Eve Szabados

METABOLISM OF ADENOSINE AND DEOXYADENOSINE BY HUMAN ERYTHROCYTES AND CCRF-CEM LEUKEMIA CELLS

Human lymphocytes lacking adenosine deaminase die and T-cell leukemias are killed by deoxycoformycin (dCf), an inhibitor of adenosine deaminase, due to impaired metabolism of dAdo. The initial metabolism of exogenous adenosine (Ado) and deoxyadenosine (dAdo) has been compared in human erythrocytes and CCRF-CEM leukemia cells and the data obtained have been simulated using kinetic constants obtained in vitro for the enzymes involved. Cells were mixed with 3H-labelled Ado and dAdo, samples were taken at 3 sec intervals and progress curves for the 3H-labelled metabolites formed were determined by quantitative two-dimensional thin layer chromatography. Erythrocytes rapidly take up Ado and the predominant metabolite after 60 sec is hypoxanthine (Hyp), while for dAdo, deoxyinosine (dIno) predominates. By contrast, leukemia cells convert to Ado predominantly to AMP, while dAdo is converted first to Hyp and the to AMP. The presence of dCf had little effect upon Ado metabolism by induced accumulation of dAdo. Erythrocytes rapidly degrade Ado and dAdo to Hyp, although the phosphorolysis of dIno is relatively slow. Human CCRF-CEM leukemia cells convert most of the Ado or dAdo to AMP after 60 sec. For dAdo, the sequence of reactions would be dAdo-->dIno-->Hyp-->IMP-->sAMP-->AMP. dCf does not significantly affect the conversion of Ado-->AMP, but dCf blocks AMP accumulation from dAdo, consistent with the reaction sequence shown above. A computer model has been developed for the metabolism of Ado and dAdo, but some of the kinetic constants determined in vitro for this model do not pertain to intact cells.

Inosine‐5′‐monophosphate analogues as inhibitors of human IMP cyclohydrolase and cellular growth

The catalytic mechanism for the enzyme, IMP cyclohydrolase, may involve a reaction intermediate with negative charge in the 2‐position of the purine ring (Szabados, E., Hindmarsh, E., Phillips, L., Duggleby, R.G. & Christopherson, R.I. (1994) Biochemistry 33, 14237‐14245). Threeanalogues of IMP have been synthesised where fluorine, chlorine or bromine has been substituted in the 2‐position on the purine ring. These analogues with an electronegative substituent may resemble a reaction intermediate for IMP cyclohydrolase; 2‐fluoro IMP is a potent inhibitor of the enzyme with a Ki value of 0.19 μM, while 2‐chtoro IMP has a Ki of 1.9 μM and 2‐bromo IMP is not inhibitory. However, IMP cyclohydrolase is not inhibited in human CCRF‐CEM leukaemia cells exposed to 2‐fluoro inosine although it is toxic to these cells with an IC50 value of 4.9 μM.

Substrate Channelling by Human IMP Synthase

IMP synthase is a bifunctional enzyme containing the activities, 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (EC 2.1.2.3) and IMP cyclohydrolase (EC 3.5.4.10) and catalyses the ninth and tenth reactions of the pathway for de novo biosynthesis of purine nucleotides.

Rapid radioassay for metabolites of adenosine and deoxyadenosine in erythrocytes

A radioassay has been developed to quantify the uptake and initial metabolism of adenosine (Ado) or deoxyadenosine (dAdo) by human erythrocytes. Cell suspension and [3H]Ado are mixed at 3-s intervals with a novel dual-syringe apparatus, and uptake and metabolism of Ado is stopped by centrifuging the cells through a dibutylphthalate layer into perchloric acid. The neutralized cell extract is analyzed by two-dimensional chromatography on poly(ethyleneimine)-cellulose plates by two procedures using combinations of solvents optimised for the separation of nucleosides and nucleobases, and for nucleotides derived from the exogenous [3H]Ado.