| A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate. | |
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MedLine Citation:
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PMID: 2437825 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured. |
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Authors:
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D J Hupe; N D Behrens |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Analytical biochemistry Volume: 161 ISSN: 0003-2697 ISO Abbreviation: Anal. Biochem. Publication Date: 1987 Feb |
Date Detail:
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Created Date: 1987-05-22 Completed Date: 1987-05-22 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0370535 Medline TA: Anal Biochem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 20-5 Citation Subset: IM |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Cattle Cells, Cultured Cellulose / analogs & derivatives Methods Orotate Phosphoribosyltransferase / analysis* Orotic Acid Pentosephosphates / analysis* Pentosyltransferases / analysis* Phosphoribosyl Pyrophosphate / analysis* Polyethyleneimine / analogs & derivatives Uridine Monophosphate / analogs & derivatives, analysis |
| Chemical | |
Reg. No./Substance:
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0/Pentosephosphates; 0/polyethyleneimine-cellulose; 2149-82-8/orotidylic acid; 58-97-9/Uridine Monophosphate; 65-86-1/Orotic Acid; 7540-64-9/Phosphoribosyl Pyrophosphate; 9002-98-6/Polyethyleneimine; 9004-34-6/Cellulose; EC 2.4.2.-/Pentosyltransferases; EC 2.4.2.10/Orotate Phosphoribosyltransferase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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