Rat brain adenosine deaminase after 2'-deoxycoformycin administration: biochemical properties and evidence for reduced enzyme levels detected by 2'-[3H]deoxycoformycin ligand binding.
Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2'-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r = 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Padua
R A
RA
Department of Pharmacology, University of Manitoba, Faculty of Medicine, Winnipeg, Canada.
Geiger
J D
JD
Delaney
S M
SM
Nagy
J I
JI
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000243:Adenosine Deaminase / Q000378:metabolism*; D058892:Adenosine Deaminase Inhibitors; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D007413:Intestinal Mucosa / Q000201:enzymology; D008024:Ligands; D008297:Male; D015649:Pentostatin / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D058892:Adenosine Deaminase Inhibitors; D008024:Ligands; D015649:Pentostatin; C010192:9-(2-hydroxy-3-nonyl)adenine; D000243:Adenosine Deaminase; D000225:Adenine