[Purine metabolism: determination of adenyl deaminase in human lymphocytes].
Adenylic acid (AMP) deaminase is a "catabolic enzyme" involved in nucleotide degradation, transforming AMP into inosinic acid (IMP). We present a simple method for the determination of the enzyme activity, which combines high sensitivity with requirement of low quantities of lymphocytes. Human lymphocytes were isolated with a Lymphocyte Separation Medium from FLOW and sonicated. After centrifugation at 2,000 rpm x 10 min and treatment with Norit A, the cells were incubated at 37 degrees C with ATP 0.8 mM and 14C-AMP 0.1 mM (specific activity 12 microCi/mumole) in potassium phosphate 100 mM (pH 7.4). 14C-IMP and 14C-AMP were separated through HPLC by an isocratic elution, with 20 mM KH2PO4 (pH 5.5) at a 1.5 ml/min flow rate. Identification of the nucleotides was carried out through retention time, coelution with internal standards: their evaluation by determining the radioactivity of the collected peaks. The enzyme activity is decreased in patients affected by CLL: the decrease is evident only when data are referred to the single cells and not when they are referred to the protein.
Tabucchi
A
A
Istituto di Chimica Biologica, Università di Siena.
Terzuoli
L
L
Di Stefano
A
A
Pizzichini
M
M
Leoncini
R
R
Dispensa
E
E
Marinello
E
E
D000659:AMP Deaminase / Q000032:analysis*; D006801:Humans; D007291:Inosine Monophosphate / Q000032:analysis*; D007292:Inosine Nucleotides / Q000032:analysis*; D008214:Lymphocytes / Q000201:enzymology*; D009709:Nucleotide Deaminases / Q000032:analysis*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000097:blood / Q000201:enzymology*; D011687:Purines / Q000378:metabolism
D007292:Inosine Nucleotides; D011687:Purines; D007291:Inosine Monophosphate; D009709:Nucleotide Deaminases; D000659:AMP Deaminase
Metabolismo purinico: determinazione dell'adenilico deaminasi in linfociti umani.