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Method of detecting 5'-methioadenosine phosphorylase defect type mammarian cell

A technology of polynucleotides and immune animals, applied in the direction of microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of low yield, lack of detection, laborious and other problems

Inactive Publication Date: 2004-07-21
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, purification of MTAse from natural sources for the preparation of antibodies used in immunoassays for MTAse has proven to be a laborious process with low yields (Rangione et al., J. Bid. Chem, 261: 12324- 12329, 1986)
[0009] The lack of simple, efficient methods for detecting MTAse-deficient cells is partly responsible for the lack of effective therapeutics that selectively starve MTAse-deficient malignant cells in vivo

Method used

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  • Method of detecting 5'-methioadenosine phosphorylase defect type mammarian cell
  • Method of detecting 5'-methioadenosine phosphorylase defect type mammarian cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment I

[0071] Detection of MTA enzyme catalytic activity in one sample

[0072] By detection from [methyl- 14 C] 5′-deoxy-5′-methylthioadenosine [methyl- 14 C] 5-Methylthioribose-1-phosphate can measure the phosphorolytic activity of MTA enzymes (Seidenfeld et al., Biochem. Biophys. Res. Commun. 95, 1861-1866, 1980). In a total volume of 200 μl, the standard reaction mixture contained 50 mM potassium phosphate buffer, pH 7.4, 0.5 mM [methyl- 14 C] 5'deoxy-5'-methylthioadenosine (2×10 5 CPM / mmol), 1 mM DTT and the indicated amounts of enzyme. After incubation at 37°C for 20 minutes, the reaction was terminated by adding 50 μl of 3M trichloroacetic acid, and 200 μl of the sample was added to a 0.6×2 cm “Dowex” 50-H equilibrated with water. * column. Will [methyl- 14 C] 5-Methylthioribose-1-phosphate was eluted directly into scintillation vials containing 2 ml of 0.1 M HCl.

Embodiment II

[0074] Purification of Native MTAase from Rat Liver

[0075] MTAse was isolated from rat liver using a modification of the method of Rangione et al. (J. Biol. Chem. 261, 12324-12329, 1986). 50 g of fresh rat liver was homogenized in Waring Blendor with 4 times the volume of 10 mM potassium phosphate buffer containing 1 mM DTT, pH 7.4 (buffer A). The homogenate was centrifuged (15,000 x g for 1 hour), and the resulting supernatant was subjected to ammonium sulfate fractionation. The pellet between 55 and 75% saturation was collected by centrifugation (15,000 xg for 20 minutes) and dissolved in a minimal volume of buffer A. Samples were then dialyzed overnight against three changes of 100 volumes of the same buffer.

[0076] Samples were clarified by centrifugation at 15,000 xg for 30 min and loaded onto a DEAE-Sephacyl column (1.5 x 18 cm; Pharmacia) pre-equilibrated with buffer A. After washing with 80 ml equilibration buffer, it was eluted with a linear ...

Embodiment III

[0079] Determination of Partial Amino Acid Sequence of Rat MTAzyme

[0080] The purified sample was lyophilized, dissolved in 50 μl carrier buffer (1% + sodium dialkyl sulfate (SDS), 10% glycerol, 0.1M DTT and 0.001% bromophenol blue) and loaded onto 0.5 mm thick 10% SDS poly Acrylamide gel (Bio Rad "MINIGEL" apparatus). After electrophoresis, according to Towbin et al. described (Proc.Natl.Acad.Sci.USA 76,4340-4345,1979) using transfer buffer (15mM Tris, 192mM glycine and 20% methanol, pH8.3) in a Bio-Rad The protein was electroblotted for 2 hours onto a nitrocellulose membrane (0.45 [mu]m pore size, Millipore) in a transfer system.

[0081] After transfer, proteins were reversibly stained with Ponceau S (Sigma) following a modified method described by Sailinovich and Montelaro (Anal. Biochem. 156, 341-347, 1987). The nitrocellulose membrane was then dipped for 60 seconds in a solution of 0.1% Ponsean S dye in 1% aqueous acetic acid. Excess dye was removed ...

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Abstract

A method for the detecting whether methyladenosine phosphatase (MTAse) is present in a cell sample in either a catalytically active or catalytically inactive form. In one respect, the method comprises adding oligonucleotide probes to the sample, which probes are capable of specifically hybridizing to any MTAse encoding nucleic acid in the sample under conditions favoring that hybridization. Absence of MTAse in a sample is considered to be indicative of malignancy. Polynucleotides encoding MTAse, MTAse peptides and antibodies to MTAse, as well as kits for performing the methods of the invention, are provided.

Description

[0001] This application is a divisional application of the Chinese patent application 94195031.X filed on August 22, 1996 with the title of "Method for Detecting 5'-Methylthioadenosine Phosphorylase Deficient Mammalian Cells". technical field [0002] The present invention relates to a method for detecting 5'-methylthioadenosine phosphorylase in mammalian cells, the state of which is an indicator of whether these cells are in a malignant state. Detection of cells deficient in the enzyme allows targeting of these cells in chemotherapy which takes advantage of the inability of these cells to convert 5'-methylthioadenosine to methionine. Background technique [0003] Methionine (MET) is required for normal and malignant cell growth. In certain malignant cells, this requirement is absolutely essential, i.e. without an appropriate supply of MET, the cell dies. [0004] In mammalian cells, there are three sources of MET. Either obtained from food or by biochemical synthesis of M...

Claims

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Application Information

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IPC IPC(8): G01N33/566C07H21/04C07K7/06C07K7/08C07K16/40C12N1/21C12N5/07C12N5/071C12N9/10C12N9/12C12N15/00C12N15/02C12N15/09C12P19/34C12P21/08C12Q1/00C12Q1/25C12Q1/68C12R1/19C12R1/91G01N33/50G01N33/573
CPCC12Q1/25G01N33/573C12N9/1077C12Q1/6886C07K16/40G01N33/5005C12Q2600/156A61P35/00
Inventor 楚特穆·诺伯里丹尼斯·A·卡森肯吉·塔卡巴亚什
Owner RGT UNIV OF CALIFORNIA
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