In:
European Journal of Biochemistry, Wiley, Vol. 155, No. 1 ( 1986-02), p. 157-165
Abstract:
Previous attempts to produce anti‐(ADP‐ribose) antibodies by immunization of rabbits with ADP‐ribose conjugated to serum albumin had resulted in the production of 5′AMP‐specific antibodies [Bredehorst et al. (1987) Eur. J. Biochem. 82 , 105–113]. To obtain true anti‐(ADP‐ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active β‐methylene derivative of NAD (NAD[CH 2 ]) was synthesized from ADP containing a methylene bridge (CH 2 ) instead of an oxygen in the diphosphate group. NAD[CH 2 ] was converted to its N 6 ‐[(2‐carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH 2 ]‐ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein‐bound ADP‐ribose groups, except for a cross‐reaction with FAD. AMP, ADP, ATP or poly(ADP‐ribose) interfered with [ 3 H]ADP‐ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000‐fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera‐toxin‐catalyzed reaction and the product, ADP‐ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide‐aided condensation. The purified antibodies allowed the detection of ADP‐ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [ 14 C]ADP‐ribosylated elongation factor 2. They also could be used to observe in situ , by indirect immunofluorescence, the increased mono(ADP‐ribosyl)ation of nuclear proteins in dimethyl‐sulfate‐treated cells, and to show that histone H2B was the principal histone acceptor of single ADP‐ribose groups in alkylated 3T3 cells.
Type of Medium:
Online Resource
ISSN:
0014-2956
,
1432-1033
DOI:
10.1111/ejb.1986.155.issue-1
DOI:
10.1111/j.1432-1033.1986.tb09471.x
Language:
English
Publisher:
Wiley
Publication Date:
1986
detail.hit.zdb_id:
1398347-7
detail.hit.zdb_id:
2172518-4
SSG:
12
Permalink