An NAD+:cysteine ADP-ribosyltransferase activity was purified from bovine erythrocytes on the assumption that, like pertussis toxin, the enzyme would exhibit a cysteine-dependent NAD+ glycohydrolase activity. A three-step purification procedure was developed involving (1) precipitation with 40% (NH4)2SO4, (2) binding to a cysteine-Sepharose affinity column, and (3) binding to an NAD+ affinity column. PAGE showed a single band of M(r) 45,000. The enzyme had been purified 47,000-fold and had a specific activity of 1900 nmol nicotinamide released/min per mg. A study of the kinetic properties of this enzyme showed saturation kinetics for cysteine (Km = 4.0 mM). The ability of this enzyme to ADP-ribosylate protein was investigated using re-sealed inverted bovine erythrocyte ghosts. Incubation of the purified enzyme with erythrocyte ghosts and [adenylate-32P]NAD+ led to the enhanced dose-dependent labelling of several proteins, a doublet of high M(r) and proteins of M(r) 60,000, 55,000 and 29,000, identified by autoradiography of separated proteins on SDS/PAGE. The enzyme-catalysed labelling of the major component at M(r) 55,000 was blocked by pre-treatment of the erythrocyte ghosts with N-ethymaleimide, a sulphydryl alkylating agent, and the label was released by mercuric ion, but not by hydroxylamine. These experiments suggested that a cysteine residue on the target protein had been mono-ADP-ribosylated. This supposition was further supported by identification of the mercf1p4ion-released radiolabelled product as ADP-ribose by HPLC, and the observation that free ADP-ribose was unable to modify the membrane target protein directly.
Skip Nav Destination
Follow us on Twitter @Biochem_Journal
Article navigation
September 1995
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkAdvertising
Research Article|
September 15 1995
The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity
B A Saxty;
B A Saxty
1Department of Biochemistry, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland, U.K.
Search for other works by this author on:
S van Heyningen
S van Heyningen
1Department of Biochemistry, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland, U.K.
Search for other works by this author on:
Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1995 The Biochemical Society, London
1995
Biochem J (1995) 310 (3): 931–937.
Citation
B A Saxty, S van Heyningen; The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity. Biochem J 15 September 1995; 310 (3): 931–937. doi: https://doi.org/10.1042/bj3100931
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Biochemical Society Member Sign in
Sign InSign in via your Institution
Sign in via your InstitutionGet Access To This Article
Follow us on Twitter @Biochem_Journal
Open Access for all
We offer compliant routes for all authors from 2025. With library support, there will be no author nor reader charges in 5 journals. Check here |
![]() View past webinars > |