Rat Brain Adenosine Deaminase After 2′‐Deoxycoformycin Administration: Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2′‐[3H]Deoxycoformycin Ligand Binding

R. A. Padua, J. D. Geiger, S. M. Delaney, J. I. Nagy

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10 Citations (Scopus)

Abstract

Abstract: 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. The results suggest that (1) although the biochemical properties of ADA are largely unchanged after treatment of rats with DCF, the enzyme in some brain regions has become less sensitive to this drug and (2) the prolonged reduction in ADA activity after DCF treatment is due to decreased levels of ADA protein possibly involving altered rates of degradation or production.

Original languageEnglish (US)
Pages (from-to)421-429
Number of pages9
JournalJournal of Neurochemistry
Volume58
Issue number2
DOIs
StatePublished - Feb 1992

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Pentostatin
Adenosine Deaminase
Rats
Brain
Ligands
Enzymes
rat Ada protein
Adenosine Deaminase Inhibitors
Rat control
Radioligand Assay
Level control

Keywords

  • 2′‐Deoxycoformycin
  • 2′‐[H]Deoxycoformycin binding
  • Adenosine deaminase
  • Purines
  • Rat brain

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

@article{453bef4c3b9e49f0bb2c80ec5a15290a,
title = "Rat Brain Adenosine Deaminase After 2′‐Deoxycoformycin Administration: Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2′‐[3H]Deoxycoformycin Ligand Binding",
abstract = "Abstract: 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. The results suggest that (1) although the biochemical properties of ADA are largely unchanged after treatment of rats with DCF, the enzyme in some brain regions has become less sensitive to this drug and (2) the prolonged reduction in ADA activity after DCF treatment is due to decreased levels of ADA protein possibly involving altered rates of degradation or production.",
keywords = "2′‐Deoxycoformycin, 2′‐[H]Deoxycoformycin binding, Adenosine deaminase, Purines, Rat brain",
author = "Padua, {R. A.} and Geiger, {J. D.} and Delaney, {S. M.} and Nagy, {J. I.}",
year = "1992",
month = "2",
doi = "10.1111/j.1471-4159.1992.tb09739.x",
language = "English (US)",
volume = "58",
pages = "421--429",
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T1 - Rat Brain Adenosine Deaminase After 2′‐Deoxycoformycin Administration

T2 - Biochemical Properties and Evidence for Reduced Enzyme Levels Detected by 2′‐[3H]Deoxycoformycin Ligand Binding

AU - Padua, R. A.

AU - Geiger, J. D.

AU - Delaney, S. M.

AU - Nagy, J. I.

PY - 1992/2

Y1 - 1992/2

N2 - Abstract: 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. The results suggest that (1) although the biochemical properties of ADA are largely unchanged after treatment of rats with DCF, the enzyme in some brain regions has become less sensitive to this drug and (2) the prolonged reduction in ADA activity after DCF treatment is due to decreased levels of ADA protein possibly involving altered rates of degradation or production.

AB - Abstract: 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. The results suggest that (1) although the biochemical properties of ADA are largely unchanged after treatment of rats with DCF, the enzyme in some brain regions has become less sensitive to this drug and (2) the prolonged reduction in ADA activity after DCF treatment is due to decreased levels of ADA protein possibly involving altered rates of degradation or production.

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