Quantitation of individual protein kinase activity

Details Quantitation of individual protein kinase activity Quantitation of individual protein kinase activity

1994-03-04

2002-02-19

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S007400, C435S007500, C435S015000, C435S194000

Reexamination Certificate

active

06348310

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed to a process for providing an assay protocol that measures enzymatic activity. More particularly, the invention is directed to a process to precisely and conveniently quantitate enzymatic activity of protein kinases and further to provide an assay specific for individual protein kinases in the presence of other protein kinases.
CITED REFERENCES
A bibliography of the references cited in this application can be found in the section preceding the claims.
DESCRIPTION OF THE PRIOR ART
Enzymes are large proteins that catalyze reactions in living cells. Enzymes build up or tear down other molecules. For example, enzymes catalyze the synthesis of fat from fatty acids, form complex sugars from glucose and fructose, and aid in the formation of other proteins from amino acids. Enzymes also reverse the build-up process by breaking down more complex structures. Enzymes are generally specific to certain substrates for their reactions. For example, an individual enzyme may catalyze the reaction where only one substrate is involved or it may act on a group of related substrates.
In healthy persons, most enzymes are found within cells. Some diseases however cause the release of enzymes from dying cells into the blood. The increased levels of enzymes can then be measured. An abnormal level of enzymes in the blood characterizes certain medical conditions. For example, an enzyme assay for abnormal levels of the enzyme creatine kinase in the blood is useful as a diagnostic measure of heart disease. In like manner, bone or liver diseases can be diagnosed by observing increasing levels of alkaline phosphatase in the blood stream. Prostate cancer is diagnosed by increased levels of acid phosphatase in the blood stream.
Enzymes are classified into groups according to the general kind of reaction they catalyze. The present invention refers to a specific group of enzymes called transferases, which catalyze the transfer of a group from one substrate to another. The present invention is specifically directed to the transferase subgroup called protein kinases.
Protein kinase is a generic name for all enzymes that transfer a phosphate to a protein. About three to four percent of the human genome contains transcription information for the formation of protein kinases. Currently, there are up to 200 known different protein kinases. However, because three to four percent of the human genome is a code for the formation of protein kinases, there may be many thousands of distinct and separate kinases in the human body.
Protein kinases are enzymes which catalyze the transfer of phosphorous from adenosine triphosphate (ATP), or guanosine triphosphate (GTP) to the targeted protein to yield a phosphorylated protein and adenosine diphosphate (ADP) or guanosine diphosphate (GDP), respectively. ATP or GTP is first hydrolyzed to form ADP or GDP and inorganic phosphate. The inorganic phosphate is then attached to the targeted protein. The protein substrate which is targeted by kinases may be a structural protein, found in membrane material such as a cell wall, or another enzyme which is a functional protein.
Due to their physiological relevance, variety and ubiquitousness, protein kinases have become one of the most important and widely studied family of enzymes in biochemical and medical research. Studies have shown that protein kinases are key regulators of many cell functions, including signal transduction, transcriptional regulation, cell motility, and cell division. Several oncogenes have also been shown to encode protein kinases, suggesting that kinases play a role in oncogenesis.
Protein kinases are often divided into two groups based on the amino acid residue they phosphorylate. The first group, called serine/threonine kinases, includes cyclic AMP and cyclic GMP dependent protein kinases, calcium and phospholipid dependent protein kinase, calcium and calmodulin-dependent protein kinases, casein kinases, cell division cycle protein kinases and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins.
The second group of kinases, called tyrosine kinases, phosphorylate tyrosine residues. They are present in much smaller quantities but play an equally important role in cell regulation. These kinases include several receptors for molecules such as growth factors and hormones, including epidermal growth factor receptor, insulin receptor, platelet derived growth factor receptor and others. Studies have indicated that many tyrosine kinases are transmembrane proteins with their receptor domains located on the outside of the cell and their kinase domains on the inside.
Phosphorylation of serine-, threonine- and tyrosine-containing proteins by kinases is important because the phosphorylated protein products have been implicated in a variety of cellular processes including oncogenesis, cellular transformation, cellular growth and exocytosis. Currently, much experimentation is performed involving kinases which may inhibit cancer growth or promote cancer cell death. Determining the specific kinase involved in inhibiting cancer growth or promoting cell death is important to society. Therefore, advances in recognizing kinase activity levels are extremely important.
Activity Determination
Reference is made to Robyt and White (1990) which is incorporated herein by reference, for a general description of methods for determining the activity of an enzyme. Robyt and White defines the activity of an enzyme as the amount of reaction that a certain amount of enzyme will produce in a specified period of time. The activity is determined by measuring the amount of product produced or the amount of substrate used up per unit of time under high concentrations or saturating conditions of substrate. This is usually accomplished by performing a chemical analysis for the product or substrate.
Substrates that are typically used in an assay for specific kinase activity include casein, which is isolated from milk; histones, isolated from calves; phosphovitin, isolated from egg yolks; and myelin basic proteins, isolated from bovine spinal cords. These substrates can be phosphorylated in an assay, assuming that the correct kinase has been chosen. Assays utilizing these substrates to determine kinase activity are well known in the prior art.
Radioactive Detection of Kinase Activity
Most current methods of measuring protein kinase activity are based on the radioactive detection method. In these methods, a sample containing the kinase of interest is incubated with activators and a substrate in the presence of &tgr;-
32
P-ATP or &tgr;-
32
P-GTP. Often, a general and inexpensive substrate such as histone or casein is used. After a suitable incubation period, the reaction is stopped and an aliquot of the reaction mixture is placed directly onto a filter which binds the substrate. The filter is then washed several times to remove excess radioactively-labeled free ATP, and the amount of radio-labeled phosphate incorporated into the substrate is measured by scintillation counting. This method is widely used and provides an accurate method for determining protein kinase activity in both crude and purified samples.
Babcook et al. (1991) also describe an assay using monoclonal antibodies and immunofluorescence technology for the determination of protein-tyrosine kinase and protein-tyrosine phosphatase activities. The method was performed utilizing p56
lck
or p60
src
.
Budde et al. (1991) disclose assay techniques utilizing acidic peptide substrates of protein kinases. This technology uses radioactive phosphorous placed in a substrate to be studied. After kinase activity, the phosphopeptide is eluted while the individual radioactive phosphorus, ATP and protein are impeded.
Gopalakrishna et al. (1992) disclose a method which utilizes the conventional approaches to measure protein kinase activity. The method combines the incubations and filtrations necessary to determine the protein kinase activity using multi-well plates with fitted filtrati

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