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.
Mackiewicz
M
M
Center for Sleep and Respiratory Neurobiology, University of Pennsylvania School of Medicine, 3600 Spruce Street, 991 Maloney Building, Philadelphia, PA 19104-4283, USA. mirekmm@mail.med.upenn.edu
Geiger
J D
JD
Pack
A I
AI
D015720:5'-Nucleotidase / Q000378:metabolism*; D000241:Adenosine / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D009708:Nucleotidases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D004791:Enzyme Inhibitors; C523965:alpha,beta-methyleneadenosine 5'-diphosphate; D000244:Adenosine Diphosphate; C019598:5'-adenosine monophosphate phosphohydrolase; D009708:Nucleotidases; C030287:nucleotidase; D015720:5'-Nucleotidase; D008274:Magnesium; D000241:Adenosine