In vitro ultrastructural localization of the catalytic activity of adenosine deaminase in murine C-1300 neuroblastoma.
Adenosine deaminase (ADA) activity was localized at the surface membrane of the mouse C-1300 neuroblastoma by incubation of a confluent tissue culture monolayer grown on Lux-Permanox cultureware with 6-chloropurine ribonucleoside (CPR). This substrate is dechlorinated by ADA to form Cl-. At loci of ADA activity, Cl- is precipitated with added silver ion (Ag+), and electron dense metallic silver (Ag degree) is formed upon exposure to light. The incubation was conducted in 0.2 M HEPES buffer (277 mOs) at 37 degrees C, pH 7.4, which contained 1 mM CPR, for 5 min (in this buffer, this is four times the Km); the control lacked the substrate. After completion of the incubation, the monolayer was briefly rinsed with 0.2 M HEPES and 2.5% glutaraldehyde in 0.2 M HEPES containing AgNO3 at a final concentration of 2 mM. Dehydration was accomplished in a graded series of ethanol followed by embedment in the L. R. White resin at 60 degrees C overnight. Thin sections (80 to 100 mm), cut parallel to the monolayer, showed ADA activity at the cells' surface membrane with a smaller amount of activity evenly distributed in the subadjacent ectoplasmic zone. The control lacked any silver grain localization. Since in preliminary studies of neuronal tissue the ADA activity was minimal, these findings may contribute to developing a diagnostic cancer screening test.
Bielat
K L
KL
Department of Pathology, Roswell Park Memorial Institute, Buffalo, NY 14263.
Tritsch
G
G
Chervinsky
D
D
D000243:Adenosine Deaminase / Q000032:analysis*; D000818:Animals; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000243:Adenosine Deaminase