enzyme which is involved in synthesis of storage proteins of plant seeds that are useful as an excellent protein source in food industry, etc.
For example, soybean glycinin is synthesized as a soybean glycinin precursor consisting of a single polypeptide originally in the form of an acidic subunit and a basic subunit bonded to each other. The precursor must be subjected to a subsequent action of legumaturain to be split between the subunits before it can be converted into a mature form, glycinin.
Therefore, the enzyme activity of legumaturain influences the industrial feasibility of storage proteins such as glycinin.
Under the circumstances, there has been demanded an accurate assay method for assaying the enzyme activity of legumaturain and conventionally the following methods have been used (cf. Japanese Patent Publication No. Hei 8-24576).
(1) Qualitative Measurement Method by an Immunoblot Method
This is a method in which a natural 11S type seed storage protein precursor inclusive of a precursor such as soybean glycinin is expressed in an
Escherichia coli
cell and purified, the precursor is split with legumaturain to produce an acidic subunit and a basic subunit, which are separated by SDS-PAGE and blotted to PVDF membrane and subsequently the subunits were qualitatively detected by immunoblot method at high sensitivity using an antibody to the basic subunit.
(2) Assay Method of the Activity by Fractionating Split Synthetic Peptide by High Performance Liquid Chromatography
This is a method in which legumaturain is acted on a synthetic peptide containing an asparagine residue, subsequently the substrate and resultant peptide were separated from each other using an inverse phase column connected to a high performance liquid chromatography apparatus, and its splitting activity is assayed.
However, the method (1) above takes totally about one day for the assay so that it cannot be said to be a quick assay method although it has advantages that it can assay relatively a large numbers of samples at a time and has a very high sensitivity. Also, the method (2) takes about 30 minutes per sample.
Furthermore, in the methods (1) and (2), contamination of protease other than legumaturain in samples makes it difficult to perform an accurate assay.
Therefore, the above-described conventional methods for assaying the activity of legumaturain have been defective in operability, quickness, etc.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for assaying the enzymatic activity of legumaturain which can overcome the above-described problems of the prior art and quickly assay the enzymatic activity of legumaturain specifically and in a simple and easy manner.
Absorptiometry, fluorimetry and the like are generally employed as a quick assay method of enzymatic activity.
Generally known method for assaying the enzymatic activity of endoproteases specific to an asparagine residue includes a method which assays an amidase activity, which is an activity of releasing an amino group from an amidated amino acid, instead of assaying the protease activity.
That is, there have already been reported several methods in which there is provided a peptide whose asparagine residue at the C-terminal side has an amidated carboxyl group and an asparagine residue-specific endoprotease is acted on said peptide to assay its activity.
More specifically, there have been reported (3) a method in which a peptide is synthesized whose asparagine residue has a carboxyl group to which an amino group is bonded and the amidase 2 activity of the peptide is assayed by high performance liquid chromatography (Abe Y. et al., Journal of Biological Chemistry, 268, 3525-3529 (1993)), (4) a method in which a peptide is synthesized whose asparagine residue has a carboxyl group to which a 7-amino-4-methylcoumarin is bonded and its amidase activity is assayed by fluorimetry (Asha A. Kembhavi et al., Archives of Biochemistry and Biophysics, 303, 208-213 (1993)), etc.
As in the assay methods (3) and (4), it is a simple and easy method to assay amidase activity instead of protease activity as used for trypsin, papain, etc.
Accordingly, the present inventors conducted the assay of legumaturain, whose enzymatic activity had already been confirmed by the methods (1) and (2) as described, with regard to its amidase activity by the methods (3) and (4), respectively.
However, legumaturain exhibited no amidase activity by these methods.
That is, in the assay methods (3) and (4), what is assayed is the activity of amidase, which is a contaminant, that is, the activity of releasing an amino group from an amidated amino acid, is just assayed as the effect of asparaginase, but not the activity of legumaturain, which specifically acts on asparagine residues to split its C-terminal side.
Therefore, it has been revealed that upon assaying the activity of legumaturain, a method using a peptide whose asparagine residue has an amidated carboxyl group at the C-terminal side thereof cannot be adopted.
Accordingly, the present inventors have synthesized various types of peptides and examined the properties of legumaturain. More specifically, various peptides were synthesized based on the splitting site of acidic subunit and basic subunit of A
2
B
1a
subunit from among the soybean glycinin precursor subunits and their splitting properties by legumaturain were examined.
As a result, it has now been elucidated that legumaturain shows no splitting activity to peptides whose asparagine residues exist on the N-terminal side and C-terminal side and it exhibits its activity according to a certain splitting rule. Furthermore, it has now been found that use of a fluorescence quenching substrate synthesized based on the splitting rule allows legumaturain activity to be assayed quickly and at high sensitivity. The present invention has been completed on the findings.
That is, in a first aspect, the present invention provides 1) a quick assay method for the activity of a plant-derived, asparagine residue-specific endoprotease, comprising measuring fluorescence generated by a fluorescence quenching substrate split by an asparagine residue-specific endoprotease, the fluorescence quenching substrate comprising an oligopeptide having in its amino acid sequence at least one asparagine residue, whose C-terminal side is other than an isoleucine residue, a leucine residue, or a valine residue, wherein the oligopeptide has a 7-methoxycoumarin-4-yl-acetyl group arranged on its N-terminal side and a 2,4-dinitrophenyl group arranged on its C-terminal side.
In a second aspect, the present invention provides 2) a quick assay method as described in 1) above, wherein the fluorescence quenching substrate is any one oligopeptides described in Sequence I. D. Nos. 1 to 6 of the sequence list, the oligopeptide having a 7-methoxycoumarin-4-yl-acetyl group arranged on its N-terminal side and a 2,4-dinitrophenyl group arranged on its C-terminal side.
In a third aspect, the present invention provides 3) a quick assay method as described in 1) or 2) above, wherein the fluorescence quenching substrate is any one oligopeptides described in Sequence I. D. Nos. 1 to 6 of the sequence list, the oligopeptide having a 7-methoxycoumarin-4-yl-acetyl group arranged on an amino group in a glycine residue at its N-terminal side and a 2,4-dinitrophenyl group arranged on an &egr;-amino group in a third lysine residue
Arahira Masaomi
Fukazawa Chikafusa
Director of National Food Research Institute, Ministry of Agricu
Leary Louise N.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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