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A SIMPLIFIED METHOD FOR ESTIMATING 5‐PHOSPHORIBOSYL 1‐PYROPHOSPHATE IN MOUSE LIVER AND SPLEEN
Author(s) -
Chalmers AH
Publication year - 1984
Publication title -
australian journal of experimental biology and medical science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0004-945X
DOI - 10.1038/icb.1984.28
Subject(s) - spleen , pyrophosphate , chemistry , biology , biochemistry , immunology , enzyme
Summary A simplified method for the estimation of 5‐phosphoribosyl 1‐pyrophosphate (PRPP) in mouse liver and spleen is described. The method uses the enzymic conversion of [8‐ 14 C] hypoxanthine to [8‐ 14 C] inosine 5′‐monophosphate in the presence of PRPP and has the advantage over previously published methods in that the enzyme used in this assay, hypoxanthine‐guanine phosphoribosyl‐transferase (HG‐PRTase), is present in the tissue being analyzed and does not require preparation. In addition, each assay uses as its internal standard PRPP, thus overcoming the problem of the chemical instability inherent in this molecule. The two‐minute heal treatment used in the method was found to destroy most interfering enzymes and resulted in average recovery of PRPP of 56% and 85% for the liver and spleen, respectively. The counts obtained in the assay were about ten‐fold above background (80 c.p.m) and allowed accurate measurement of the phosphoribose sugar. The levels of PRPP (± standard deviation) found for liver and spleen in twelve (12) separate Bath/C male mice were 6·3 ± 1·8 and 10·8 ± 5·0 nmoles/g wet weight tissue, respectively, and agree with the tissue concentrations already reported for this metabolite. A further advantage of this assay is that it gives an index of the HG‐PRTase activity in the tissues.