Determination of enzyme activity in single bovine adrenal medullary cells by separation of isotopically labeled catecholamines.
A microcolumn liquid chromatography method for determining norepinephrine (NE), epinephrine (E), and phenylethanolamine N-methyltransferase (PNMT) enzyme activity in single bovine adrenal medullary cells is presented. Single cells were isolated and treated with excess deuterated substrate, D3-NE (0.05 mM) for enzyme reaction. After 6 h, the reaction was quenched and the product, D3-E, was quantified along with endogenous NE and E. Separation and detection of deuterated and protiated NE and E were achieved with microcolumns (110-125 cm long, 25 microns inner diameter) packed with 3 microns octadecylsilane-modified particles and operated with amperometric detection. Of the 33 cells reported, most cells containing predominantly E have enzyme activity while cells containing predominantly NE and cells containing a mixture of both NE and E show no enzyme activity. After incubation with 10 microM hydrocortisone, of the 17 cells reported, most cells containing predominantly E and cells containing a mixture of both NE and E have enzyme activity while cells containing predominantly NE have no enzyme activity. Detection limits for NE and E were 42 and 48 amol, respectively.
Hsieh
S
S
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290, USA.
Jorgenson
J W
JW
D000313:Adrenal Medulla / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D004837:Epinephrine / Q000302:isolation & purification*; D008832:Microchemistry; D009638:Norepinephrine / Q000302:isolation & purification*; D010625:Phenylethanolamine N-Methyltransferase / Q000302:isolation & purification*
D010625:Phenylethanolamine N-Methyltransferase; D009638:Norepinephrine; D004837:Epinephrine