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Transport and Metabolism of D‐[ 3 H]Adenosine and L‐[ 3 H]Adenosine in Rat Cerebral Cortical Synaptoneurosomes
Author(s) -
Gu J. G.,
Geiger J. D.
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb10043.x
Subject(s) - adenosine , adenosine kinase , adenosine deaminase , chemistry , metabolism , purine metabolism , biochemistry , enzyme
The relationship between transport and metabolism in synaptoneurosomes was examined to determine the metabolic stability of rapidly accumulated D‐[ 3 H]adenosine and L‐[ 3 H]adenosine and the degree to which metabolism of the accumulated purines affected measurements of apparent K T and V max values for adenosine transport. For D‐[ 3 H]adenosine, high‐ and low‐affinity accumulation processes were present. For the high‐affinity system an inverse relationship was found between transport reaction times and K T and V max values. For incubations of 5, 15, and 600 s, which corresponded to 24, 32, and 76% phosphorylation of accumulated D‐[ 3 H]adenosine to nucleotides, apparent K T values were 9.4, 8.4, and 4.5 μ M , respectively, and V max values were 850, 70, and 12 pmol/min/mg of protein, respectively. Pretreatment with 10 μ M erythro ‐9‐(2‐hydroxy‐3‐nonyl)adenine, an adenosine deaminase inhibitor, and 5′‐iodotubercidin, an adenosine kinase inhibitor, decreased the phosphorylation of accumulated D‐[ 3 H]adenosine to 6% with 5‐s and 9% with 15‐s incubations. This resulted in significantly higher K T values: 36 μ M at 5 s and 44 μ M at 5 s. At 10‐min incubations in the presence of these inhibitors, metabolism of accumulated D‐[ 3 H]adenosine was 32%, and apparent K T and V max values at this time were not significantly different from those obtained without inhibitors. For L‐[ 3 H]adenosine, apparent K T and V max values for 20‐s incubations were 38.7 μ M and 330 pmol/min/mg of protein, respectively. Metabolism (mainly phosphorylation) of accumulated L‐[ 3 H]adenosine was observed only at incubations of >30 s. Taken together, these results demonstrate that adenosine transport is significantly faster than subsequent metabolism; that accumulated D‐adenosine is rapidly incorporated into and trapped intracellularly as adenine nucleotides, thereby affecting measured kinetic parameters for adenosine transport and giving an “appearance” of concentrative accumulations; and that the apparent K T T value of 39 μ M for D‐adenosine transport conducted in the presence of the enzyme inhibitors was the same as the apparent K T value for L‐adenosine transport.