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Methods for carboxypeptidasesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving TransferaseMethods for carboxypeptidases description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080057527, Methods for carboxypeptidases. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of U.S. patent application Ser. No. 10/860,372, filed Jun. 3, 2004, which is a divisional of U.S. patent application Ser. No. 10/199,970, filed Jul. 19, 2002 (now U.S. Pat. No. 7,195,884), which are incorporated herein by reference in their entireties. FIELD OF THE INVENTION [0002] The invention relates to enzyme assays. More specifically, the invention relates to the detection of transferase activity, such as kinase activity and phosphatase activity. Furthermore, the invention relates to a process for screening potential inhibitors, activators, and other modifiers of transferases, such as kinases and phosphatases. Moreover, the invention is directed to kits for that can be used for detecting enzymatic activity of transferases, such as kinases and phosphatases, and for detecting inhibitors and activators of transferases. DESCRIPTION OF THE RELATED ART [0003] Enzymes are classified into groups according to the general kind of reaction they catalyze. Transferases catalyze the transfer of a group from one substrate to another and include kinases and phosphatases. Protein kinases transfer a phosphomoiety from a donor such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to an acceptor such as a peptide or protein to yield a phosphorylated peptide or protein and adenosine diphosphate (ADP) or guanosine diphosphate (GDP), respectively. Protein phosphatases are enzymes that transfer a phosphate group from a phosphopeptide or a phosphoprotein donor to an acceptor such as water. [0004] About two to five percent of the eukaryotic genome encodes for protein kinases and protein phosphatases. Although approximately 870 different protein kinases have been identified in the human genome, there may be many thousands of distinct and separate enzymes. In addition, protein substrates for these enzymes may amount to one-third of all cellular proteins. An understanding of these enzymes and their targets is crucial to understanding cellular regulation and cellular pathology. [0005] Protein kinases are often divided into two major groups based on the amino acid residue that is phosphorylated. The first group is serine/threonine kinases, which includes cyclic AMP-dependent protein kinases (PKA), cyclic GMP-dependent protein kinases (PKG), calcium and phospholipid dependent protein kinases (PKC), calcium and calmodulin-dependent protein kinases (CaMK), casein kinases, cell cycle protein kinases (cdc or cdk), protein kinase B (Akt), and others. These kinases are usually cytoplasmic or associated with the particulate fractions of cells, possibly by anchoring proteins. Protein serine/threonine kinases are the most common type of cytosolic kinases, and are thought to be responsible for the majority of phosphorylation events in the cell. In addition, there are some receptor kinases of the serine/threonine type, such as transforming growth factor beta (TGF-.beta.). Overall, serine/threonine kinases represent over 70% of cellular protein kinases. [0006] The second group of kinases, called tyrosine kinases, phosphorylate tyrosine residues. Overall, over 10% of kinases are tyrosine kinases. There are fewer tyrosine kinases, but they play an equally important role in cell regulation. 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 of the cell. More than 50 receptor tyrosine kinases are known. These kinases include several receptors for molecules such as growth factors and hormones, cytokines, and neurotransmitters. Examples of these include epidermal growth factor receptor (EGFR), insulin receptor (IR) and platelet derived growth factor receptor (PDGFR). There are also cytosolic tyrosine kinases, such as src, src-N1, fyn, lyk, lynA, lck. In addition, other kinases phosphorylate proteins or peptides containing histidine or aspartic acid residues. [0007] Protein phosphatases are enzymes that catalyze the removal of phosphate moieties from proteins or peptides that contain such modifications. As with kinases, classes of phosphatases are distinguished by their substrate specificity and dependence on other molecules for activation. Three major classes of phosphatases have been identified. The first class includes type 1 protein phosphatase (protein phosphatase-1 or PP1) and type 2 protein phosphatases (PP2A, PP2B, and PP2C). The second class includes tyrosine phosphatases such as PTP-1B, and YOP-51. Some phosphatases in this class are soluble but others comprise parts of a larger molecule, such as the receptor CD45. The third major class of phosphatases includes dual-specificity protein phosphatases that remove phosphate groups from both phosphoserine/phosphothreonine and phosphotyrosine. [0008] Protein kinases and protein phosphatases play very important roles in many cell functions, including, but not limited to, cellular metabolism, signal transduction, transcriptional regulation, cell motility, cell division, cellular signaling processes, cellular proliferation, cellular differentiation, apoptosis, and secretion. These processes are mediated by phosphorylation or dephosphorylation of enzymes, protein substrates, transcription factors, hormone or growth factor receptors, and other cellular proteins. [0009] In addition, protein kinases and protein phosphatases are involved in mediating the response to naturally occurring toxins and pathogens, which alter the phosphorylation states of proteins. Additionally, protein kinases are related to many epidemiologically relevant oncogenes and tumor suppressor genes. [0010] Notably, there are over 400 human diseases in which kinases are implicated. Examples include neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In myotonic dystrophy, a genetic defect in one form of the disorder is characterized by an amplified trinucleotide repeat in the 3' untranslated region of a protein kinase gene on chromosome 19. These modifications may someday elucidate many of the unusual features of the disorder. [0011] Because of this role of kinases and phosphatases in human pathology, modulators of kinases and phosphatases are potential drug targets. Currently, many inhibitors of kinases and phosphatases are available for treating a variety of diseases, while others are being tested for such use. One such inhibitor is Gleevec.TM. (Imatinib mesylate) (Novartis, Basel, Switzerland), which is a protein tyrosine kinase inhibitor of the Bcr-Abl tyrosine kinase. The abnormal constitutive expression of this tyrosine kinase is created by the "Philadelphia chromosome" abnormality in chronic myelogenous leukemia (CML). Gleevec.TM. inhibits proliferation and induces apoptosis in Bcr-Abl positive cell lines as well as fresh leukemic cells from Philadelphia chromosome positive CML patients. [0012] Fasudil (Eril.RTM. Injection S, Asahi Kasei Corp.) is potent inhibitor of Rho-kinase. Eril.RTM. has been approved in Japan for the treatment of cerebral vaspasm and an oral formulation is now is in clinical trials for the treatment of angina. [0013] An exemplary inhibitor of a clinically relevant phosphatase is cyclosporine A (CSA), which is used to prevent and treat ongoing acute rejection of transplanted organs. CSA inhibits the production of interleukin IL-2 by helper T-cells, thereby blocking T cell activation and proliferation (and inhibiting amplification of the immune response). The current model for the mechanism of action of CSA suggests that it blocks a phosphatase called calcineurin (PP2B). [0014] Further, phosphotyrosine phosphatase (PTP-1B) is currently under investigation as a target for the treatment of type II diabetes. [0015] These examples illustrate the importance of modulating kinases and phosphatases for clinically relevant circumstances. [0016] Current types of assays used to measure kinase and phosphatase activity and to detect potential kinase and phosphatase inhibitors and activators include Fluorescence Resonance Energy Transfer (FRET) assays, Fluorescent Polarization (FP) assays, and assays based on radioactivity such as Scintillation Proximity Assay (SPA). FRET assays used to detect kinase activity utilize a protein substrate that has two linked fluorescent molecules. The two molecules are in close proximity, separated by a fixed distance. The energy of an excited electron in one molecule (the donor) is passed to an adjacent molecule (the acceptor) through resonance. The ability of a higher energy donor flourophore to transfer energy directly to a lower energy acceptor molecule causes sensitized fluorescence of the acceptor molecule and simultaneously quenches the donor fluorescence. In this case, the fluorescence of the donor is "quenched" by the proximity to the acceptor and the energy of the donor is transferred to the acceptor in a non-radiative manner. The efficiency of energy transfer is dependent on the distance between the donor and acceptor chromophores according to the Forster equation. In most cases, no FRET is observed at distances greater than 100 angstroms and thus the presence of FRET is a good indicator of close proximity. [0017] In order for FRET to be useful, the fluorescence of the acceptor molecule must be significantly different from the fluorescence of the donor. A useful FRET based protein substrate may include a separation of the two fluorescent molecules via a peptide linker that maintains specificity for an endopeptidase that is capable of cleaving the peptide linker between the two fluorophores. If the peptide is phosphorylated, then the enzyme may not cleave the protein or may cleave it at a reduced rate, keeping the fluorescent molecules in close proximity such that quenching occurs. On the other hand, if the protein is not phosphorylated, then the endopeptidase cleaves the protein substrate, releasing the two fluorescent molecules such that the quenching is alleviated, and the two fluorescent molecules fluoresce independently. The FRET assay requires peptide substrates that must be carefully engineered to meet these requirements. That is, the peptide substrates must contain the enzyme recognition site required for the endopeptidase, the distance between the two fluorophores must be within the range to allow FRET to occur and the fluorescent molecules must be paired in such a way that donor fluorescence is significantly quenched, minimizing background fluorescence from the donor. Furthermore, the fluorescence of the starting material (the "quenched" substrate) must be significantly different from the product (the "released" non-quenched product). These requirements make a FRET based assay cumbersome and costly. [0018] FP assays are based on binding of a high affinity binding reagent, such as an antibody, a chelating agent, or the like, to a fluorescently labeled molecule. For example, an antibody that binds to a phosphorylated fluorescently labeled peptide but not to a non-phosphorylated fluorescently labeled peptide can be used for a kinase assay. When the fluorescent label is excited with plane polarized light, it emits light in the same polarized plane as long as the fluorescent label remains stationary throughout the excited state (duration of the excited state varies with fluorophore, and is 4 nanoseconds for fluoroscein). However, if the excited fluorescent label rotates or tumbles out of the plane of polarization during the excited state, then light is emitted in a different plane from that of the initial excitation state. If polarized light is used to excite the fluorophore, the emission light intensity can be monitored in both the plane parallel to the plane of polarization (the excitation plane) and in the plane perpendicular to the plane of polarization. The degree to which the emission intensity moves from the parallel to the perpendicular plane is related to the mobility of the fluorescently labeled molecule. If the fluorescently labeled molecules are large, such as when they are bound to the binding reagent, the fluorescently labeled molecules move little during the excited state interval, and emitted light remains highly polarized with respect to the excitation plane. If the fluorescently labeled molecules are small, such as when no binding reagent is bound to the fluorescently labeled molecules, the fluorescently labeled molecules rotate or tumble faster, and the resulting emitted light is depolarized relative to the excitation plane. Thus, an FP assay requires a high affinity binding reagent, e.g., an antibody, capable of binding with high specificity to the fluorescently labeled molecule. The time consuming and costly optimization of antibody binding with the specific fluorescently labeled molecules such as peptides is required where antibodies are used. Additionally, with FP assay there is the potential for a phosphorylated protein and other reaction components, e.g., lipids and detergents, to interfere with the polarization. [0019] Kinase assays that use radioactive labels include SPA. In SPA, modified ligand-specific or ligand-capturing molecules are coupled to fluoromicrospheres, which are solid-phase support particles or beads impregnated with substances that emit energy when excited by radioactively labeled molecules. When added to a modified ligand such as radiolabeled phosphopeptide in a mixture with the nonphosphorylated peptide, only the phosphopeptide is captured on a fluoromicrosphere, bringing any bound radiolabeled peptide close enough to allow the radiation energy emitted to activate the fluoromicrosphere and emit light energy. If the concentration of fluoromicrospheres is optimized, only the signal from the radiolabeled ligand bound to the target is detected, eliminating the need for any separation of bound and free ligand. The level of the light energy emitted may be measured in a liquid scintillation counter and is indicative of the extent to which the ligand is bound to the target. However, a SPA requires radiolabeled ligands, which have high disposal costs and possible health risks. In addition, a SPA requires the fluoromicrospheres to settle by gravity or be centrifuged, adding an additional step and time to the assay. [0020] With phosphorylation and dephosphorylation events involved in so many cell functions and diseases, identifying kinase and phosphatase activity is tremendously important. Thus, there is a need for alternative enzyme assays for detecting transferase activity, such as protein kinase and protein phosphatase activity, that do not require large amounts of costly or highly specialized starting materials and that do not require a large amount of time to complete. Additionally, there is a need for alternative assays to identify activators and inhibitors of kinases and phosphatases. In addition, it would also be desirable to provide kits for carrying out such assays. SUMMARY OF THE INVENTION Continue reading about Methods for carboxypeptidases... Full patent description for Methods for carboxypeptidases Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods for carboxypeptidases patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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