[Purine metabolism: determination of adenyl deaminase in human lymphocytes]

Boll Soc Ital Biol Sper. 1990 Apr;66(4):349-55.
[Article in Italian]

Abstract

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.

Publication types

  • English Abstract

MeSH terms

  • AMP Deaminase / analysis*
  • Humans
  • Inosine Monophosphate / analysis*
  • Inosine Nucleotides / analysis*
  • Lymphocytes / enzymology*
  • Nucleotide Deaminases / analysis*
  • Precursor Cell Lymphoblastic Leukemia-Lymphoma / blood
  • Precursor Cell Lymphoblastic Leukemia-Lymphoma / enzymology*
  • Purines / metabolism

Substances

  • Inosine Nucleotides
  • Purines
  • Inosine Monophosphate
  • Nucleotide Deaminases
  • AMP Deaminase