The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity

Abstract

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.

Interaction of Two Classes of ADP-ribose Transfer Reactions in Immune Signaling

“…The concentrate was applied to a high performance liquid chromatography gel filtration column and eluted with PBS. Cysteine-specific ADPRT activity was measured as described (27). One unit of the enzyme was expressed as nanomole/min.…”

O-ADP-Ribosylation in the NAD/NADase System: 2-Alkanols as Efficient Substrates.

“…The isolated mass was further subjected to chromatography and the appropriate fractions were repeatedly lyophilized to provide 9 (307 mg, 41% yield) as its white ammonium salt, and as a single spot by TLC analysis. An analytical sample was obtained by further drying over P 2 Conversion of 2-Butanol (2) to O-Ribosylation Product (10) b-NAD (900 mg, 1.34 mmol) and 2 (3.0 g, 41 mmol) were incubated with NADase (10 ml, 4 U) in 0.1 M Tris-HCl (80 ml, pH 7.2) at 37°C for 30 h. The reaction mixture was treated similarly as described above to provide a crude mass. The crude mass was repeatedly purified by column chromatography on DEAE Sephadex A-25 in a similar manner as described above to give 10 (236 mg, 29% yield).…”

“…[1][2][3] ADP-ribosylation occurs, depending upon the chemicoenvironmental conditions, on the appropriate ring nitrogen atom of various nitrogen-containing heterocyclic compounds. 4) In general, the enzymatic reaction has been shown to be susceptible to the stereochemical and electronic environment of the substrate.…”

Unusual Enzymatic Hydrolysis of NAD by Solubilized Form of NAD+ Glycohydrolase.

“…There have been many reports [1][2][3][4][5][6][7][8][9] concerning substrates for the transferaselike activity of NADase, including synthetic low molecular compounds [2][3][4][5] and certain antibiotics components 6,7) as well as in vivo high molecular constituents. [8][9][10] NADase is a membrane-bound ecto-enzyme that is widely distributed in the organism, the richest source generally being the spleen, brain, and liver.…”

“…There have been many reports [1][2][3][4][5][6][7][8][9] concerning substrates for the transferaselike activity of NADase, including synthetic low molecular compounds [2][3][4][5] and certain antibiotics components 6,7) as well as in vivo high molecular constituents. [8][9][10] NADase is a membrane-bound ecto-enzyme that is widely distributed in the organism, the richest source generally being the spleen, brain, and liver.11) From the established viewpoint that the catalytic domain of the enzyme occurs in the extracellular region of the molecule, 12) it is of interest to examine whether enzyme activities of the parent particulate NADase (pNADase) are similarly observed with a solubilized extracellular portion (sNADase) of the pNADase.Thus, we undertook the solubilization of the porcine brain pNADase according to the method for bovine spleen NADase of Augustin et al,13) since no procedure has not been established for porcine brain NADase. The crude sNADase thus obtained, however, showed unexpected catalytic properties of a phosphatase-like function rather than the usual hydrolase activity.…”