Studies of mono- and poly(ADP-ribose) metabolism in cultured cells using in vivo labelling methods

Conference · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5331539

Jacobson, M K; Aboul-Ela, N; Jacobson, E L

NAD is consumed in a metabolism involving protein bound monomers and polymers of ADP-ribose. The lack of suitable methods to radiolabel and quantify ADP-ribosyl residues in intact cells has been an obstacle to the study of this metabolism. The authors have developed analytical methodology that allows the labelling and rapid, sensitive, and selective quantification of NAD, protein bound mono(ADP-ribose), and poly(ADP-ribose) in intact cultured cells. Confluent cultures are radiolabelled overnight with high specific activity /sup 3/H-adenine which results in the efficient labelling of ATP and NAD pools to constant specific radioactivity. For analysis, cells are extracted with trichloroacetic acid. The acid soluble pool can be directly analyzed for radiolabelled NAD using dihydroxyboronyl sepharose (DHB-S) affinity chromatography. Radiolabel in poly(ADP-ribose) can be specifically quantified from the acid insoluble fraction by alkaline treatment to destroy monomers of ADP-ribose followed by DHB-S chromatography. Radiolabel in protein bound monomers of ADP-ribose can be quantified utilizing selective release of ADP-ribose, and boronate affinity chromatography. These methods should facilitate the detailed study of the ADP-ribose metabolism in intact cultured cells.

• Research Organization: North Texas State Univ., Ft. Worth
• OSTI ID: 5331539
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

550501* -- Metabolism-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADENINES
ADP
ALDEHYDES
AMINES
ANTIMETABOLITES
AROMATICS
AZAARENES
CARBOHYDRATES
CELL CULTURES
CHROMATOGRAPHY
COENZYMES
DRUGS
HETEROCYCLIC COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
METABOLISM
MONOSACCHARIDES
NAD
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PENTOSES
PURINES
RIBOSE
SACCHARIDES
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS