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Title: The purification and characterisation of a cysteine specific mono-ADP-ribosyltransferase from bovine erythrocytes
Author: Saxty, Barbara Ann
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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A cysteine specific ADP-ribosyltransferase activity was found in bovine erythrocytes. A three step purification procedure was developed involving (1) precipitation with 40% ammonium sulphate, (2) binding to a cysteine Sepharose affinity column, and (3) binding to a NAD+ affinity column. Polyacrylamide gel electrophoresis showed a single band at 45,000. The enzyme had been purified 47000 fold and had a specific activity of 1900 nmol nicotinamide released min-1 mg-1. A study of the kinetic properties of this enzyme showed that apparent substrate kinetics for cysteine were observed (Km of 4.4 mM) and a putative ADP-ribosyl-cysteine product was identified by HPLC. 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 labelling of a 55 kDa protein identified by autoradiography of separated proteins on SDS-polyacrylamide gels. Pertussis toxin also caused enhanced labelling of this 55 kDa protein as well as labelling of a 42 kDa protein, Giα. The 55 kDa protein was not recognised by antibodies raised against Gi or Gs and has not been identified. However evidence that mono-ADP-ribosylation of a cysteine residue on this 55 kDa protein had been catalysed was supported in three ways. (1) the labelling was blocked by pre-treatment of the erythrocyte ghosts with N-ethylmaleimide (NEM), a sulphydryl alkylating agent. (2) the labelling was insensitive to hydroxylamine, but was released by mercuric ion, and (3) the released radiolabelled product was identified as ADP-ribose on HPLC.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available