Simultaneous assessment of ecto- and cytosolic-5'-nucleotidase activities in brain micropunches

We propose a new methodology for simultaneous assessment of ecto- and cytosolic-5'-nucleotidase that can be utilized in brain to measure the activity of these enzymes in micropunches of tissues. It is based on the differential sensitivity of both enzymes to alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP) and the requirements for magnesium as a co-factor. The design of assay protocol contains an internal validation by allowing comparisons between total level of 5'-nucleotidase activity with that calculated from the sum of individual activities of the ecto- and cytosolic-5'-nucleotidases. We have applied this new approach to assess the activity of ecto- and cytosolic-5'-nucleotidase in the brain regions relevant to sleep regulation. The level of both enzymes was significantly lower in the cerebral cortex than other brain regions tested.