Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1999-05-26
2001-08-28
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S007400, C435S021000
Reexamination Certificate
active
06280965
ABSTRACT:
BACKGROUND OF THE INVENTION
Protein Kinase C (PKC) is a family of serine-threonine kinases which play an important role in many cellular functions. So far, at least ten different isoforms of PKC have been identified and broadly classified into three major families named classical PKC (cPKC), novel PKC (nPKC) and atypical PKC (aPKC). Of these three families, the classical type consisting of the &agr;, &bgr; (I and II), and &ggr; is the best characterized and most abundant. Enzymes in the cPKC family are activated by diacylglycerol in the presence of phosphatidylserine and Ca
++
subsequent to various cell activation mechanisms. However, enzymes in the nPKC family are independent of Ca
++
and the enzymes in the aPKC family are not readily activated by any of the standard second messengers such as phorbol esters and Ca
++
. Since different isoforms of PKC are widely distributed in various tissues and regulate different intracellular functions, estimation of PKC activity is an important step in signal transduction research.
Normally, PKC is assayed by measuring incorporation of
32
P from (&ggr;-
32
P)ATP into suitable substrates, usually histone (type III-SS), in a reaction mixture that contains magnesium, (&ggr;-
32
P)ATP, diacylglycerol, L-&agr;-phosphatidyl L-serine and Ca
++
. In most assays, the PKC reaction is run at 37° C. and at the end of reaction phosphorylated (
32
P) substrate is removed from the rest of the reaction mixture either by using precipitation methods or by using acidic phosphocellulose papers that bind only the basic phosphorylated histone while anionic (&ggr;-
32
P)ATP may be washed away. Both of these methods involve (&ggr;-
32
P)ATP and relatively expensive radioactive measurement techniques.
A procedure has been developed in which the PKC activity is measurable using non-radiolabeled ATP and a 96-well micro-plate. Our method uses a simple substrate immobilization technique and a colorimetric analysis to determine the extent of substrate phosphorylation. The method is simple, sensitive, rapid, and specific for PKC. This method may also be used for various other protein kinases by immobilizing suitable substrates and adjusting the incubation conditions.
The second type of enzymes that can be assayed by this invention, protein phosphatases, are regulatory enzymes that antagonize the action of protein kinases within the cell. Protein phosphatases fall into two general categories: those with specificity for phosphoserine or phosphothreonine residues and those with specificity for phosphotyrosine residues. Protein tyrosine phosphatases (PTPases) play a critical role in lymphocyte signaling, cell cycling, bacterial virulence and tumorigenesis. The serine/threonine class of protein phosphatases are also involved in many aspects of cellular regulation including immunosuppression, shellfish poisoning and cell cycle control. The serine/threonine-specific protein phosphatases are divided into two groups, type-1 and type-2. The type-1 protein phosphatases (PP-1) preferentially dephosphorylate the &bgr; subunit of phosphorylase kinase. Type-2 protein phosphatases termed PP-2A, PP-2B and PP-2C dephosphorylate the &agr; subunit of phosphorylase kinase. Protein phosphatases activities are normally measured using radiolabeled substrates or by using anti-phosphotyrosine antibodies to estimate the level of dephosphorylation from the specific substrates. The above mentioned techniques involve costly equipment or hazardous radioisotopes. A need exists for a simple, non-radioactive assay for estimating protein phosphatases. The present invention is a procedure in which the PP-1 activity is measured using non-radiolabeled substrate (phosphorylase-b) and a 96-well ELISA plate reader. The method uses a simple substrate immobilization technique and a colorimetric analysis to determine the extent of dephosphorylation. The method is simple, sensitive, rapid and specific for pp-1. This method may also be used for various other protein phosphatases by immobilizing suitable substrates and adjusting the incubation conditions.
SUMMARY OF THE INVENTION
The present invention is a simple and a rapid non-radioactive procedure for estimating the activity of protein kinase C (PKC) and protein phosphatase-1 (PP-1). The PKC assay involves immobilizing histone (type III-SS), the substrate for PKC, and then determining the extent of phosphorylation by using ammonium molybdate and 1-amino-2-naphthol-4-sulfonic acid based reaction for inorganic phosphate quantitation. Histone was attached to the assay plate by incubating 200 &mgr;l of 5 mg/ml solution at 4° C. for 12 hours. After removing non-adsorbed histone from the wells the PKC mediated phosphorylation reaction was initiated by adding a reaction mixture that contained PKC enzyme preparation, phorbol 12,13-dibutyrate (PDBu), L-&agr;-phosphatidyl-L-serine (PS), Ca
++
and ATP (adenosine triphosphate) in 50 mM Tris HCl (pH 7.5) buffer. At the end of 15 min incubation the reaction mixture was removed and the wells were washed twice with ice cold 50 mM Tris-HCl (pH 7.5) buffer. After two washings, 200 &mgr;l of alkaline phosphatase enzyme in 1.0 M diethanolamine buffer (pH 9.8) was added to the wells to release phosphates that have been transferred from ATP to histone by PKC. At the end of alkaline phosphatase hydrolysis of phosphorylated histone, the amount of phosphates present in the incubation medium was estimated using 1-amino-2-naphthol-4-sulfonic acid. The assay method is suitable for estimating PKC activity in tissue and cell samples without using any radiolabeled ATP.
The PP-1 assay involves immobilizing phosphorylase-b substrate to the 96-well assay plate and then phosphorylating the substrate using phosphorylase kinase. The activity of PP-1 is determined by quantitating the amount of phosphates released from the substrate by using ammonium molybdate and 1-amino-2-naphthol-4-sulfonic acid based reaction for inorganic phosphate quantitation. Phosphorylase-b is attached to the assay plates by incubating 200 &mgr;l of 5.0 mg/ml solution at 4° C. for 12 hours. After removing non-adsorbed phosphorylase-b from the wells, the PP-1 mediated dephosphorylation reaction is initiated by adding a reaction mixture that contains PP-1 enzyme preparation, 1 mM EDTA, 1 mM EGTA in 50 mM Tris HCl (pH 7.5) buffer. After a 15 minute incubation at 37° C., 50 &mgr;l of the reaction mixture is transferred to wells in a new plate that contains ammonium molybdate and then the amount of phosphates released into the incubation medium is estimated using 1-amino-2-naphthol-4-sulfonic acid. The assay method is suitable for estimating PP-1 activity in tissue and cell samples without using any radiolabeled substrate.
These, and further, and other objects and features of the invention are apparent in the disclosure, which includes the above and ongoing written specification, with the drawings.
REFERENCES:
patent: 4087331 (1978-05-01), Bucolo et al.
patent: 4923802 (1990-05-01), Gallis
patent: 5141852 (1992-08-01), Egan et al.
patent: 5527688 (1996-06-01), Mallia
patent: 5580747 (1996-12-01), Shulz et al.
patent: 5759787 (1998-06-01), Strulovici
Malave Andres
Rathinavelu Appu
Fronda Christian L.
Malin Haley & DiMaggio, P.A.
Nashed Nashaat T.
Nova Southeastern University
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